Katharina RöltgenpersName, Abigail E. PowellpersName, Oliver F. WirzpersName, Bryan StevenspersName, Catherine A. HoganpersName, Javaria NajeebpersName, Molly HunterpersName, Hannah WangpersName, Malaya K. SahoopersName, ChunHong HuangpersName, Fumiko YamamotopersName, Monali ManoharpersName, Justin ManalacpersName, Ana Rita Otrelo-CardosopersName, Tho D. PhampersName, Arjun RustagipersName, Angela J. RogerspersName, Nigam H. ShahpersName, Catherine A. BlishpersName, Jennifer R. CochranpersName, Theodore S. JardetzkypersName, James L. ZehnderpersName, Taia T. WangpersName, Balasubramanian NarasimhanpersName, Saurabh GombarpersName, Robert TibshiranipersName, Kari C. NadeaupersName, Peter S. KimpersName, Benjamin A. PinskypersName, Scott D. BoydpersName (2020date). “Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome.” Science Immunologytitle·venue.
Object of study — AN ANTIBODY RESPONSE MEASURED SIX WAYS OVER FIVE MONTHS - 983 plasma samples from 254 people, from asymptomatic to fatal, with the isotypesterm measured separately because summing them hides the decline
Research question — which features of the serological response track disease severityterm and outcome, and how long the response lasts
Contribution — isotype-resolved and antigen-resolved antibody kineticsterm across the full severity range, showing a decline from the first month and a spike-to-nucleocapsid ratio that separates mild from severe illness
Cross-disciplinary analysis
content-tags & facets:
phenomenon · claim-type · cross-domain-transfer · mechanism · claim-relation · replication-status · limitation — grounded in the full text| phenomenon | a biological disagreement in the literature that turns out to be partly a disagreement about instrument resolution — “We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately” | |
| claim-type | existence — “There is an urgent need to understand how long antibody titers against the virus persist after infection, now that the pandemic has been underway for more than half a year in many countries, and initial case reports of proven reinfection by SARS-CoV-2 have begun to appear ( 37 , 38 ). Studies differing in their patient populations, disease severity, and serological assays, have disagreed on the duration of SARS-CoV-2 antibody responses ( 14 , 16 , 32 ). Our data derived from inpatients, outpatients and asymptomatic individuals, with an additional asymptomatic validation cohort, show clearly that not only IgM and IgA, but also IgG titers to RBD, S1 and N antigens, RBD-ACE2 blocking activity and spike pseudotyped viral neutralization titers all begin to decrease in patients after approximately the first month post-onset of symptoms.” | |
| method — evidence | — “Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD.” | |
| object of study — evidence | — “We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness.” | |
| research question — evidence | — “Reports about the longevity of antibody titers to SARS-CoV-2 are not in full agreement, with some finding a rapid waning of virus-specific IgG antibodies by approximately 3 months after infection ( 14 , 15 ), and others emphasizing stable titers detected over several weeks or several months” | |
| contribution — evidence | — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood.” | |
| mechanism | THE LITERATURE DISAGREED ABOUT WHETHER IMMUNITY WANES, AND THE PAPER NAMES THE MEASUREMENT CHOICE THAT EXPLAINS IT: `reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on `PAN-Ig` assays that cannot evaluate each isotype separately`. An instrument that sums the isotypes cannot see one of them falling — “We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately” | mechanisms[0] |
| mechanism | and the disagreement is stated as a property of the studies rather than of the virus: `Studies differing in their patient populations, disease severity, and serological assays, have disagreed on the duration of SARS-CoV-2 antibody responses` — “There is an urgent need to understand how long antibody titers against the virus persist after infection, now that the pandemic has been underway for more than half a year in many countries, and initial case reports of proven reinfection by SARS-CoV-2 have begun to appear ( 37 , 38 ). Studies differing in their patient populations, disease severity, and serological assays, have disagreed on the duration of SARS-CoV-2 antibody responses ( 14 , 16 , 32 ). Our data derived from inpatients, outpatients and asymptomatic individuals, with an additional asymptomatic validation cohort, show clearly that not only IgM and IgA, but also IgG titers to RBD, S1 and N antigens, RBD-ACE2 blocking activity and spike pseudotyped viral neutralization titers all begin to decrease in patients after approximately the first month post-onset of symptoms.” | mechanisms[1] |
| mechanism | AND TWO FUNCTIONAL ASSAYS FOR ONE PROPERTY DISAGREE, WITH A MECHANISM: `RBD-ACE2 blocking was less sensitive than the neutralization assay, potentially because of antibodies that can neutralize by binding to non-RBD regions of the spike, or lower affinity antibodies that can neutralize in the cell culture assay but do not compete as well with binding of ACE2 under the blocking assay conditions` — “Neutralization, RBD-ACE2 blocking, and RBD-specific IgG were all highly correlated in patients with high antibody levels, but RBD-ACE2 blocking was less sensitive than the neutralization assay, potentially because of antibodies that can neutralize by binding to non-RBD regions of the spike, or lower affinity antibodies that can neutralize in the cell culture assay but do not compete as well with binding of ACE2 under the blocking assay conditions.” | mechanisms[2] |
| mechanism | and there is nothing to score them against: `It is currently unclear, however, which of these assays will be the best predictor of in vivo immunological protection from SARS-CoV-2 infection or reinfection`. Third record this session to say so outright — “It is currently unclear, however, which of these assays will be the best predictor of in vivo immunological protection from SARS-CoV-2 infection or reinfection in vaccinated or previously infected individuals.” | mechanisms[3] |
| mechanism | AND A RATIO CARRIES WHAT NEITHER SIDE OF IT DOES: `outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms`. Not the amount of antibody but WHICH TARGET IT PREFERS — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood.” | mechanisms[4] |
| mechanism | AND ONE MARKER WILL SEPARATE TWO CAUSES OF THE SAME SIGNAL: `Assuming spike-targeting vaccines ... become widely used, monitoring N-specific antibodies may be of utility in distinguishing vaccine-related antibodies from those stimulated by infection`. The antigen the vaccine omits becomes the discriminator — “Assuming spike-targeting vaccines are shown to be safe and effective and become widely used, monitoring N-specific antibodies may be of utility in distinguishing vaccine-related antibodies from those stimulated by infection.” | mechanisms[5] |
| contrasts-with | the reports finding stable SARS-CoV-2 antibody titers over months, which this paper attributes to pan-Ig assays that cannot resolve the isotypes — “We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately” | claim_relations[0] |
| supports | the finding that spike-targeting responses are higher in patients who recover, reported from a single-time-point antibody panel — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood.” | claim_relations[1] |
| sense | `antibody response duration` - CONTESTED, and the contest is partly instrumental: studies using `PAN-Ig` ASSAYS THAT CANNOT EVALUATE EACH ISOTYPE SEPARATELY report no decrease; isotype-resolved measurement finds one from the first month — “We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately” | senses[0] |
| sense | `neutralizing activity` - measured two ways that do not agree: a cell-culture neutralization assay and an RBD-ACE2 blocking assay, the latter `less sensitive` for named mechanistic reasons — “Neutralization, RBD-ACE2 blocking, and RBD-specific IgG were all highly correlated in patients with high antibody levels, but RBD-ACE2 blocking was less sensitive than the neutralization assay, potentially because of antibodies that can neutralize by binding to non-RBD regions of the spike, or lower affinity antibodies that can neutralize in the cell culture assay but do not compete as well with binding of ACE2 under the blocking assay conditions.” | senses[1] |
| sense | `seropositive` - a status that DECAYS, so `seroprevalence studies may, over time, underestimate the proportion ... previously infected`. The label expires while the history does not — “One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2.” | senses[2] |
| sense | Disease-severity specificity - mild outpatient illness is associated with relatively spike-focused IgG, while absolute antibody levels become highest in severe disease — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood. Outpatients and admitted non-ICU patients had the lowest viral loads in their nasopharyngeal swabs, but whether this is due to their antibody responses cannot be determined from this observational study. Associations of mortality in COVID-19 patients with SARS-CoV-2 viral load assessed by rRT-PCR applied to nasopharyngeal swab specimens have also been found in other studies ( 34 , 35 ). Our data are consistent with results reported from a panel of antibody assays applied to single time point samples from COVID-19 patients who recovered or died of their disease, which found higher values of spike-targeting responses in the convalescents ( 19 ). Patients with more severe illness in our study eventually raised higher antibody titers than those with milder disease, consistent with prior publications ( 32 , 36 ) and reports of other coronavirus infections ( 21 – 23 ). Patients with more severe disease also had somewhat higher viral loads than patients with milder illness, suggesting that larger initial amounts of viral antigen may contribute to their greater serological responses” | senses[3] |
| sense | Antibody quantity versus outcome - longitudinal inpatient response patterns span recovery and death and cannot by themselves explain clinical outcome — “In the detailed serological time courses of the hospitalized patients in this study it was evident that the patterns of antibody responses could not fully explain patient outcomes, including death. Substantial numbers of patients recovered from their illness and were discharged from hospital before they had formed detectable antibody responses, but minimal serological responses were also seen in patients who died of COVID-19 at early times post-onset of symptoms. Similarly, individuals with moderate antibody production were seen across the full spectrum of inpatient disease severity, and many patients who died of their disease generated high levels of antibodies, RBD-ACE2 blocking activity, and neutralizing titers. Differences between individuals in other aspects of the immune response or disease course, such as production of inflammatory mediators, T cell responses, host cell and tissue vulnerability to the damage during viral infection, coagulopathy, and secondary infections, are all likely to contribute to patient outcomes” | senses[4] |
| sense | Functional-assay scalability - live or pseudotyped-virus neutralization is physiologically relevant but harder to scale and standardize than purified-protein ELISA — “Neutralizing antibodies are increased in inpatients compared to outpatients, and correlate with RBD-ACE2 blocking and RBD IgG titers Antibody neutralization of live SARS-CoV-2, or of pseudotyped viruses such as lentiviruses expressing the SARS-CoV-2 spike may represent the most physiologically relevant surrogates for humoral immunity in vivo, but are poorly scalable due to restrictive biosafety requirements for SARS-CoV-2 and less easily standardized assay components and protocols, compared to tests using purified proteins. We tested SARS-CoV-2 spike-pseudotyped lentivirus neutralization ( 27 ) in HeLa cells overexpressing ACE2 ( 28 ) by inpatient and outpatient samples, and evaluated correlations with the more scalable ELISAs for RBD, S1 and RBD-ACE2 blocking ( Fig. 3A, B ). As with RBD-ACE2 blocking, neutralizing antibody activity was higher in inpatients compared to outpatients, and began to decrease after about one month after symptom onset. Neutralization was well-correlated with RBD IgG titers and RBD-ACE2 blocking (linear regression coefficient of determination R 2 of 0.6995 and 0.6824 for inpatients; 0.7338 and 0.6839 for outpatients). The RBD-ACE2 blocking assay was less sensitive than neutralization or RBD IgG ELISA. RBD IgM and IgA ELISA results were much more variable and did not correlate as well with neutralization or RBD-ACE2 blocking compared to RBD IgG” | senses[5] |
| sense | RNAemia - an operational plasma-RNA definition rather than proof of culturable virus — “A volume of 400 μL of EDTA-anticoagulated plasma was extracted by Qiagen EZ1 Virus Mini Kit v2.0 (Qiagen Germantown, MD). Molecular testing for the presence of SARS-CoV-2 RNA in plasma was performed with a modification of a published rRT-PCR assay targeting the envelope ( E ) gene ( 46 , 47 ). The standard Ct values of positive tests with this assay range from Ct <20 to 45 cycles. Testing of plasma samples with a Ct value of 40 or greater was repeated to ensure reproducibility of the positive result. As viral culture was not performed as part of this study, presence of SARS-CoV-2 in tested plasma was defined as RNAemia. Statistics GraphPad Prism version 8.0 (GraphPad Software, San Diego, California, USA) software was used to visualize ELISA data, analyze for differences in antibody responses between disease categories by Wilcoxon rank sum testing, and carry out linear regression of pseudotyped virus neutralization, RBD-ACE2 blocking and antibody ELISA results. The goodness of fit for linear regression analyses was reported as the coefficient of determination, R 2 . Locally estimated scatterplot smoothing for the development and decrease of antibody responses over time was performed using the loess method in the R statistical package version 3.6.1 ( 50 ). Correlation between antibody OD 450 values, RNAemia, and ACE2-RBD blocking assay OD 450 values were calculated as Spearman correlations with the R cor function” | senses[6] |
| sense | Waning antibody levels - binding, blocking, and neutralization measures all decline after roughly the first month, especially after mild or asymptomatic infection — “There is an urgent need to understand how long antibody titers against the virus persist after infection, now that the pandemic has been underway for more than half a year in many countries, and initial case reports of proven reinfection by SARS-CoV-2 have begun to appear ( 37 , 38 ). Studies differing in their patient populations, disease severity, and serological assays, have disagreed on the duration of SARS-CoV-2 antibody responses ( 14 , 16 , 32 ). Our data derived from inpatients, outpatients and asymptomatic individuals, with an additional asymptomatic validation cohort, show clearly that not only IgM and IgA, but also IgG titers to RBD, S1 and N antigens, RBD-ACE2 blocking activity and spike pseudotyped viral neutralization titers all begin to decrease in patients after approximately the first month post-onset of symptoms. The decline in antibody titers is most evident in individuals who had asymptomatic infection or mild illness, who produce lower levels of antibodies at the peak of their responses. We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately ( 16 ). Our data do not permit us to predict what fraction of the population will be susceptible to reinfection at a given time after their initial illness, or whether individuals will maintain sustained plateaus of lower antibody levels after an initial decrease; additional time and follow-up will be required to obtain this information” | senses[7] |
| sense | Serum-antibody versus immunity boundary - declining circulating antibody does not establish loss of mucosal, B-cell, or T-cell protection — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | senses[8] |
| sense | Seroprevalence undercount - antibody waning makes prior infection progressively less observable in population surveys — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | senses[9] |
| sense | Observational causal boundary - lower viral loads and spike-focused responses co-occur in milder disease, but this study cannot determine that antibodies caused the difference — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood. Outpatients and admitted non-ICU patients had the lowest viral loads in their nasopharyngeal swabs, but whether this is due to their antibody responses cannot be determined from this observational study. Associations of mortality in COVID-19 patients with SARS-CoV-2 viral load assessed by rRT-PCR applied to nasopharyngeal swab specimens have also been found in other studies ( 34 , 35 ). Our data are consistent with results reported from a panel of antibody assays applied to single time point samples from COVID-19 patients who recovered or died of their disease, which found higher values of spike-targeting responses in the convalescents ( 19 ). Patients with more severe illness in our study eventually raised higher antibody titers than those with milder disease, consistent with prior publications ( 32 , 36 ) and reports of other coronavirus infections ( 21 – 23 ). Patients with more severe disease also had somewhat higher viral loads than patients with milder illness, suggesting that larger initial amounts of viral antigen may contribute to their greater serological responses” | senses[10] |
| control | measuring IgM, IgG and IgA separately against three antigens rather than as a summed pan-Ig signal — held — “There is an urgent need to understand how long antibody titers against the virus persist after infection, now that the pandemic has been underway for more than half a year in many countries, and initial case reports of proven reinfection by SARS-CoV-2 have begun to appear ( 37 , 38 ). Studies differing in their patient populations, disease severity, and serological assays, have disagreed on the duration of SARS-CoV-2 antibody responses ( 14 , 16 , 32 ). Our data derived from inpatients, outpatients and asymptomatic individuals, with an additional asymptomatic validation cohort, show clearly that not only IgM and IgA, but also IgG titers to RBD, S1 and N antigens, RBD-ACE2 blocking activity and spike pseudotyped viral neutralization titers all begin to decrease in patients after approximately the first month post-onset of symptoms.” — rules out the decline being invisible - 'reported results FAILING TO FIND A DECREASE ... HAVE RELIED ON PAN-Ig ASSAYS THAT CANNOT EVALUATE EACH ISOTYPE SEPARATELY' — “We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately” | controls[0] |
| control | a prospective validation cohort with monthly sampling, independent of hospital stay — partial — “We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness.” — rules out partially, the confound the authors name: 'apart from the prospective validation cohort, the sampling time points ... were determined by their length of hospital stay' — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness.” | controls[1] |
| control | an in vivo standard of immunological protection to score the assays against — absent-not-in-staged-text — “It is currently unclear, however, which of these assays will be the best predictor of in vivo immunological protection from SARS-CoV-2 infection or reinfection in vaccinated or previously infected individuals.” — rules out nothing - 'It is currently unclear ... which of these assays will be the best predictor of in vivo immunological protection' — “It is currently unclear, however, which of these assays will be the best predictor of in vivo immunological protection from SARS-CoV-2 infection or reinfection in vaccinated or previously infected individuals.” | controls[2] |
| control | the antibody response as an explanation of who died — failed — “Differences between individuals in other aspects of the immune response or disease course, such as production of inflammatory mediators, T cell responses, host cell and tissue vulnerability to the damage during viral infection, coagulopathy, and secondary infections, are all likely to contribute to patient outcomes.” — rules out nothing, and the failure is reported in both directions: patients recovered 'before they had formed detectable antibody responses' and 'many patients who died ... generated high levels of antibodies' — “In the detailed serological time courses of the hospitalized patients in this study it was evident that the patterns of antibody responses could not fully explain patient outcomes, including death. Substantial numbers of patients recovered from their illness and were discharged from hospital before they had formed detectable antibody responses, but minimal serological responses were also seen in patients who died of COVID-19 at early times post-onset of symptoms. Similarly, individuals with moderate antibody production were seen across the full spectrum of inpatient disease severity, and many patients who died of their disease generated high levels of antibodies, RBD-ACE2 blocking activity, and neutralizing titers.” | controls[3] |
| weight | the reports finding stable SARS-CoV-2 antibody titers over months, which this paper attributes to pan-Ig assays that cannot resolve the isotypes — 0.9 — “We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately” | claim_relations[0] |
| weight | the finding that spike-targeting responses are higher in patients who recover, reported from a single-time-point antibody panel — 0.85 — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood.” | claim_relations[1] |
| note | RBD-ACE2 blocking —distinct-from→ neutralization assay — 'RBD-ACE2 blocking was less sensitive than the neutralization assay, potentially because of antibodies that can neutralize by binding to non-RBD regions of the spike, or lower affinity antibodies that can neutralize in the cell culture assay but do not compete as well with binding of ACE2'. Two instruments for one property, and the paper says WHY they diverge rather than only that they do — “Neutralization, RBD-ACE2 blocking, and RBD-specific IgG were all highly correlated in patients with high antibody levels, but RBD-ACE2 blocking was less sensitive than the neutralization assay, potentially because of antibodies that can neutralize by binding to non-RBD regions of the spike, or lower affinity antibodies that can neutralize in the cell culture assay but do not compete as well with binding of ACE2 under the blocking assay conditions.” | concept_edges[0] |
| weight | RBD-ACE2 blocking —distinct-from→ neutralization assay — 0.95 — “Neutralization, RBD-ACE2 blocking, and RBD-specific IgG were all highly correlated in patients with high antibody levels, but RBD-ACE2 blocking was less sensitive than the neutralization assay, potentially because of antibodies that can neutralize by binding to non-RBD regions of the spike, or lower affinity antibodies that can neutralize in the cell culture assay but do not compete as well with binding of ACE2 under the blocking assay conditions.” | concept_edges[0] |
| note | SARS-CoV-2 —part-of→ Sarbecovirus subgenus — The virus is stated to belong to the named subgenus. — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2” | concept_edges[3] |
| note | receptor binding domain (RBD) —part-of→ spike surface glycoprotein — The spike glycoprotein is explicitly said to contain RBD. — “The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors” | concept_edges[4] |
| note | spike —part-of→ structural proteins — Spike is included in the four-protein coronavirus structural set. — “Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment” | concept_edges[5] |
| note | SARS-CoV RBD —distinct-from→ SARS-CoV-2 RBD — Failure of most monoclonals to cross-bind indicates distinct antigenicity. — “Most monoclonal antibodies targeting SARS-CoV RBD fail to bind SARS-CoV-2 RBD, indicating distinct antigenicity despite sequence and structural similarity of the two proteins” | concept_edges[6] |
| note | RBD IgM and IgA —contrasted-with→ RBD IgG — IgM/IgA variability and correlations are directly compared with IgG. — “RBD IgM and IgA ELISA results were much more variable and did not correlate as well with neutralization or RBD-ACE2 blocking compared to RBD IgG” | concept_edges[7] |
| note | RNAemia —operationalised-as→ presence of SARS-CoV-2 in tested plasma — Direct defined-as statement, explicitly conditioned on absence of viral culture. — “presence of SARS-CoV-2 in tested plasma was defined as RNAemia.” | concept_edges[8] |
| relation | RNAemia —operationalised-as→ presence of SARS-CoV-2 in tested plasma — presence of SARS-CoV-2 in tested plasma was defined as RNAemia. Statistics GraphPad Prism version 8.0 — “presence of SARS-CoV-2 in tested plasma was defined as RNAemia.” | concept_edges[8] |
| note | vaccination strategies —contrasted-with→ natural infection — The paper says vaccine strategies differ from natural infection in multiple named ways. — “The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants” | concept_edges[9] |
| audit | decreasing antibody levels —contrasted-with→ all immunity will be lost — Audit 2026-09-28: DOUBTFUL - read against its full span and kept, but the span says decreasing antibody levels do NOT NECESSARILY indicate lost immunity; contrasted-with is loose. — “decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure” | concept_edges[10] |
| note | decreasing antibody levels —contrasted-with→ all immunity will be lost — The paper explicitly rejects equating serum decline with total immunity loss. — “decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure” | concept_edges[10] |
| evidence status note | concept — A FIGURE/TABLE CAPTION IS JOINED INTO THE 'evidence' QUOTE, beginning at character 1411: 'Fig. 4 Development of anti-SARS-CoV-2 spike RBD antibody responses in …'. The page prints the caption apart from the paragraph; read the quote as the paragraph, then the caption. — “Most individuals who become infected with SARS-CoV-2 infection do not require hospitalization to recover from their illness, and a sizeable fraction (approximately 40-45%) ( 29 ) remain asymptomatic. We carried out further analysis of antibody responses in a larger set of samples from 136 outpatients and asymptomatic individuals who tested positive by rRT-PCR for SARS-CoV-2 RNA in nasopharyngeal swabs and had serological testing conducted in the Stanford Health Care Clinical Laboratory. We also tested an independent validation set of 45 plasma samples from an additional 14 asymptomatic or mildly ill individuals. As seen for outpatients in Fig. 2B , the asymptomatic and outpatient individuals showed relatively low titers and rapid decline of RBD IgM and IgG ( Fig. 4A , table S3). The timing of infection in asymptomatic individuals is less certain than for symptomatic individuals, whose symptoms usually develop within 2 to 14 days after exposure to the virus ( 30 ). Plotting of serological responses of outpatients and asymptomatic individuals relative to the date of their first positive rRT-PCR test for infection nonetheless showed a similar time course to that seen for outpatients plotted as days post onset of symptoms ( Fig. 2B ). Notably, the amount of viral RNA detected in diagnostic nasopharyngeal swabs was correlated with the antibody titers measured in these individuals ( Fig. 4B ). Fig. 4 Development of anti-SARS-CoV-2 spike RBD antibody responses in SARS-CoV-2 rRT-PCR+ asymptomatic individuals and outpatients over time. 176 plasma samples from 136 SARS-CoV-2 rRT-PCR+ individuals were tested for RBD IgM and IgG. The x-axis indicates the time following the first positive rRT-PCR test ( A ). Dotted lines denote the assay cutoff for positive results. RBD IgM and IgG are shown for the latest available timepoint for subjects with low (Ct >12-25), middle (Ct >25-35) and high (Ct >35-40) SARS-CoV-2 rRT-PCR Ct at diagnosis ( B ). 45 plasma samples collected from 14 rRT-PCR+ asymptomatic individuals and outpatients sampled at monthly intervals (V1 = enrollment, V2 to V5 = months 1 to 4 post-enrollment) were tested for RBD IgM, IgG, IgA at a dilution of 1:100, as well as RBD-ACE2 blocking antibodies at a dilution of 1:10 ( C ). The 45 plasma samples were further tested for pseudoviral neutralization at a dilution of 1:1250 ( D ). Box-whisker ELISA OD 450 and blocking/neutralization percent plots show the interquartile range as the box and the minimum and maximum values as the ends of the whiskers. Correlations between virus neutralization and RBD IgM, IgG, IgA, and RBD-ACE2 blocking are shown with superimposed simple linear regression and 95% confidence bands (grey shading) of the best-fit line ( E ). Plots show mean ELISA OD 450 values of duplicate measurements and average percent neutralization from duplicate testing in each of two replicate experiments. Plasma specimens from the independent validation cohort of asymptomatic or outpatient individuals were collected prospectively during monthly visits up to four months following recruitment ( Fig. 4C, D , fig. S9). RBD IgM, IgG and IgA, RBD-ACE2 blocking and neutralization assays all showed a progressive decrease during the months sampled. Neutralizing antibodies were best correlated with RBD IgG titers, and somewhat less well with RBD-ACE2 blocking ELISA in these individuals” | concepts[52] |
| evidence status note | concept — A FIGURE/TABLE CAPTION IS JOINED INTO THE 'evidence' QUOTE, beginning at character 1411: 'Fig. 4 Development of anti-SARS-CoV-2 spike RBD antibody responses in …'. The page prints the caption apart from the paragraph; read the quote as the paragraph, then the caption. — “Most individuals who become infected with SARS-CoV-2 infection do not require hospitalization to recover from their illness, and a sizeable fraction (approximately 40-45%) ( 29 ) remain asymptomatic. We carried out further analysis of antibody responses in a larger set of samples from 136 outpatients and asymptomatic individuals who tested positive by rRT-PCR for SARS-CoV-2 RNA in nasopharyngeal swabs and had serological testing conducted in the Stanford Health Care Clinical Laboratory. We also tested an independent validation set of 45 plasma samples from an additional 14 asymptomatic or mildly ill individuals. As seen for outpatients in Fig. 2B , the asymptomatic and outpatient individuals showed relatively low titers and rapid decline of RBD IgM and IgG ( Fig. 4A , table S3). The timing of infection in asymptomatic individuals is less certain than for symptomatic individuals, whose symptoms usually develop within 2 to 14 days after exposure to the virus ( 30 ). Plotting of serological responses of outpatients and asymptomatic individuals relative to the date of their first positive rRT-PCR test for infection nonetheless showed a similar time course to that seen for outpatients plotted as days post onset of symptoms ( Fig. 2B ). Notably, the amount of viral RNA detected in diagnostic nasopharyngeal swabs was correlated with the antibody titers measured in these individuals ( Fig. 4B ). Fig. 4 Development of anti-SARS-CoV-2 spike RBD antibody responses in SARS-CoV-2 rRT-PCR+ asymptomatic individuals and outpatients over time. 176 plasma samples from 136 SARS-CoV-2 rRT-PCR+ individuals were tested for RBD IgM and IgG. The x-axis indicates the time following the first positive rRT-PCR test ( A ). Dotted lines denote the assay cutoff for positive results. RBD IgM and IgG are shown for the latest available timepoint for subjects with low (Ct >12-25), middle (Ct >25-35) and high (Ct >35-40) SARS-CoV-2 rRT-PCR Ct at diagnosis ( B ). 45 plasma samples collected from 14 rRT-PCR+ asymptomatic individuals and outpatients sampled at monthly intervals (V1 = enrollment, V2 to V5 = months 1 to 4 post-enrollment) were tested for RBD IgM, IgG, IgA at a dilution of 1:100, as well as RBD-ACE2 blocking antibodies at a dilution of 1:10 ( C ). The 45 plasma samples were further tested for pseudoviral neutralization at a dilution of 1:1250 ( D ). Box-whisker ELISA OD 450 and blocking/neutralization percent plots show the interquartile range as the box and the minimum and maximum values as the ends of the whiskers. Correlations between virus neutralization and RBD IgM, IgG, IgA, and RBD-ACE2 blocking are shown with superimposed simple linear regression and 95% confidence bands (grey shading) of the best-fit line ( E ). Plots show mean ELISA OD 450 values of duplicate measurements and average percent neutralization from duplicate testing in each of two replicate experiments. Plasma specimens from the independent validation cohort of asymptomatic or outpatient individuals were collected prospectively during monthly visits up to four months following recruitment ( Fig. 4C, D , fig. S9). RBD IgM, IgG and IgA, RBD-ACE2 blocking and neutralization assays all showed a progressive decrease during the months sampled. Neutralizing antibodies were best correlated with RBD IgG titers, and somewhat less well with RBD-ACE2 blocking ELISA in these individuals” | concepts[71] |
| outcome | control — held — “We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately” — “There is an urgent need to understand how long antibody titers against the virus persist after infection, now that the pandemic has been underway for more than half a year in many countries, and initial case reports of proven reinfection by SARS-CoV-2 have begun to appear ( 37 , 38 ). Studies differing in their patient populations, disease severity, and serological assays, have disagreed on the duration of SARS-CoV-2 antibody responses ( 14 , 16 , 32 ). Our data derived from inpatients, outpatients and asymptomatic individuals, with an additional asymptomatic validation cohort, show clearly that not only IgM and IgA, but also IgG titers to RBD, S1 and N antigens, RBD-ACE2 blocking activity and spike pseudotyped viral neutralization titers all begin to decrease in patients after approximately the first month post-onset of symptoms.” | controls[0] |
| outcome | control — partial — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness.” — “We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness.” | controls[1] |
| outcome | control — absent-not-in-staged-text — “It is currently unclear, however, which of these assays will be the best predictor of in vivo immunological protection from SARS-CoV-2 infection or reinfection in vaccinated or previously infected individuals.” | controls[2] |
| outcome | control — failed — “In the detailed serological time courses of the hospitalized patients in this study it was evident that the patterns of antibody responses could not fully explain patient outcomes, including death. Substantial numbers of patients recovered from their illness and were discharged from hospital before they had formed detectable antibody responses, but minimal serological responses were also seen in patients who died of COVID-19 at early times post-onset of symptoms. Similarly, individuals with moderate antibody production were seen across the full spectrum of inpatient disease severity, and many patients who died of their disease generated high levels of antibodies, RBD-ACE2 blocking activity, and neutralizing titers.” — “Differences between individuals in other aspects of the immune response or disease course, such as production of inflammatory mediators, T cell responses, host cell and tissue vulnerability to the damage during viral infection, coagulopathy, and secondary infections, are all likely to contribute to patient outcomes.” | controls[3] |
| weight | larger initial amounts of viral antigen → greater serological responses — 0.6 — “larger initial amounts of viral antigen may contribute to their greater serological responses. Functional blocking of RBD-ACE2 interaction” | unhoused_relations[1] |
| weight | waning antibody levels → seroprevalence studies — 0.6 — “One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2” | unhoused_relations[2] |
| weight | higher ratios of spike RBD and S1 domains compared to N antigen → mildest illness — 0.8 — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood. Outpatients and admitted non-ICU patients had the lowest viral loads in their nasopharyngeal swabs, but whether this is due to their antibody responses cannot be determined from this observational study. Associations of mortality in COVID-19 patients with SARS-CoV-2 viral load assessed by rRT-PCR applied to nasopharyngeal swab specimens have also been found in other studies ( 34 , 35 ). Our data are consistent with results reported from a panel of antibody assays applied to single time point samples from COVID-19 patients who recovered or died of their disease, which found higher values of spike-targeting responses in the convalescents ( 19 ). Patients with more severe illness in our study eventually raised higher antibody titers than those with milder disease, consistent with prior publications ( 32 , 36 ) and reports of other coronavirus infections ( 21 – 23 ). Patients with more severe disease also had somewhat higher viral loads than patients with milder illness, suggesting that larger initial amounts of viral antigen may contribute to their greater serological responses” | unhoused_relations[6] |
| weight | local mucosal antibody production → SARS-CoV-2-infection upon re-exposure — 0.5 — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | unhoused_relations[10] |
| no type for this | cutoff value for seroconversion → historical negative control samples — The seroconversion cutoff is calculated from the historical-negative mean plus three standard deviations; calculated-from is absent and the gate does not admit the wording as operationalised-as. — “The cutoff value for seroconversion was calculated by adding 3 standard deviations to mean ELISA ODs of 94 historical negative control samples from healthy blood donors (collected before the pandemic for an unrelated seroprevalence study) obtained by testing the samples in all protein/isotype assays” | unhoused_relations[0] |
| no type for this | larger initial amounts of viral antigen → greater serological responses — Larger initial antigen amounts may contribute to greater serological responses; contributes-to is absent, and the explicitly hedged observational proposal is not forced into causes. — “larger initial amounts of viral antigen may contribute to their greater serological responses. Functional blocking of RBD-ACE2 interaction” | unhoused_relations[1] |
| no type for this | waning antibody levels → seroprevalence studies — Waning levels may make seroprevalence studies underestimate prior infection; biases-or-undercounts is absent from the governed types. — “One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2” | unhoused_relations[2] |
| no type for this | spike surface glycoprotein → viral entry into host cells — Spike plays a major role in viral attachment, membrane fusion, and entry; mediates is absent from the eight governed types. — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | unhoused_relations[3] |
| no type for this | clinical serology assays → spike — Clinical serology assays use spike and N as antigens; uses-as-antigen is absent from the eight governed types. — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | unhoused_relations[4] |
| no type for this | Reduction in RNAemia → appearance of plasma antibodies — RNAemia reduction is strongly correlated with antibody appearance; correlated-with is absent and is not forced into causation. — “Reduction in RNAemia was strongly correlated with the appearance of plasma antibodies (Spearman’s correlation coefficients of -0.47 for IgM, -0.43 for IgG, and -0.44 for IgA, p<0.001 for each)” | unhoused_relations[5] |
| no type for this | higher ratios of spike RBD and S1 domains compared to N antigen → mildest illness — Higher spike-to-N response ratios are associated with milder outpatient illness; associated-with is absent and observational causation is explicitly unresolved. — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood. Outpatients and admitted non-ICU patients had the lowest viral loads in their nasopharyngeal swabs, but whether this is due to their antibody responses cannot be determined from this observational study. Associations of mortality in COVID-19 patients with SARS-CoV-2 viral load assessed by rRT-PCR applied to nasopharyngeal swab specimens have also been found in other studies ( 34 , 35 ). Our data are consistent with results reported from a panel of antibody assays applied to single time point samples from COVID-19 patients who recovered or died of their disease, which found higher values of spike-targeting responses in the convalescents ( 19 ). Patients with more severe illness in our study eventually raised higher antibody titers than those with milder disease, consistent with prior publications ( 32 , 36 ) and reports of other coronavirus infections ( 21 – 23 ). Patients with more severe disease also had somewhat higher viral loads than patients with milder illness, suggesting that larger initial amounts of viral antigen may contribute to their greater serological responses” | unhoused_relations[6] |
| no type for this | Neutralization → RBD IgG titers — Neutralization is well correlated with RBD IgG titers; correlated-with is absent from the governed edge types. — “Neutralization was well-correlated with RBD IgG titers and RBD-ACE2 blocking (linear regression coefficient of determination R 2 of 0.6995 and 0.6824 for inpatients; 0.7338 and 0.6839 for outpatients). The RBD-ACE2 blocking assay was less sensitive than neutralization or RBD IgG ELISA” | unhoused_relations[7] |
| no type for this | sampling time points → length of hospital stay and subsequent healthcare visits — Retrospective sampling times were determined by hospital stay and later visits; determined-by is absent from the eight types. — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | unhoused_relations[8] |
| no type for this | three main patterns of response → disease categories — The response patterns were shared across disease categories rather than diagnostic of outcome; shared-across is absent from the eight types. — “Inpatient SARS-CoV-2 antibody time course patterns are associated with decreases in RNAemia, but not disease outcome We hypothesized that detailed examination of the time course of SARS-CoV-2 antibody responses could identify distinct patterns associated with admitted non-ICU patients ( Fig. 6 , fig. S10), ICU patients who recovered from their illness ( Fig. 7 , fig. S11A), or those who died ( Fig. 8 , fig. S11B). We identified three main patterns of response: at the time point closest to their discharge from hospital, or death, Group 1 individuals had SARS-CoV-2 antibody responses with no RBD IgG or RBD-ACE2 blocking activity; Group 2 individuals had up to 25% RBD-ACE2 blocking activity; and Group 3 individuals developed high levels of antibodies and over 25% RBD-ACE2 blocking activity. These patterns of response were not strongly associated with disease severity, but instead were shared across disease categories. Group 1 patients included admitted patients who recovered from their illness rapidly and were discharged before they had developed detectable antibody responses, but also included patients who recovered from their illness without antibody production after prolonged ICU stays, and patients who died of COVID-19 prior to antibody development. Similarly, Group 2 patterns with low levels of RBD-ACE2 blocking were shared across all three patient categories. In Group 3 (high levels of antibody production and >25% RBD-ACE2 blocking activity) admitted non-ICU patients differed from ICU or deceased patients in that they developed their robust antibody responses during short hospital stays prior to discharge, whereas ICU or deceased patients typically had prolonged hospital courses. In patients tested for neutralizing antibodies (13 admitted non-ICU patients, 16 ICU surviving patients, and 16 deceased patients), the results were closely correlated with IgG titers to RBD, or RBD-ACE2 blocking” | unhoused_relations[9] |
| no type for this | local mucosal antibody production → SARS-CoV-2-infection upon re-exposure — Local airway antibodies could help prevent or impede infection after re-exposure; protective-against is absent, and the claim is explicitly possible rather than established. — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | unhoused_relations[10] |
| limitation | THE SAMPLING SCHEDULE IS CONFOUNDED WITH THE OUTCOME, DECLARED: `the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness`. Sicker patients were measured more often because they were there to be measured — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness.” | limitations[0] |
| limitation | AND THE RESPONSE DOES NOT EXPLAIN THE OUTCOME: `the patterns of antibody responses could not fully explain patient outcomes, including death. Substantial numbers of patients recovered ... before they had formed detectable antibody responses, but minimal serological responses were also seen in patients who died ... and many patients who died of their disease generated high levels of antibodies` — “In the detailed serological time courses of the hospitalized patients in this study it was evident that the patterns of antibody responses could not fully explain patient outcomes, including death. Substantial numbers of patients recovered from their illness and were discharged from hospital before they had formed detectable antibody responses, but minimal serological responses were also seen in patients who died of COVID-19 at early times post-onset of symptoms. Similarly, individuals with moderate antibody production were seen across the full spectrum of inpatient disease severity, and many patients who died of their disease generated high levels of antibodies, RBD-ACE2 blocking activity, and neutralizing titers.” | limitations[1] |
| limitation | and the alternatives are named rather than left implicit: `production of inflammatory mediators, T cell responses, host cell and tissue vulnerability ..., coagulopathy, and secondary infections, are all likely to contribute to patient outcomes` — “Differences between individuals in other aspects of the immune response or disease course, such as production of inflammatory mediators, T cell responses, host cell and tissue vulnerability to the damage during viral infection, coagulopathy, and secondary infections, are all likely to contribute to patient outcomes.” | limitations[2] |
| limitation | AND THE MEASUREMENT DECAYS, SO THE POPULATION ESTIMATE DECAYS WITH IT: `One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected` — “One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2.” | limitations[3] |
| limitation | and a falling number is not the thing it appears to measure: `decreasing antibody levels do not necessarily indicate that all immunity will be lost ... Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response` — “It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure.” | limitations[4] |
Content tags
content-observations:
finding · quantity · variable · definition · key-term — grounded in the full text| finding | — “We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately” | content_tags.findings[0] |
| finding | — “There is an urgent need to understand how long antibody titers against the virus persist after infection, now that the pandemic has been underway for more than half a year in many countries, and initial case reports of proven reinfection by SARS-CoV-2 have begun to appear ( 37 , 38 ). Studies differing in their patient populations, disease severity, and serological assays, have disagreed on the duration of SARS-CoV-2 antibody responses ( 14 , 16 , 32 ). Our data derived from inpatients, outpatients and asymptomatic individuals, with an additional asymptomatic validation cohort, show clearly that not only IgM and IgA, but also IgG titers to RBD, S1 and N antigens, RBD-ACE2 blocking activity and spike pseudotyped viral neutralization titers all begin to decrease in patients after approximately the first month post-onset of symptoms.” | content_tags.findings[1] |
| finding | — “Neutralization, RBD-ACE2 blocking, and RBD-specific IgG were all highly correlated in patients with high antibody levels, but RBD-ACE2 blocking was less sensitive than the neutralization assay, potentially because of antibodies that can neutralize by binding to non-RBD regions of the spike, or lower affinity antibodies that can neutralize in the cell culture assay but do not compete as well with binding of ACE2 under the blocking assay conditions.” | content_tags.findings[2] |
| finding | — “It is currently unclear, however, which of these assays will be the best predictor of in vivo immunological protection from SARS-CoV-2 infection or reinfection in vaccinated or previously infected individuals.” | content_tags.findings[3] |
| finding | — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood.” | content_tags.findings[4] |
| finding | — “Assuming spike-targeting vaccines are shown to be safe and effective and become widely used, monitoring N-specific antibodies may be of utility in distinguishing vaccine-related antibodies from those stimulated by infection.” | content_tags.findings[5] |
| finding | — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness.” | content_tags.findings[6] |
| finding | — “In the detailed serological time courses of the hospitalized patients in this study it was evident that the patterns of antibody responses could not fully explain patient outcomes, including death. Substantial numbers of patients recovered from their illness and were discharged from hospital before they had formed detectable antibody responses, but minimal serological responses were also seen in patients who died of COVID-19 at early times post-onset of symptoms. Similarly, individuals with moderate antibody production were seen across the full spectrum of inpatient disease severity, and many patients who died of their disease generated high levels of antibodies, RBD-ACE2 blocking activity, and neutralizing titers.” | content_tags.findings[7] |
| finding | — “One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2.” | content_tags.findings[8] |
| finding | — “It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure.” | content_tags.findings[9] |
| quantity | 983 plasma samples — longitudinal samples from 254 people - 79 hospitalised, 175 outpatient or asymptomatic, 25 of whom died — “We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness.” | content_tags.quantities[0] |
| quantity | 5 months post-infection — the observation window over which antibodies in outpatients and asymptomatic individuals progressively decreased — “Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.” | content_tags.quantities[1] |
| variable | N-specific antibodies — “Assuming spike-targeting vaccines are shown to be safe and effective and become widely used, monitoring N-specific antibodies may be of utility in distinguishing vaccine-related antibodies from those stimulated by infection.” | content_tags.variables[0] |
| definition | pan-Ig — “We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately” | content_tags.definitions[0] |
Concepts
terms this paper carries, each with the sentence it was cut from — the layer that joins one paper to another. Terms with no quote yet are on CONCEPTS.html.
| concept | RBD-ACE2 blocking — not defined in this paper's own words; the scalable assay measuring antibodies that block receptor binding - less sensitive than neutralization — “Neutralization, RBD-ACE2 blocking, and RBD-specific IgG were all highly correlated in patients with high antibody levels, but RBD-ACE2 blocking was less sensitive than the neutralization assay, potentially because of antibodies that can neutralize by binding to non-RBD regions of the spike, or lower affinity antibodies that can neutralize in the cell culture assay but do not compete as well with binding of ACE2 under the blocking assay conditions.” | concepts[0] |
| concept | neutralization assay — not defined in this paper's own words; the cell-culture assay against which the blocking assay is compared — “Neutralization, RBD-ACE2 blocking, and RBD-specific IgG were all highly correlated in patients with high antibody levels, but RBD-ACE2 blocking was less sensitive than the neutralization assay, potentially because of antibodies that can neutralize by binding to non-RBD regions of the spike, or lower affinity antibodies that can neutralize in the cell culture assay but do not compete as well with binding of ACE2 under the blocking assay conditions.” | concepts[1] |
| concept | vaccine-related antibodies — not defined in this paper's own words; antibodies raised by a spike-targeting vaccine, lacking the nucleocapsid response — “Assuming spike-targeting vaccines are shown to be safe and effective and become widely used, monitoring N-specific antibodies may be of utility in distinguishing vaccine-related antibodies from those stimulated by infection.” | concepts[2] |
| concept | N-specific antibodies — not defined in this paper's own words; the marker proposed to tell vaccination from infection, because the vaccines omit that antigen — “Assuming spike-targeting vaccines are shown to be safe and effective and become widely used, monitoring N-specific antibodies may be of utility in distinguishing vaccine-related antibodies from those stimulated by infection.” | concepts[3] |
| concept | pan-Ig — not defined in this paper's own words; assays summing the isotypes, which therefore cannot see one of them falling — “We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately” | concepts[4] |
| concept | SARS-CoV-2-specific antibodies — not defined in this paper's own words; virus-specific antibodies whose levels and functions were followed longitudinally — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[5] |
| concept | viral spike receptor binding domain (RBD) — not defined in this paper's own words; spike domain binding the host ACE2 receptor — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[6] |
| concept | angiotensin-converting enzyme 2 (ACE2) receptor — not defined in this paper's own words; host receptor bound by spike RBD — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[7] |
| concept | 983 longitudinal plasma samples — not defined in this paper's own words; serial sample collection used for the principal analysis — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[8] |
| concept | hospitalized COVID-19 patients — not defined in this paper's own words; inpatient cohort component — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[9] |
| concept | outpatients and asymptomatic individuals — not defined in this paper's own words; mild or symptom-free cohort component — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[10] |
| concept | S1 — not defined in this paper's own words; spike subdomain used as a serological antigen — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[11] |
| concept | RBD domains — not defined in this paper's own words; spike receptor-binding targets compared with nucleocapsid — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[12] |
| concept | nucleocapsid antigen — not defined in this paper's own words; non-spike antigen used in specificity ratios — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[13] |
| concept | viral RNAemia — not defined in this paper's own words; viral RNA detected in plasma — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[14] |
| concept | Pseudovirus neutralization assays — not defined in this paper's own words; functional assay using spike-pseudotyped virus — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[15] |
| concept | scalable ELISA — not defined in this paper's own words; protein-based assay measuring RBD-ACE2 blocking antibodies — “Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients. SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection” | concepts[16] |
| concept | RBD-specific IgG — not defined in this paper's own words; RBD-binding IgG correlated with both functional assays — “Functional blocking of RBD-ACE2 interaction, and spike-pseudotyped virus neutralization by patient antibodies appeared with a similar time course to IgM, IgG, and IgA, but were most closely correlated in magnitude with IgG titers. Neutralization, RBD-ACE2 blocking, and RBD-specific IgG were all highly correlated in patients with high antibody levels, but RBD-ACE2 blocking was less sensitive than the neutralization assay, potentially because of antibodies that can neutralize by binding to non-RBD regions of the spike, or lower affinity antibodies that can neutralize in the cell culture assay but do not compete as well with binding of ACE2 under the blocking assay conditions. It is currently unclear, however, which of these assays will be the best predictor of in vivo immunological protection from SARS-CoV-2 infection or reinfection in vaccinated or previously infected individuals” | concepts[17] |
| concept | antibody titers — not defined in this paper's own words; quantitative antibody levels followed over time — “Current information on the role of antibodies in viral clearance and modulation of disease severity as well as the durability of these responses following primary infection is limited or controversial. Improved understanding of humoral immunity to SARS-CoV-2 is needed to inform strategies for vaccination and the use of therapeutics in the form of neutralizing antibodies or convalescent plasma. Reports about the longevity of antibody titers to SARS-CoV-2 are not in full agreement, with some finding a rapid waning of virus-specific IgG antibodies by approximately 3 months after infection ( 14 , 15 ), and others emphasizing stable titers detected over several weeks or several months ( 16 – 18 ). Virus-specific antibody responses appear to be elevated in COVID-19 patients with severe disease as opposed to asymptomatic or mildly ill individuals, raising concerns about the effectiveness of antibody responses to SARS-CoV-2. A suggestion that the quality rather than quantity of antibodies may predict the outcome of infection is provided by a recent report applying a panel of serological assays to COVID-19 patients who convalesced or died ( 19 ). Nonhuman primates challenged with SARS-CoV-2 after vaccination with spike-based DNA vaccines developed neutralizing antibodies and immune correlates of protection, suggesting that antibody responses may be more effective in preventing than resolving disease” | concepts[18] |
| concept | asymptomatic infection — not defined in this paper's own words; least severe point on the clinical spectrum — “The clinical spectrum of COVID-19 ranges from asymptomatic infection and mild upper respiratory tract illness in the majority of patients, to severe viral pneumonia with respiratory failure, multiorgan failure, and death ( 2 – 4 ). Older adults and people with serious underlying health conditions are at greatest risk for severe illness and death” | concepts[19] |
| concept | severe viral pneumonia — not defined in this paper's own words; severe COVID-19 manifestation — “The clinical spectrum of COVID-19 ranges from asymptomatic infection and mild upper respiratory tract illness in the majority of patients, to severe viral pneumonia with respiratory failure, multiorgan failure, and death ( 2 – 4 ). Older adults and people with serious underlying health conditions are at greatest risk for severe illness and death” | concepts[20] |
| concept | respiratory failure — not defined in this paper's own words; severe clinical outcome — “The clinical spectrum of COVID-19 ranges from asymptomatic infection and mild upper respiratory tract illness in the majority of patients, to severe viral pneumonia with respiratory failure, multiorgan failure, and death ( 2 – 4 ). Older adults and people with serious underlying health conditions are at greatest risk for severe illness and death” | concepts[21] |
| concept | multiorgan failure — not defined in this paper's own words; severe clinical outcome — “The clinical spectrum of COVID-19 ranges from asymptomatic infection and mild upper respiratory tract illness in the majority of patients, to severe viral pneumonia with respiratory failure, multiorgan failure, and death ( 2 – 4 ). Older adults and people with serious underlying health conditions are at greatest risk for severe illness and death” | concepts[22] |
| concept | Sarbecovirus subgenus — not defined in this paper's own words; taxonomic subgenus containing SARS-CoV-2 — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[23] |
| concept | Betacoronavirus — not defined in this paper's own words; genus containing Sarbecovirus — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[24] |
| concept | SARS-CoV — not defined in this paper's own words; related severe acute respiratory syndrome coronavirus — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[25] |
| concept | structural proteins — not defined in this paper's own words; coronavirus spike, envelope, membrane, and nucleocapsid proteins — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[26] |
| concept | spike — not defined in this paper's own words; surface structural protein containing RBD — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[27] |
| concept | envelope — not defined in this paper's own words; coronavirus structural protein — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[28] |
| concept | membrane — not defined in this paper's own words; coronavirus structural protein — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[29] |
| concept | nucleocapsid (N) proteins — not defined in this paper's own words; coronavirus structural protein and serology antigen — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[30] |
| concept | spike surface glycoprotein — not defined in this paper's own words; surface glycoprotein mediating entry — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[31] |
| concept | receptor binding domain (RBD) — not defined in this paper's own words; ACE2-binding portion of spike — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[32] |
| concept | viral attachment — not defined in this paper's own words; first spike-mediated entry function — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[33] |
| concept | fusion of viral and host membranes — not defined in this paper's own words; membrane-fusion stage of viral entry — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[34] |
| concept | viral entry into host cells — not defined in this paper's own words; entry blocked by neutralizing antibodies — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[35] |
| concept | clinical serology assays — not defined in this paper's own words; assays using spike and N antigens — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[36] |
| concept | neutralizing antibodies — not defined in this paper's own words; antibodies capable of preventing viral entry — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[37] |
| concept | viral clearance — not defined in this paper's own words; removal of virus whose antibody contribution was uncertain — “Current information on the role of antibodies in viral clearance and modulation of disease severity as well as the durability of these responses following primary infection is limited or controversial. Improved understanding of humoral immunity to SARS-CoV-2 is needed to inform strategies for vaccination and the use of therapeutics in the form of neutralizing antibodies or convalescent plasma. Reports about the longevity of antibody titers to SARS-CoV-2 are not in full agreement, with some finding a rapid waning of virus-specific IgG antibodies by approximately 3 months after infection ( 14 , 15 ), and others emphasizing stable titers detected over several weeks or several months ( 16 – 18 ). Virus-specific antibody responses appear to be elevated in COVID-19 patients with severe disease as opposed to asymptomatic or mildly ill individuals, raising concerns about the effectiveness of antibody responses to SARS-CoV-2. A suggestion that the quality rather than quantity of antibodies may predict the outcome of infection is provided by a recent report applying a panel of serological assays to COVID-19 patients who convalesced or died ( 19 ). Nonhuman primates challenged with SARS-CoV-2 after vaccination with spike-based DNA vaccines developed neutralizing antibodies and immune correlates of protection, suggesting that antibody responses may be more effective in preventing than resolving disease” | concepts[38] |
| concept | disease severity — not defined in this paper's own words; clinical severity dimension compared with serological responses — “Current information on the role of antibodies in viral clearance and modulation of disease severity as well as the durability of these responses following primary infection is limited or controversial. Improved understanding of humoral immunity to SARS-CoV-2 is needed to inform strategies for vaccination and the use of therapeutics in the form of neutralizing antibodies or convalescent plasma. Reports about the longevity of antibody titers to SARS-CoV-2 are not in full agreement, with some finding a rapid waning of virus-specific IgG antibodies by approximately 3 months after infection ( 14 , 15 ), and others emphasizing stable titers detected over several weeks or several months ( 16 – 18 ). Virus-specific antibody responses appear to be elevated in COVID-19 patients with severe disease as opposed to asymptomatic or mildly ill individuals, raising concerns about the effectiveness of antibody responses to SARS-CoV-2. A suggestion that the quality rather than quantity of antibodies may predict the outcome of infection is provided by a recent report applying a panel of serological assays to COVID-19 patients who convalesced or died ( 19 ). Nonhuman primates challenged with SARS-CoV-2 after vaccination with spike-based DNA vaccines developed neutralizing antibodies and immune correlates of protection, suggesting that antibody responses may be more effective in preventing than resolving disease” | concepts[39] |
| concept | durability — not defined in this paper's own words; persistence of responses after primary infection — “Current information on the role of antibodies in viral clearance and modulation of disease severity as well as the durability of these responses following primary infection is limited or controversial. Improved understanding of humoral immunity to SARS-CoV-2 is needed to inform strategies for vaccination and the use of therapeutics in the form of neutralizing antibodies or convalescent plasma. Reports about the longevity of antibody titers to SARS-CoV-2 are not in full agreement, with some finding a rapid waning of virus-specific IgG antibodies by approximately 3 months after infection ( 14 , 15 ), and others emphasizing stable titers detected over several weeks or several months ( 16 – 18 ). Virus-specific antibody responses appear to be elevated in COVID-19 patients with severe disease as opposed to asymptomatic or mildly ill individuals, raising concerns about the effectiveness of antibody responses to SARS-CoV-2. A suggestion that the quality rather than quantity of antibodies may predict the outcome of infection is provided by a recent report applying a panel of serological assays to COVID-19 patients who convalesced or died ( 19 ). Nonhuman primates challenged with SARS-CoV-2 after vaccination with spike-based DNA vaccines developed neutralizing antibodies and immune correlates of protection, suggesting that antibody responses may be more effective in preventing than resolving disease” | concepts[40] |
| concept | humoral immunity — not defined in this paper's own words; antibody-mediated immune response to SARS-CoV-2 — “Current information on the role of antibodies in viral clearance and modulation of disease severity as well as the durability of these responses following primary infection is limited or controversial. Improved understanding of humoral immunity to SARS-CoV-2 is needed to inform strategies for vaccination and the use of therapeutics in the form of neutralizing antibodies or convalescent plasma. Reports about the longevity of antibody titers to SARS-CoV-2 are not in full agreement, with some finding a rapid waning of virus-specific IgG antibodies by approximately 3 months after infection ( 14 , 15 ), and others emphasizing stable titers detected over several weeks or several months ( 16 – 18 ). Virus-specific antibody responses appear to be elevated in COVID-19 patients with severe disease as opposed to asymptomatic or mildly ill individuals, raising concerns about the effectiveness of antibody responses to SARS-CoV-2. A suggestion that the quality rather than quantity of antibodies may predict the outcome of infection is provided by a recent report applying a panel of serological assays to COVID-19 patients who convalesced or died ( 19 ). Nonhuman primates challenged with SARS-CoV-2 after vaccination with spike-based DNA vaccines developed neutralizing antibodies and immune correlates of protection, suggesting that antibody responses may be more effective in preventing than resolving disease” | concepts[41] |
| concept | vaccination — not defined in this paper's own words; preventive strategy informed by humoral-immunity findings — “Current information on the role of antibodies in viral clearance and modulation of disease severity as well as the durability of these responses following primary infection is limited or controversial. Improved understanding of humoral immunity to SARS-CoV-2 is needed to inform strategies for vaccination and the use of therapeutics in the form of neutralizing antibodies or convalescent plasma. Reports about the longevity of antibody titers to SARS-CoV-2 are not in full agreement, with some finding a rapid waning of virus-specific IgG antibodies by approximately 3 months after infection ( 14 , 15 ), and others emphasizing stable titers detected over several weeks or several months ( 16 – 18 ). Virus-specific antibody responses appear to be elevated in COVID-19 patients with severe disease as opposed to asymptomatic or mildly ill individuals, raising concerns about the effectiveness of antibody responses to SARS-CoV-2. A suggestion that the quality rather than quantity of antibodies may predict the outcome of infection is provided by a recent report applying a panel of serological assays to COVID-19 patients who convalesced or died ( 19 ). Nonhuman primates challenged with SARS-CoV-2 after vaccination with spike-based DNA vaccines developed neutralizing antibodies and immune correlates of protection, suggesting that antibody responses may be more effective in preventing than resolving disease” | concepts[42] |
| concept | convalescent plasma — not defined in this paper's own words; antibody-containing therapeutic product — “Current information on the role of antibodies in viral clearance and modulation of disease severity as well as the durability of these responses following primary infection is limited or controversial. Improved understanding of humoral immunity to SARS-CoV-2 is needed to inform strategies for vaccination and the use of therapeutics in the form of neutralizing antibodies or convalescent plasma. Reports about the longevity of antibody titers to SARS-CoV-2 are not in full agreement, with some finding a rapid waning of virus-specific IgG antibodies by approximately 3 months after infection ( 14 , 15 ), and others emphasizing stable titers detected over several weeks or several months ( 16 – 18 ). Virus-specific antibody responses appear to be elevated in COVID-19 patients with severe disease as opposed to asymptomatic or mildly ill individuals, raising concerns about the effectiveness of antibody responses to SARS-CoV-2. A suggestion that the quality rather than quantity of antibodies may predict the outcome of infection is provided by a recent report applying a panel of serological assays to COVID-19 patients who convalesced or died ( 19 ). Nonhuman primates challenged with SARS-CoV-2 after vaccination with spike-based DNA vaccines developed neutralizing antibodies and immune correlates of protection, suggesting that antibody responses may be more effective in preventing than resolving disease” | concepts[43] |
| concept | virus-specific IgG antibodies — not defined in this paper's own words; IgG whose reported longevity differed across studies — “Current information on the role of antibodies in viral clearance and modulation of disease severity as well as the durability of these responses following primary infection is limited or controversial. Improved understanding of humoral immunity to SARS-CoV-2 is needed to inform strategies for vaccination and the use of therapeutics in the form of neutralizing antibodies or convalescent plasma. Reports about the longevity of antibody titers to SARS-CoV-2 are not in full agreement, with some finding a rapid waning of virus-specific IgG antibodies by approximately 3 months after infection ( 14 , 15 ), and others emphasizing stable titers detected over several weeks or several months ( 16 – 18 ). Virus-specific antibody responses appear to be elevated in COVID-19 patients with severe disease as opposed to asymptomatic or mildly ill individuals, raising concerns about the effectiveness of antibody responses to SARS-CoV-2. A suggestion that the quality rather than quantity of antibodies may predict the outcome of infection is provided by a recent report applying a panel of serological assays to COVID-19 patients who convalesced or died ( 19 ). Nonhuman primates challenged with SARS-CoV-2 after vaccination with spike-based DNA vaccines developed neutralizing antibodies and immune correlates of protection, suggesting that antibody responses may be more effective in preventing than resolving disease” | concepts[44] |
| concept | spike-pseudotyped lentivirus neutralization — not defined in this paper's own words; functional neutralization assay in the study — “We performed a comprehensive analysis of SARS-CoV-2 RBD, S1 and N protein specific antibodies, and RBD-ACE2 blocking antibodies, spike-pseudotyped virus neutralization, and viral RNA measurements in nasopharyngeal swab and plasma samples of individuals infected with SARS-CoV-2. A total of 254 individuals (79 inpatients and 175 outpatients or asymptomatic individuals) were studied. RBD-ACE2 blocking antibodies and spike-pseudotyped lentivirus neutralization were well-correlated with IgG specific for RBD. Notably, higher ratios of IgG antibodies targeting S1 or RBD compared to N were strongly associated with clinically milder infection. Viral RNAemia decreased to undetectable levels rapidly once plasma antibodies and RBD-ACE2 blocking activity appeared. Outpatients and asymptomatic individuals show substantial and progressive decreases in SARS-CoV-2 specific antibodies after the first month of documented infection” | concepts[45] |
| concept | nasopharyngeal swab — not defined in this paper's own words; specimen source for diagnostic viral RNA measurement — “We performed a comprehensive analysis of SARS-CoV-2 RBD, S1 and N protein specific antibodies, and RBD-ACE2 blocking antibodies, spike-pseudotyped virus neutralization, and viral RNA measurements in nasopharyngeal swab and plasma samples of individuals infected with SARS-CoV-2. A total of 254 individuals (79 inpatients and 175 outpatients or asymptomatic individuals) were studied. RBD-ACE2 blocking antibodies and spike-pseudotyped lentivirus neutralization were well-correlated with IgG specific for RBD. Notably, higher ratios of IgG antibodies targeting S1 or RBD compared to N were strongly associated with clinically milder infection. Viral RNAemia decreased to undetectable levels rapidly once plasma antibodies and RBD-ACE2 blocking activity appeared. Outpatients and asymptomatic individuals show substantial and progressive decreases in SARS-CoV-2 specific antibodies after the first month of documented infection” | concepts[46] |
| concept | viral RNA measurements — not defined in this paper's own words; measurements in swab and plasma specimens — “We performed a comprehensive analysis of SARS-CoV-2 RBD, S1 and N protein specific antibodies, and RBD-ACE2 blocking antibodies, spike-pseudotyped virus neutralization, and viral RNA measurements in nasopharyngeal swab and plasma samples of individuals infected with SARS-CoV-2. A total of 254 individuals (79 inpatients and 175 outpatients or asymptomatic individuals) were studied. RBD-ACE2 blocking antibodies and spike-pseudotyped lentivirus neutralization were well-correlated with IgG specific for RBD. Notably, higher ratios of IgG antibodies targeting S1 or RBD compared to N were strongly associated with clinically milder infection. Viral RNAemia decreased to undetectable levels rapidly once plasma antibodies and RBD-ACE2 blocking activity appeared. Outpatients and asymptomatic individuals show substantial and progressive decreases in SARS-CoV-2 specific antibodies after the first month of documented infection” | concepts[47] |
| concept | real-time RT-PCR — not defined in this paper's own words; diagnostic assay confirming cohort infection — “A total of 254 individuals with a positive SARS-CoV-2 real-time RT-PCR (rRT-PCR) nasopharyngeal swab test were included in the study (fig. S1). Study subjects were identified either after routine serology testing or occupational health screening in the Stanford Health Care Clinical Laboratories for anti-SARS-CoV-2 RBD IgM and IgG antibodies (136 asymptomatic individuals or outpatients), or after they reported to Stanford Health Care-associated clinical sites with symptoms of COVID-19. This included 24 outpatients, 35 hospitalized patients who were not admitted to the intensive care unit (ICU), and 20 ICU inpatients who survived their illness. To evaluate serological responses associated with patient mortality, we also analyzed specimens from 25 patients who died of COVID-19 (one outpatient, seven admitted non-ICU, and seventeen admitted ICU patients). Of patients who were treated in the ICU, 26 of 37 (70%) required mechanical ventilation, including 15 patients who died. Demographic and clinical characteristics of patients stratified by disease status are presented in Table 1 . The percentage of males, and those with comorbidities of hypertension or diabetes mellitus increased with patient disease severity. Outpatients and asymptomatic individuals were younger and had the lowest levels of obesity compared to more severely ill patients. Notably, levels of viral RNA measured by rRT-PCR of nasopharyngeal swabs at diagnosis showed a progressive increase (lower rRT-PCR cycle threshold, Ct) with disease severity; patients who died had the highest viral loads (fig. S2). Plasma samples for in-depth serological testing were available from all inpatients and 86 of the 160 outpatient survivors. Demographic and clinical characteristics of outpatients and asymptomatic individuals with and without plasma availability are presented in table S1. A total of 828 samples were analyzed with ELISAs measuring IgM, IgG and IgA specific for SARS-CoV-2 RBD, S1 or N, as well as the RBD-ACE2 blocking assay ( Fig. 1A-C ). Representative samples were also tested for antibody cross-reactivity to SARS-CoV RBD, viral RNA in the blood (RNAemia), and neutralization of spike-pseudotyped lentivirus” | concepts[48] |
| concept | intensive care unit (ICU) — not defined in this paper's own words; highest-acuity inpatient setting — “A total of 254 individuals with a positive SARS-CoV-2 real-time RT-PCR (rRT-PCR) nasopharyngeal swab test were included in the study (fig. S1). Study subjects were identified either after routine serology testing or occupational health screening in the Stanford Health Care Clinical Laboratories for anti-SARS-CoV-2 RBD IgM and IgG antibodies (136 asymptomatic individuals or outpatients), or after they reported to Stanford Health Care-associated clinical sites with symptoms of COVID-19. This included 24 outpatients, 35 hospitalized patients who were not admitted to the intensive care unit (ICU), and 20 ICU inpatients who survived their illness. To evaluate serological responses associated with patient mortality, we also analyzed specimens from 25 patients who died of COVID-19 (one outpatient, seven admitted non-ICU, and seventeen admitted ICU patients). Of patients who were treated in the ICU, 26 of 37 (70%) required mechanical ventilation, including 15 patients who died. Demographic and clinical characteristics of patients stratified by disease status are presented in Table 1 . The percentage of males, and those with comorbidities of hypertension or diabetes mellitus increased with patient disease severity. Outpatients and asymptomatic individuals were younger and had the lowest levels of obesity compared to more severely ill patients. Notably, levels of viral RNA measured by rRT-PCR of nasopharyngeal swabs at diagnosis showed a progressive increase (lower rRT-PCR cycle threshold, Ct) with disease severity; patients who died had the highest viral loads (fig. S2). Plasma samples for in-depth serological testing were available from all inpatients and 86 of the 160 outpatient survivors. Demographic and clinical characteristics of outpatients and asymptomatic individuals with and without plasma availability are presented in table S1. A total of 828 samples were analyzed with ELISAs measuring IgM, IgG and IgA specific for SARS-CoV-2 RBD, S1 or N, as well as the RBD-ACE2 blocking assay ( Fig. 1A-C ). Representative samples were also tested for antibody cross-reactivity to SARS-CoV RBD, viral RNA in the blood (RNAemia), and neutralization of spike-pseudotyped lentivirus” | concepts[49] |
| concept | mechanical ventilation — not defined in this paper's own words; respiratory support received by many ICU patients — “A total of 254 individuals with a positive SARS-CoV-2 real-time RT-PCR (rRT-PCR) nasopharyngeal swab test were included in the study (fig. S1). Study subjects were identified either after routine serology testing or occupational health screening in the Stanford Health Care Clinical Laboratories for anti-SARS-CoV-2 RBD IgM and IgG antibodies (136 asymptomatic individuals or outpatients), or after they reported to Stanford Health Care-associated clinical sites with symptoms of COVID-19. This included 24 outpatients, 35 hospitalized patients who were not admitted to the intensive care unit (ICU), and 20 ICU inpatients who survived their illness. To evaluate serological responses associated with patient mortality, we also analyzed specimens from 25 patients who died of COVID-19 (one outpatient, seven admitted non-ICU, and seventeen admitted ICU patients). Of patients who were treated in the ICU, 26 of 37 (70%) required mechanical ventilation, including 15 patients who died. Demographic and clinical characteristics of patients stratified by disease status are presented in Table 1 . The percentage of males, and those with comorbidities of hypertension or diabetes mellitus increased with patient disease severity. Outpatients and asymptomatic individuals were younger and had the lowest levels of obesity compared to more severely ill patients. Notably, levels of viral RNA measured by rRT-PCR of nasopharyngeal swabs at diagnosis showed a progressive increase (lower rRT-PCR cycle threshold, Ct) with disease severity; patients who died had the highest viral loads (fig. S2). Plasma samples for in-depth serological testing were available from all inpatients and 86 of the 160 outpatient survivors. Demographic and clinical characteristics of outpatients and asymptomatic individuals with and without plasma availability are presented in table S1. A total of 828 samples were analyzed with ELISAs measuring IgM, IgG and IgA specific for SARS-CoV-2 RBD, S1 or N, as well as the RBD-ACE2 blocking assay ( Fig. 1A-C ). Representative samples were also tested for antibody cross-reactivity to SARS-CoV RBD, viral RNA in the blood (RNAemia), and neutralization of spike-pseudotyped lentivirus” | concepts[50] |
| concept | cycle threshold, Ct — not defined in this paper's own words; inverse rRT-PCR indicator of viral RNA quantity — “A total of 254 individuals with a positive SARS-CoV-2 real-time RT-PCR (rRT-PCR) nasopharyngeal swab test were included in the study (fig. S1). Study subjects were identified either after routine serology testing or occupational health screening in the Stanford Health Care Clinical Laboratories for anti-SARS-CoV-2 RBD IgM and IgG antibodies (136 asymptomatic individuals or outpatients), or after they reported to Stanford Health Care-associated clinical sites with symptoms of COVID-19. This included 24 outpatients, 35 hospitalized patients who were not admitted to the intensive care unit (ICU), and 20 ICU inpatients who survived their illness. To evaluate serological responses associated with patient mortality, we also analyzed specimens from 25 patients who died of COVID-19 (one outpatient, seven admitted non-ICU, and seventeen admitted ICU patients). Of patients who were treated in the ICU, 26 of 37 (70%) required mechanical ventilation, including 15 patients who died. Demographic and clinical characteristics of patients stratified by disease status are presented in Table 1 . The percentage of males, and those with comorbidities of hypertension or diabetes mellitus increased with patient disease severity. Outpatients and asymptomatic individuals were younger and had the lowest levels of obesity compared to more severely ill patients. Notably, levels of viral RNA measured by rRT-PCR of nasopharyngeal swabs at diagnosis showed a progressive increase (lower rRT-PCR cycle threshold, Ct) with disease severity; patients who died had the highest viral loads (fig. S2). Plasma samples for in-depth serological testing were available from all inpatients and 86 of the 160 outpatient survivors. Demographic and clinical characteristics of outpatients and asymptomatic individuals with and without plasma availability are presented in table S1. A total of 828 samples were analyzed with ELISAs measuring IgM, IgG and IgA specific for SARS-CoV-2 RBD, S1 or N, as well as the RBD-ACE2 blocking assay ( Fig. 1A-C ). Representative samples were also tested for antibody cross-reactivity to SARS-CoV RBD, viral RNA in the blood (RNAemia), and neutralization of spike-pseudotyped lentivirus” | concepts[51] |
| concept | validation cohort — not defined in this paper's own words; independent prospectively sampled mild/asymptomatic group — “Most individuals who become infected with SARS-CoV-2 infection do not require hospitalization to recover from their illness, and a sizeable fraction (approximately 40-45%) ( 29 ) remain asymptomatic. We carried out further analysis of antibody responses in a larger set of samples from 136 outpatients and asymptomatic individuals who tested positive by rRT-PCR for SARS-CoV-2 RNA in nasopharyngeal swabs and had serological testing conducted in the Stanford Health Care Clinical Laboratory. We also tested an independent validation set of 45 plasma samples from an additional 14 asymptomatic or mildly ill individuals. As seen for outpatients in Fig. 2B , the asymptomatic and outpatient individuals showed relatively low titers and rapid decline of RBD IgM and IgG ( Fig. 4A , table S3). The timing of infection in asymptomatic individuals is less certain than for symptomatic individuals, whose symptoms usually develop within 2 to 14 days after exposure to the virus ( 30 ). Plotting of serological responses of outpatients and asymptomatic individuals relative to the date of their first positive rRT-PCR test for infection nonetheless showed a similar time course to that seen for outpatients plotted as days post onset of symptoms ( Fig. 2B ). Notably, the amount of viral RNA detected in diagnostic nasopharyngeal swabs was correlated with the antibody titers measured in these individuals ( Fig. 4B ). Fig. 4 Development of anti-SARS-CoV-2 spike RBD antibody responses in SARS-CoV-2 rRT-PCR+ asymptomatic individuals and outpatients over time. 176 plasma samples from 136 SARS-CoV-2 rRT-PCR+ individuals were tested for RBD IgM and IgG. The x-axis indicates the time following the first positive rRT-PCR test ( A ). Dotted lines denote the assay cutoff for positive results. RBD IgM and IgG are shown for the latest available timepoint for subjects with low (Ct >12-25), middle (Ct >25-35) and high (Ct >35-40) SARS-CoV-2 rRT-PCR Ct at diagnosis ( B ). 45 plasma samples collected from 14 rRT-PCR+ asymptomatic individuals and outpatients sampled at monthly intervals (V1 = enrollment, V2 to V5 = months 1 to 4 post-enrollment) were tested for RBD IgM, IgG, IgA at a dilution of 1:100, as well as RBD-ACE2 blocking antibodies at a dilution of 1:10 ( C ). The 45 plasma samples were further tested for pseudoviral neutralization at a dilution of 1:1250 ( D ). Box-whisker ELISA OD 450 and blocking/neutralization percent plots show the interquartile range as the box and the minimum and maximum values as the ends of the whiskers. Correlations between virus neutralization and RBD IgM, IgG, IgA, and RBD-ACE2 blocking are shown with superimposed simple linear regression and 95% confidence bands (grey shading) of the best-fit line ( E ). Plots show mean ELISA OD 450 values of duplicate measurements and average percent neutralization from duplicate testing in each of two replicate experiments. Plasma specimens from the independent validation cohort of asymptomatic or outpatient individuals were collected prospectively during monthly visits up to four months following recruitment ( Fig. 4C, D , fig. S9). RBD IgM, IgG and IgA, RBD-ACE2 blocking and neutralization assays all showed a progressive decrease during the months sampled. Neutralizing antibodies were best correlated with RBD IgG titers, and somewhat less well with RBD-ACE2 blocking ELISA in these individuals” | concepts[52] |
| concept | ACE2 competition ELISA — not defined in this paper's own words; assay quantifying antibody blockade of RBD-ACE2 binding — “Fig. 1 Serological testing of plasma from SARS-CoV-2 rRT-PCR+ individuals. Plasma samples from SARS-CoV-2 rRT-PCR-positive individuals ( A ) were analyzed for the presence of antibodies binding to SARS-CoV-2 spike RBD ( B ). *Plasma was also tested for antibodies specific for SARS-CoV-2 S1 and N proteins, and SARS-CoV RBD. In addition, samples were tested for antibodies blocking the interaction of ACE2 and RBD in an ACE2 competition ELISA ( C ). Spike SARS-CoV-2 pseudotyped lentiviral neutralization assays were performed on selected plasma samples ( D ). Spike-mediated virus entry into HeLa cells overexpressing the ACE2 receptor was measured via luciferase reporter activity” | concepts[53] |
| concept | pseudotyped lentiviral neutralization assays — not defined in this paper's own words; cell-based functional neutralization tests — “Fig. 1 Serological testing of plasma from SARS-CoV-2 rRT-PCR+ individuals. Plasma samples from SARS-CoV-2 rRT-PCR-positive individuals ( A ) were analyzed for the presence of antibodies binding to SARS-CoV-2 spike RBD ( B ). *Plasma was also tested for antibodies specific for SARS-CoV-2 S1 and N proteins, and SARS-CoV RBD. In addition, samples were tested for antibodies blocking the interaction of ACE2 and RBD in an ACE2 competition ELISA ( C ). Spike SARS-CoV-2 pseudotyped lentiviral neutralization assays were performed on selected plasma samples ( D ). Spike-mediated virus entry into HeLa cells overexpressing the ACE2 receptor was measured via luciferase reporter activity” | concepts[54] |
| concept | HeLa cells overexpressing the ACE2 receptor — not defined in this paper's own words; target cells used for pseudovirus entry — “Fig. 1 Serological testing of plasma from SARS-CoV-2 rRT-PCR+ individuals. Plasma samples from SARS-CoV-2 rRT-PCR-positive individuals ( A ) were analyzed for the presence of antibodies binding to SARS-CoV-2 spike RBD ( B ). *Plasma was also tested for antibodies specific for SARS-CoV-2 S1 and N proteins, and SARS-CoV RBD. In addition, samples were tested for antibodies blocking the interaction of ACE2 and RBD in an ACE2 competition ELISA ( C ). Spike SARS-CoV-2 pseudotyped lentiviral neutralization assays were performed on selected plasma samples ( D ). Spike-mediated virus entry into HeLa cells overexpressing the ACE2 receptor was measured via luciferase reporter activity” | concepts[55] |
| concept | luciferase reporter activity — not defined in this paper's own words; readout of spike-mediated viral entry — “Fig. 1 Serological testing of plasma from SARS-CoV-2 rRT-PCR+ individuals. Plasma samples from SARS-CoV-2 rRT-PCR-positive individuals ( A ) were analyzed for the presence of antibodies binding to SARS-CoV-2 spike RBD ( B ). *Plasma was also tested for antibodies specific for SARS-CoV-2 S1 and N proteins, and SARS-CoV RBD. In addition, samples were tested for antibodies blocking the interaction of ACE2 and RBD in an ACE2 competition ELISA ( C ). Spike SARS-CoV-2 pseudotyped lentiviral neutralization assays were performed on selected plasma samples ( D ). Spike-mediated virus entry into HeLa cells overexpressing the ACE2 receptor was measured via luciferase reporter activity” | concepts[56] |
| concept | seroconverted by week 2 — not defined in this paper's own words; typical timing of detectable RBD-binding antibodies — “Anti-RBD, S1 and N antibody responses and duration are associated with disease severity Lower antibody responses in patients with mild symptoms compared to those with more severe disease have been reported for other coronavirus infections, such as MERS-CoV ( 21 – 23 ) as well as for SARS-CoV-2 ( 15 , 24 ). The detection rate of RBD-binding antibodies at one-week intervals after symptom onset are shown in table S2; most individuals seroconverted by week 2 post-onset of symptoms ( Fig. 2A, B ). Positivity rates for RBD IgM, IgG and IgA reached their maximum at weeks 4, 6 and 5, respectively, with most patients negative for IgM and IgA after 12 weeks, while RBD IgG levels showed a slower, but progressive decline, or a continued negative result in those who failed to generate IgG at earlier time points ( Fig. 2A , fig. S3, and table S2). Outpatients had lower titers of RBD IgM, IgG, and IgA compared to inpatients, and a more rapid decline of titers ( Fig. 2B , fig. S4). Patients who required ICU care, and those who died, developed and maintained the highest levels of IgM, IgG and IgA, as well as RBD-ACE2 blocking antibodies, throughout the time course” | concepts[57] |
| concept | RBD IgM — not defined in this paper's own words; RBD-specific IgM response — “Anti-RBD, S1 and N antibody responses and duration are associated with disease severity Lower antibody responses in patients with mild symptoms compared to those with more severe disease have been reported for other coronavirus infections, such as MERS-CoV ( 21 – 23 ) as well as for SARS-CoV-2 ( 15 , 24 ). The detection rate of RBD-binding antibodies at one-week intervals after symptom onset are shown in table S2; most individuals seroconverted by week 2 post-onset of symptoms ( Fig. 2A, B ). Positivity rates for RBD IgM, IgG and IgA reached their maximum at weeks 4, 6 and 5, respectively, with most patients negative for IgM and IgA after 12 weeks, while RBD IgG levels showed a slower, but progressive decline, or a continued negative result in those who failed to generate IgG at earlier time points ( Fig. 2A , fig. S3, and table S2). Outpatients had lower titers of RBD IgM, IgG, and IgA compared to inpatients, and a more rapid decline of titers ( Fig. 2B , fig. S4). Patients who required ICU care, and those who died, developed and maintained the highest levels of IgM, IgG and IgA, as well as RBD-ACE2 blocking antibodies, throughout the time course” | concepts[58] |
| concept | RBD IgG — not defined in this paper's own words; RBD-specific IgG response with slower decline — “Anti-RBD, S1 and N antibody responses and duration are associated with disease severity Lower antibody responses in patients with mild symptoms compared to those with more severe disease have been reported for other coronavirus infections, such as MERS-CoV ( 21 – 23 ) as well as for SARS-CoV-2 ( 15 , 24 ). The detection rate of RBD-binding antibodies at one-week intervals after symptom onset are shown in table S2; most individuals seroconverted by week 2 post-onset of symptoms ( Fig. 2A, B ). Positivity rates for RBD IgM, IgG and IgA reached their maximum at weeks 4, 6 and 5, respectively, with most patients negative for IgM and IgA after 12 weeks, while RBD IgG levels showed a slower, but progressive decline, or a continued negative result in those who failed to generate IgG at earlier time points ( Fig. 2A , fig. S3, and table S2). Outpatients had lower titers of RBD IgM, IgG, and IgA compared to inpatients, and a more rapid decline of titers ( Fig. 2B , fig. S4). Patients who required ICU care, and those who died, developed and maintained the highest levels of IgM, IgG and IgA, as well as RBD-ACE2 blocking antibodies, throughout the time course” | concepts[59] |
| concept | antibody kinetics — not defined in this paper's own words; time-varying antibody trajectories — “S1-specific IgM, IgG, and IgA showed antibody kinetics that were very similar to those seen for RBD (figs. S3 to S5 and table S2). While N-specific IgG responses showed high positivity rates with antibody kinetics similar to those for RBD and S1, IgM antibody responses to N were strikingly low in most patients (figs. S4 and S6 and table S2). There was no consistent difference in RBD, S1, and N antibody titers or RBD-ACE2 blocking antibodies between ICU patients who survived compared to patients who died (fig. S7). We further evaluated the breadth of antibody responses in different disease severity categories by testing for SARS-CoV RBD binding. Most monoclonal antibodies targeting SARS-CoV RBD fail to bind SARS-CoV-2 RBD, indicating distinct antigenicity despite sequence and structural similarity of the two proteins ( 25 , 26 ). Nine of 13 ICU patients, three of 25 admitted non-ICU patients (fig. S8A) and five of 82 outpatients (fig. S8B), developed SARS-CoV RBD IgG titers during the course of their infection. The time course of anti-SARS-CoV RBD positivity in serial samples from individual patients did not always mirror anti-SARS-CoV-2 RBD IgG responses, suggesting limited clonal or oligoclonal B cell responses with this broader reactivity within the overall polyclonal anti-SARS-CoV-2 serological response” | concepts[60] |
| concept | distinct antigenicity — not defined in this paper's own words; antigenic difference despite SARS-CoV sequence and structural similarity — “S1-specific IgM, IgG, and IgA showed antibody kinetics that were very similar to those seen for RBD (figs. S3 to S5 and table S2). While N-specific IgG responses showed high positivity rates with antibody kinetics similar to those for RBD and S1, IgM antibody responses to N were strikingly low in most patients (figs. S4 and S6 and table S2). There was no consistent difference in RBD, S1, and N antibody titers or RBD-ACE2 blocking antibodies between ICU patients who survived compared to patients who died (fig. S7). We further evaluated the breadth of antibody responses in different disease severity categories by testing for SARS-CoV RBD binding. Most monoclonal antibodies targeting SARS-CoV RBD fail to bind SARS-CoV-2 RBD, indicating distinct antigenicity despite sequence and structural similarity of the two proteins ( 25 , 26 ). Nine of 13 ICU patients, three of 25 admitted non-ICU patients (fig. S8A) and five of 82 outpatients (fig. S8B), developed SARS-CoV RBD IgG titers during the course of their infection. The time course of anti-SARS-CoV RBD positivity in serial samples from individual patients did not always mirror anti-SARS-CoV-2 RBD IgG responses, suggesting limited clonal or oligoclonal B cell responses with this broader reactivity within the overall polyclonal anti-SARS-CoV-2 serological response” | concepts[61] |
| concept | cross-reactivity — not defined in this paper's own words; binding response spanning SARS-CoV and SARS-CoV-2 antigens — “A total of 254 individuals with a positive SARS-CoV-2 real-time RT-PCR (rRT-PCR) nasopharyngeal swab test were included in the study (fig. S1). Study subjects were identified either after routine serology testing or occupational health screening in the Stanford Health Care Clinical Laboratories for anti-SARS-CoV-2 RBD IgM and IgG antibodies (136 asymptomatic individuals or outpatients), or after they reported to Stanford Health Care-associated clinical sites with symptoms of COVID-19. This included 24 outpatients, 35 hospitalized patients who were not admitted to the intensive care unit (ICU), and 20 ICU inpatients who survived their illness. To evaluate serological responses associated with patient mortality, we also analyzed specimens from 25 patients who died of COVID-19 (one outpatient, seven admitted non-ICU, and seventeen admitted ICU patients). Of patients who were treated in the ICU, 26 of 37 (70%) required mechanical ventilation, including 15 patients who died. Demographic and clinical characteristics of patients stratified by disease status are presented in Table 1 . The percentage of males, and those with comorbidities of hypertension or diabetes mellitus increased with patient disease severity. Outpatients and asymptomatic individuals were younger and had the lowest levels of obesity compared to more severely ill patients. Notably, levels of viral RNA measured by rRT-PCR of nasopharyngeal swabs at diagnosis showed a progressive increase (lower rRT-PCR cycle threshold, Ct) with disease severity; patients who died had the highest viral loads (fig. S2). Plasma samples for in-depth serological testing were available from all inpatients and 86 of the 160 outpatient survivors. Demographic and clinical characteristics of outpatients and asymptomatic individuals with and without plasma availability are presented in table S1. A total of 828 samples were analyzed with ELISAs measuring IgM, IgG and IgA specific for SARS-CoV-2 RBD, S1 or N, as well as the RBD-ACE2 blocking assay ( Fig. 1A-C ). Representative samples were also tested for antibody cross-reactivity to SARS-CoV RBD, viral RNA in the blood (RNAemia), and neutralization of spike-pseudotyped lentivirus” | concepts[62] |
| concept | clonal or oligoclonal B cell responses — not defined in this paper's own words; limited B-cell sources proposed for broader reactivity — “S1-specific IgM, IgG, and IgA showed antibody kinetics that were very similar to those seen for RBD (figs. S3 to S5 and table S2). While N-specific IgG responses showed high positivity rates with antibody kinetics similar to those for RBD and S1, IgM antibody responses to N were strikingly low in most patients (figs. S4 and S6 and table S2). There was no consistent difference in RBD, S1, and N antibody titers or RBD-ACE2 blocking antibodies between ICU patients who survived compared to patients who died (fig. S7). We further evaluated the breadth of antibody responses in different disease severity categories by testing for SARS-CoV RBD binding. Most monoclonal antibodies targeting SARS-CoV RBD fail to bind SARS-CoV-2 RBD, indicating distinct antigenicity despite sequence and structural similarity of the two proteins ( 25 , 26 ). Nine of 13 ICU patients, three of 25 admitted non-ICU patients (fig. S8A) and five of 82 outpatients (fig. S8B), developed SARS-CoV RBD IgG titers during the course of their infection. The time course of anti-SARS-CoV RBD positivity in serial samples from individual patients did not always mirror anti-SARS-CoV-2 RBD IgG responses, suggesting limited clonal or oligoclonal B cell responses with this broader reactivity within the overall polyclonal anti-SARS-CoV-2 serological response” | concepts[63] |
| concept | polyclonal anti-SARS-CoV-2 serological response — not defined in this paper's own words; overall antibody response containing broader-reactivity clones — “S1-specific IgM, IgG, and IgA showed antibody kinetics that were very similar to those seen for RBD (figs. S3 to S5 and table S2). While N-specific IgG responses showed high positivity rates with antibody kinetics similar to those for RBD and S1, IgM antibody responses to N were strikingly low in most patients (figs. S4 and S6 and table S2). There was no consistent difference in RBD, S1, and N antibody titers or RBD-ACE2 blocking antibodies between ICU patients who survived compared to patients who died (fig. S7). We further evaluated the breadth of antibody responses in different disease severity categories by testing for SARS-CoV RBD binding. Most monoclonal antibodies targeting SARS-CoV RBD fail to bind SARS-CoV-2 RBD, indicating distinct antigenicity despite sequence and structural similarity of the two proteins ( 25 , 26 ). Nine of 13 ICU patients, three of 25 admitted non-ICU patients (fig. S8A) and five of 82 outpatients (fig. S8B), developed SARS-CoV RBD IgG titers during the course of their infection. The time course of anti-SARS-CoV RBD positivity in serial samples from individual patients did not always mirror anti-SARS-CoV-2 RBD IgG responses, suggesting limited clonal or oligoclonal B cell responses with this broader reactivity within the overall polyclonal anti-SARS-CoV-2 serological response” | concepts[64] |
| concept | live SARS-CoV-2 — not defined in this paper's own words; virus used in physiologically relevant but restrictive neutralization — “Neutralizing antibodies are increased in inpatients compared to outpatients, and correlate with RBD-ACE2 blocking and RBD IgG titers Antibody neutralization of live SARS-CoV-2, or of pseudotyped viruses such as lentiviruses expressing the SARS-CoV-2 spike may represent the most physiologically relevant surrogates for humoral immunity in vivo, but are poorly scalable due to restrictive biosafety requirements for SARS-CoV-2 and less easily standardized assay components and protocols, compared to tests using purified proteins. We tested SARS-CoV-2 spike-pseudotyped lentivirus neutralization ( 27 ) in HeLa cells overexpressing ACE2 ( 28 ) by inpatient and outpatient samples, and evaluated correlations with the more scalable ELISAs for RBD, S1 and RBD-ACE2 blocking ( Fig. 3A, B ). As with RBD-ACE2 blocking, neutralizing antibody activity was higher in inpatients compared to outpatients, and began to decrease after about one month after symptom onset. Neutralization was well-correlated with RBD IgG titers and RBD-ACE2 blocking (linear regression coefficient of determination R 2 of 0.6995 and 0.6824 for inpatients; 0.7338 and 0.6839 for outpatients). The RBD-ACE2 blocking assay was less sensitive than neutralization or RBD IgG ELISA. RBD IgM and IgA ELISA results were much more variable and did not correlate as well with neutralization or RBD-ACE2 blocking compared to RBD IgG” | concepts[65] |
| concept | pseudotyped viruses — not defined in this paper's own words; safer virus surrogates expressing SARS-CoV-2 spike — “Neutralizing antibodies are increased in inpatients compared to outpatients, and correlate with RBD-ACE2 blocking and RBD IgG titers Antibody neutralization of live SARS-CoV-2, or of pseudotyped viruses such as lentiviruses expressing the SARS-CoV-2 spike may represent the most physiologically relevant surrogates for humoral immunity in vivo, but are poorly scalable due to restrictive biosafety requirements for SARS-CoV-2 and less easily standardized assay components and protocols, compared to tests using purified proteins. We tested SARS-CoV-2 spike-pseudotyped lentivirus neutralization ( 27 ) in HeLa cells overexpressing ACE2 ( 28 ) by inpatient and outpatient samples, and evaluated correlations with the more scalable ELISAs for RBD, S1 and RBD-ACE2 blocking ( Fig. 3A, B ). As with RBD-ACE2 blocking, neutralizing antibody activity was higher in inpatients compared to outpatients, and began to decrease after about one month after symptom onset. Neutralization was well-correlated with RBD IgG titers and RBD-ACE2 blocking (linear regression coefficient of determination R 2 of 0.6995 and 0.6824 for inpatients; 0.7338 and 0.6839 for outpatients). The RBD-ACE2 blocking assay was less sensitive than neutralization or RBD IgG ELISA. RBD IgM and IgA ELISA results were much more variable and did not correlate as well with neutralization or RBD-ACE2 blocking compared to RBD IgG” | concepts[66] |
| concept | biosafety requirements — not defined in this paper's own words; constraints limiting live-virus assay scalability — “Neutralizing antibodies are increased in inpatients compared to outpatients, and correlate with RBD-ACE2 blocking and RBD IgG titers Antibody neutralization of live SARS-CoV-2, or of pseudotyped viruses such as lentiviruses expressing the SARS-CoV-2 spike may represent the most physiologically relevant surrogates for humoral immunity in vivo, but are poorly scalable due to restrictive biosafety requirements for SARS-CoV-2 and less easily standardized assay components and protocols, compared to tests using purified proteins. We tested SARS-CoV-2 spike-pseudotyped lentivirus neutralization ( 27 ) in HeLa cells overexpressing ACE2 ( 28 ) by inpatient and outpatient samples, and evaluated correlations with the more scalable ELISAs for RBD, S1 and RBD-ACE2 blocking ( Fig. 3A, B ). As with RBD-ACE2 blocking, neutralizing antibody activity was higher in inpatients compared to outpatients, and began to decrease after about one month after symptom onset. Neutralization was well-correlated with RBD IgG titers and RBD-ACE2 blocking (linear regression coefficient of determination R 2 of 0.6995 and 0.6824 for inpatients; 0.7338 and 0.6839 for outpatients). The RBD-ACE2 blocking assay was less sensitive than neutralization or RBD IgG ELISA. RBD IgM and IgA ELISA results were much more variable and did not correlate as well with neutralization or RBD-ACE2 blocking compared to RBD IgG” | concepts[67] |
| concept | purified proteins — not defined in this paper's own words; components enabling scalable ELISA alternatives — “Neutralizing antibodies are increased in inpatients compared to outpatients, and correlate with RBD-ACE2 blocking and RBD IgG titers Antibody neutralization of live SARS-CoV-2, or of pseudotyped viruses such as lentiviruses expressing the SARS-CoV-2 spike may represent the most physiologically relevant surrogates for humoral immunity in vivo, but are poorly scalable due to restrictive biosafety requirements for SARS-CoV-2 and less easily standardized assay components and protocols, compared to tests using purified proteins. We tested SARS-CoV-2 spike-pseudotyped lentivirus neutralization ( 27 ) in HeLa cells overexpressing ACE2 ( 28 ) by inpatient and outpatient samples, and evaluated correlations with the more scalable ELISAs for RBD, S1 and RBD-ACE2 blocking ( Fig. 3A, B ). As with RBD-ACE2 blocking, neutralizing antibody activity was higher in inpatients compared to outpatients, and began to decrease after about one month after symptom onset. Neutralization was well-correlated with RBD IgG titers and RBD-ACE2 blocking (linear regression coefficient of determination R 2 of 0.6995 and 0.6824 for inpatients; 0.7338 and 0.6839 for outpatients). The RBD-ACE2 blocking assay was less sensitive than neutralization or RBD IgG ELISA. RBD IgM and IgA ELISA results were much more variable and did not correlate as well with neutralization or RBD-ACE2 blocking compared to RBD IgG” | concepts[68] |
| concept | linear regression coefficient of determination R 2 — not defined in this paper's own words; reported correlation-fit quantity — “Neutralizing antibodies are increased in inpatients compared to outpatients, and correlate with RBD-ACE2 blocking and RBD IgG titers Antibody neutralization of live SARS-CoV-2, or of pseudotyped viruses such as lentiviruses expressing the SARS-CoV-2 spike may represent the most physiologically relevant surrogates for humoral immunity in vivo, but are poorly scalable due to restrictive biosafety requirements for SARS-CoV-2 and less easily standardized assay components and protocols, compared to tests using purified proteins. We tested SARS-CoV-2 spike-pseudotyped lentivirus neutralization ( 27 ) in HeLa cells overexpressing ACE2 ( 28 ) by inpatient and outpatient samples, and evaluated correlations with the more scalable ELISAs for RBD, S1 and RBD-ACE2 blocking ( Fig. 3A, B ). As with RBD-ACE2 blocking, neutralizing antibody activity was higher in inpatients compared to outpatients, and began to decrease after about one month after symptom onset. Neutralization was well-correlated with RBD IgG titers and RBD-ACE2 blocking (linear regression coefficient of determination R 2 of 0.6995 and 0.6824 for inpatients; 0.7338 and 0.6839 for outpatients). The RBD-ACE2 blocking assay was less sensitive than neutralization or RBD IgG ELISA. RBD IgM and IgA ELISA results were much more variable and did not correlate as well with neutralization or RBD-ACE2 blocking compared to RBD IgG” | concepts[69] |
| concept | independent validation set — not defined in this paper's own words; additional longitudinal mild/asymptomatic samples — “Most individuals who become infected with SARS-CoV-2 infection do not require hospitalization to recover from their illness, and a sizeable fraction (approximately 40-45%) ( 29 ) remain asymptomatic. We carried out further analysis of antibody responses in a larger set of samples from 136 outpatients and asymptomatic individuals who tested positive by rRT-PCR for SARS-CoV-2 RNA in nasopharyngeal swabs and had serological testing conducted in the Stanford Health Care Clinical Laboratory. We also tested an independent validation set of 45 plasma samples from an additional 14 asymptomatic or mildly ill individuals. As seen for outpatients in Fig. 2B , the asymptomatic and outpatient individuals showed relatively low titers and rapid decline of RBD IgM and IgG ( Fig. 4A , table S3). The timing of infection in asymptomatic individuals is less certain than for symptomatic individuals, whose symptoms usually develop within 2 to 14 days after exposure to the virus ( 30 ). Plotting of serological responses of outpatients and asymptomatic individuals relative to the date of their first positive rRT-PCR test for infection nonetheless showed a similar time course to that seen for outpatients plotted as days post onset of symptoms ( Fig. 2B ). Notably, the amount of viral RNA detected in diagnostic nasopharyngeal swabs was correlated with the antibody titers measured in these individuals ( Fig. 4B ).” | concepts[70] |
| concept | progressive decrease — not defined in this paper's own words; decline observed across binding, blocking, and neutralization assays — “Most individuals who become infected with SARS-CoV-2 infection do not require hospitalization to recover from their illness, and a sizeable fraction (approximately 40-45%) ( 29 ) remain asymptomatic. We carried out further analysis of antibody responses in a larger set of samples from 136 outpatients and asymptomatic individuals who tested positive by rRT-PCR for SARS-CoV-2 RNA in nasopharyngeal swabs and had serological testing conducted in the Stanford Health Care Clinical Laboratory. We also tested an independent validation set of 45 plasma samples from an additional 14 asymptomatic or mildly ill individuals. As seen for outpatients in Fig. 2B , the asymptomatic and outpatient individuals showed relatively low titers and rapid decline of RBD IgM and IgG ( Fig. 4A , table S3). The timing of infection in asymptomatic individuals is less certain than for symptomatic individuals, whose symptoms usually develop within 2 to 14 days after exposure to the virus ( 30 ). Plotting of serological responses of outpatients and asymptomatic individuals relative to the date of their first positive rRT-PCR test for infection nonetheless showed a similar time course to that seen for outpatients plotted as days post onset of symptoms ( Fig. 2B ). Notably, the amount of viral RNA detected in diagnostic nasopharyngeal swabs was correlated with the antibody titers measured in these individuals ( Fig. 4B ). Fig. 4 Development of anti-SARS-CoV-2 spike RBD antibody responses in SARS-CoV-2 rRT-PCR+ asymptomatic individuals and outpatients over time. 176 plasma samples from 136 SARS-CoV-2 rRT-PCR+ individuals were tested for RBD IgM and IgG. The x-axis indicates the time following the first positive rRT-PCR test ( A ). Dotted lines denote the assay cutoff for positive results. RBD IgM and IgG are shown for the latest available timepoint for subjects with low (Ct >12-25), middle (Ct >25-35) and high (Ct >35-40) SARS-CoV-2 rRT-PCR Ct at diagnosis ( B ). 45 plasma samples collected from 14 rRT-PCR+ asymptomatic individuals and outpatients sampled at monthly intervals (V1 = enrollment, V2 to V5 = months 1 to 4 post-enrollment) were tested for RBD IgM, IgG, IgA at a dilution of 1:100, as well as RBD-ACE2 blocking antibodies at a dilution of 1:10 ( C ). The 45 plasma samples were further tested for pseudoviral neutralization at a dilution of 1:1250 ( D ). Box-whisker ELISA OD 450 and blocking/neutralization percent plots show the interquartile range as the box and the minimum and maximum values as the ends of the whiskers. Correlations between virus neutralization and RBD IgM, IgG, IgA, and RBD-ACE2 blocking are shown with superimposed simple linear regression and 95% confidence bands (grey shading) of the best-fit line ( E ). Plots show mean ELISA OD 450 values of duplicate measurements and average percent neutralization from duplicate testing in each of two replicate experiments. Plasma specimens from the independent validation cohort of asymptomatic or outpatient individuals were collected prospectively during monthly visits up to four months following recruitment ( Fig. 4C, D , fig. S9). RBD IgM, IgG and IgA, RBD-ACE2 blocking and neutralization assays all showed a progressive decrease during the months sampled. Neutralizing antibodies were best correlated with RBD IgG titers, and somewhat less well with RBD-ACE2 blocking ELISA in these individuals” | concepts[71] |
| concept | RBD/N — not defined in this paper's own words; ratio of RBD-targeting to nucleocapsid-targeting responses — “Outpatients and non-ICU inpatients show preferential antibody targeting of RBD and S1 compared to N It is an open question whether antibody responses in the initial weeks of SARS-CoV-2 infection have a role in modulating disease severity. Having found that patients with milder illness or asymptomatic infection had lower levels of SARS-CoV-2 antibodies to RBD, S1 and N compared to severely ill patients, we hypothesized that the relative targeting of the antibody response between antigens might be associated with different disease severity. In a recent study, serological analysis of samples from patients who died of COVID-19 compared to individuals who convalesced suggested an enrichment of an aggregate measure of spike antibodies or antibody functional activities in the convalescent ( 19 ). We calculated the ratios of RBD to N ( Fig. 5A ) and S1 to N ( Fig. 5B ) ELISA results for IgM, IgG and IgA for all specimens that had detectable antibodies. Notably, in weeks 1 and 2 post-onset of symptoms, outpatient IgG RBD/N and S1/N ratios were higher than those of admitted non-ICU patients, and patients who died of their illness; admitted and ICU patients also had higher RBD/N ratios compared to patients who died. In weeks 3 and 4, and after 4 weeks, outpatients and admitted patients had higher S1/N IgG ratios compared to ICU patients and those who died. IgA responses showed a similar pattern to IgG in weeks 3 and 4, and after week 4 post-onset of symptoms. The IgM ratios to these antigens was more variable and did not show a consistent relationship to disease severity” | concepts[72] |
| concept | S1/N — not defined in this paper's own words; ratio of S1-targeting to nucleocapsid-targeting responses — “Outpatients and non-ICU inpatients show preferential antibody targeting of RBD and S1 compared to N It is an open question whether antibody responses in the initial weeks of SARS-CoV-2 infection have a role in modulating disease severity. Having found that patients with milder illness or asymptomatic infection had lower levels of SARS-CoV-2 antibodies to RBD, S1 and N compared to severely ill patients, we hypothesized that the relative targeting of the antibody response between antigens might be associated with different disease severity. In a recent study, serological analysis of samples from patients who died of COVID-19 compared to individuals who convalesced suggested an enrichment of an aggregate measure of spike antibodies or antibody functional activities in the convalescent ( 19 ). We calculated the ratios of RBD to N ( Fig. 5A ) and S1 to N ( Fig. 5B ) ELISA results for IgM, IgG and IgA for all specimens that had detectable antibodies. Notably, in weeks 1 and 2 post-onset of symptoms, outpatient IgG RBD/N and S1/N ratios were higher than those of admitted non-ICU patients, and patients who died of their illness; admitted and ICU patients also had higher RBD/N ratios compared to patients who died. In weeks 3 and 4, and after 4 weeks, outpatients and admitted patients had higher S1/N IgG ratios compared to ICU patients and those who died. IgA responses showed a similar pattern to IgG in weeks 3 and 4, and after week 4 post-onset of symptoms. The IgM ratios to these antigens was more variable and did not show a consistent relationship to disease severity” | concepts[73] |
| concept | preferential antibody targeting — not defined in this paper's own words; relative antigen specificity associated with milder illness — “Outpatients and non-ICU inpatients show preferential antibody targeting of RBD and S1 compared to N It is an open question whether antibody responses in the initial weeks of SARS-CoV-2 infection have a role in modulating disease severity. Having found that patients with milder illness or asymptomatic infection had lower levels of SARS-CoV-2 antibodies to RBD, S1 and N compared to severely ill patients, we hypothesized that the relative targeting of the antibody response between antigens might be associated with different disease severity. In a recent study, serological analysis of samples from patients who died of COVID-19 compared to individuals who convalesced suggested an enrichment of an aggregate measure of spike antibodies or antibody functional activities in the convalescent ( 19 ). We calculated the ratios of RBD to N ( Fig. 5A ) and S1 to N ( Fig. 5B ) ELISA results for IgM, IgG and IgA for all specimens that had detectable antibodies. Notably, in weeks 1 and 2 post-onset of symptoms, outpatient IgG RBD/N and S1/N ratios were higher than those of admitted non-ICU patients, and patients who died of their illness; admitted and ICU patients also had higher RBD/N ratios compared to patients who died. In weeks 3 and 4, and after 4 weeks, outpatients and admitted patients had higher S1/N IgG ratios compared to ICU patients and those who died. IgA responses showed a similar pattern to IgG in weeks 3 and 4, and after week 4 post-onset of symptoms. The IgM ratios to these antigens was more variable and did not show a consistent relationship to disease severity” | concepts[74] |
| concept | three main patterns of response — not defined in this paper's own words; inpatient antibody-pattern grouping — “Inpatient SARS-CoV-2 antibody time course patterns are associated with decreases in RNAemia, but not disease outcome We hypothesized that detailed examination of the time course of SARS-CoV-2 antibody responses could identify distinct patterns associated with admitted non-ICU patients ( Fig. 6 , fig. S10), ICU patients who recovered from their illness ( Fig. 7 , fig. S11A), or those who died ( Fig. 8 , fig. S11B). We identified three main patterns of response: at the time point closest to their discharge from hospital, or death, Group 1 individuals had SARS-CoV-2 antibody responses with no RBD IgG or RBD-ACE2 blocking activity; Group 2 individuals had up to 25% RBD-ACE2 blocking activity; and Group 3 individuals developed high levels of antibodies and over 25% RBD-ACE2 blocking activity. These patterns of response were not strongly associated with disease severity, but instead were shared across disease categories. Group 1 patients included admitted patients who recovered from their illness rapidly and were discharged before they had developed detectable antibody responses, but also included patients who recovered from their illness without antibody production after prolonged ICU stays, and patients who died of COVID-19 prior to antibody development. Similarly, Group 2 patterns with low levels of RBD-ACE2 blocking were shared across all three patient categories. In Group 3 (high levels of antibody production and >25% RBD-ACE2 blocking activity) admitted non-ICU patients differed from ICU or deceased patients in that they developed their robust antibody responses during short hospital stays prior to discharge, whereas ICU or deceased patients typically had prolonged hospital courses. In patients tested for neutralizing antibodies (13 admitted non-ICU patients, 16 ICU surviving patients, and 16 deceased patients), the results were closely correlated with IgG titers to RBD, or RBD-ACE2 blocking” | concepts[75] |
| concept | Group 1 — not defined in this paper's own words; no RBD IgG or blocking at final inpatient time point — “Inpatient SARS-CoV-2 antibody time course patterns are associated with decreases in RNAemia, but not disease outcome We hypothesized that detailed examination of the time course of SARS-CoV-2 antibody responses could identify distinct patterns associated with admitted non-ICU patients ( Fig. 6 , fig. S10), ICU patients who recovered from their illness ( Fig. 7 , fig. S11A), or those who died ( Fig. 8 , fig. S11B). We identified three main patterns of response: at the time point closest to their discharge from hospital, or death, Group 1 individuals had SARS-CoV-2 antibody responses with no RBD IgG or RBD-ACE2 blocking activity; Group 2 individuals had up to 25% RBD-ACE2 blocking activity; and Group 3 individuals developed high levels of antibodies and over 25% RBD-ACE2 blocking activity. These patterns of response were not strongly associated with disease severity, but instead were shared across disease categories. Group 1 patients included admitted patients who recovered from their illness rapidly and were discharged before they had developed detectable antibody responses, but also included patients who recovered from their illness without antibody production after prolonged ICU stays, and patients who died of COVID-19 prior to antibody development. Similarly, Group 2 patterns with low levels of RBD-ACE2 blocking were shared across all three patient categories. In Group 3 (high levels of antibody production and >25% RBD-ACE2 blocking activity) admitted non-ICU patients differed from ICU or deceased patients in that they developed their robust antibody responses during short hospital stays prior to discharge, whereas ICU or deceased patients typically had prolonged hospital courses. In patients tested for neutralizing antibodies (13 admitted non-ICU patients, 16 ICU surviving patients, and 16 deceased patients), the results were closely correlated with IgG titers to RBD, or RBD-ACE2 blocking” | concepts[76] |
| concept | Group 2 — not defined in this paper's own words; up to 25 percent RBD-ACE2 blocking — “Inpatient SARS-CoV-2 antibody time course patterns are associated with decreases in RNAemia, but not disease outcome We hypothesized that detailed examination of the time course of SARS-CoV-2 antibody responses could identify distinct patterns associated with admitted non-ICU patients ( Fig. 6 , fig. S10), ICU patients who recovered from their illness ( Fig. 7 , fig. S11A), or those who died ( Fig. 8 , fig. S11B). We identified three main patterns of response: at the time point closest to their discharge from hospital, or death, Group 1 individuals had SARS-CoV-2 antibody responses with no RBD IgG or RBD-ACE2 blocking activity; Group 2 individuals had up to 25% RBD-ACE2 blocking activity; and Group 3 individuals developed high levels of antibodies and over 25% RBD-ACE2 blocking activity. These patterns of response were not strongly associated with disease severity, but instead were shared across disease categories. Group 1 patients included admitted patients who recovered from their illness rapidly and were discharged before they had developed detectable antibody responses, but also included patients who recovered from their illness without antibody production after prolonged ICU stays, and patients who died of COVID-19 prior to antibody development. Similarly, Group 2 patterns with low levels of RBD-ACE2 blocking were shared across all three patient categories. In Group 3 (high levels of antibody production and >25% RBD-ACE2 blocking activity) admitted non-ICU patients differed from ICU or deceased patients in that they developed their robust antibody responses during short hospital stays prior to discharge, whereas ICU or deceased patients typically had prolonged hospital courses. In patients tested for neutralizing antibodies (13 admitted non-ICU patients, 16 ICU surviving patients, and 16 deceased patients), the results were closely correlated with IgG titers to RBD, or RBD-ACE2 blocking” | concepts[77] |
| concept | Group 3 — not defined in this paper's own words; high antibodies and over 25 percent RBD-ACE2 blocking — “Inpatient SARS-CoV-2 antibody time course patterns are associated with decreases in RNAemia, but not disease outcome We hypothesized that detailed examination of the time course of SARS-CoV-2 antibody responses could identify distinct patterns associated with admitted non-ICU patients ( Fig. 6 , fig. S10), ICU patients who recovered from their illness ( Fig. 7 , fig. S11A), or those who died ( Fig. 8 , fig. S11B). We identified three main patterns of response: at the time point closest to their discharge from hospital, or death, Group 1 individuals had SARS-CoV-2 antibody responses with no RBD IgG or RBD-ACE2 blocking activity; Group 2 individuals had up to 25% RBD-ACE2 blocking activity; and Group 3 individuals developed high levels of antibodies and over 25% RBD-ACE2 blocking activity. These patterns of response were not strongly associated with disease severity, but instead were shared across disease categories. Group 1 patients included admitted patients who recovered from their illness rapidly and were discharged before they had developed detectable antibody responses, but also included patients who recovered from their illness without antibody production after prolonged ICU stays, and patients who died of COVID-19 prior to antibody development. Similarly, Group 2 patterns with low levels of RBD-ACE2 blocking were shared across all three patient categories. In Group 3 (high levels of antibody production and >25% RBD-ACE2 blocking activity) admitted non-ICU patients differed from ICU or deceased patients in that they developed their robust antibody responses during short hospital stays prior to discharge, whereas ICU or deceased patients typically had prolonged hospital courses. In patients tested for neutralizing antibodies (13 admitted non-ICU patients, 16 ICU surviving patients, and 16 deceased patients), the results were closely correlated with IgG titers to RBD, or RBD-ACE2 blocking” | concepts[78] |
| concept | RNAemia — not defined in this paper's own words; defined presence of SARS-CoV-2 RNA in plasma — “Viral RNAemia is detected in up to a third of COVID-19 patients, most often in patients with severe disease ( 3 , 31 ). RNAemia was evaluated in a subset of patients, and detected in 15 of 25 admitted patients, 13 of 15 ICU patients who recovered, and 2 of 2 patients who died of their disease. Reduction in RNAemia was strongly correlated with the appearance of plasma antibodies (Spearman’s correlation coefficients of -0.47 for IgM, -0.43 for IgG, and -0.44 for IgA, p<0.001 for each)” | concepts[79] |
| concept | Spearman’s correlation coefficients — not defined in this paper's own words; rank correlations relating RNAemia decline to antibody appearance — “Viral RNAemia is detected in up to a third of COVID-19 patients, most often in patients with severe disease ( 3 , 31 ). RNAemia was evaluated in a subset of patients, and detected in 15 of 25 admitted patients, 13 of 15 ICU patients who recovered, and 2 of 2 patients who died of their disease. Reduction in RNAemia was strongly correlated with the appearance of plasma antibodies (Spearman’s correlation coefficients of -0.47 for IgM, -0.43 for IgG, and -0.44 for IgA, p<0.001 for each)” | concepts[80] |
| concept | vaccine-induced immune responses — not defined in this paper's own words; responses to compare with natural infection and reinfection — “Key clinical questions in the ongoing COVID-19 pandemic are the role of antibodies in modulating disease severity during infection, the duration of individuals’ serological responses, and the extent to which patient antibody responses may be protective against reinfection. As SARS-CoV-2 vaccine candidates progress through clinical trials, comparison of vaccine-induced immune responses to those stimulated by viral infection, and those of individuals who become reinfected, will help clarify immunological correlates of protection. More comprehensive understanding of the role of antibodies in acute COVID-19 illness will guide effective use of therapeutic convalescent plasma products and recombinant antibodies targeting SARS-CoV-2. Assuming spike-targeting vaccines are shown to be safe and effective and become widely used, monitoring N-specific antibodies may be of utility in distinguishing vaccine-related antibodies from those stimulated by infection. Here, we have analyzed serological responses to SARS-CoV-2 in 254 individuals ranging from asymptomatic individuals to ICU patients, with detailed analysis of antibody responses to SARS-CoV-2 RBD, S1 and N antigens, and functional assays testing antibody blocking of RBD-ACE2 binding or spike-pseudotyped virus neutralization” | concepts[81] |
| concept | immunological correlates of protection — not defined in this paper's own words; immune features predictive of protection — “Key clinical questions in the ongoing COVID-19 pandemic are the role of antibodies in modulating disease severity during infection, the duration of individuals’ serological responses, and the extent to which patient antibody responses may be protective against reinfection. As SARS-CoV-2 vaccine candidates progress through clinical trials, comparison of vaccine-induced immune responses to those stimulated by viral infection, and those of individuals who become reinfected, will help clarify immunological correlates of protection. More comprehensive understanding of the role of antibodies in acute COVID-19 illness will guide effective use of therapeutic convalescent plasma products and recombinant antibodies targeting SARS-CoV-2. Assuming spike-targeting vaccines are shown to be safe and effective and become widely used, monitoring N-specific antibodies may be of utility in distinguishing vaccine-related antibodies from those stimulated by infection. Here, we have analyzed serological responses to SARS-CoV-2 in 254 individuals ranging from asymptomatic individuals to ICU patients, with detailed analysis of antibody responses to SARS-CoV-2 RBD, S1 and N antigens, and functional assays testing antibody blocking of RBD-ACE2 binding or spike-pseudotyped virus neutralization” | concepts[82] |
| concept | recombinant antibodies — not defined in this paper's own words; therapeutic antibodies targeting SARS-CoV-2 — “Key clinical questions in the ongoing COVID-19 pandemic are the role of antibodies in modulating disease severity during infection, the duration of individuals’ serological responses, and the extent to which patient antibody responses may be protective against reinfection. As SARS-CoV-2 vaccine candidates progress through clinical trials, comparison of vaccine-induced immune responses to those stimulated by viral infection, and those of individuals who become reinfected, will help clarify immunological correlates of protection. More comprehensive understanding of the role of antibodies in acute COVID-19 illness will guide effective use of therapeutic convalescent plasma products and recombinant antibodies targeting SARS-CoV-2. Assuming spike-targeting vaccines are shown to be safe and effective and become widely used, monitoring N-specific antibodies may be of utility in distinguishing vaccine-related antibodies from those stimulated by infection. Here, we have analyzed serological responses to SARS-CoV-2 in 254 individuals ranging from asymptomatic individuals to ICU patients, with detailed analysis of antibody responses to SARS-CoV-2 RBD, S1 and N antigens, and functional assays testing antibody blocking of RBD-ACE2 binding or spike-pseudotyped virus neutralization” | concepts[83] |
| concept | memory B cell populations — not defined in this paper's own words; potential long-term recall immune compartment — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[84] |
| concept | seroconversion — not defined in this paper's own words; appearance of detectable serum antibodies — “The ELISA procedure in this study was modified from a protocol published by Stadlbauer ( 48 ). 96-well Corning Costar high binding plates (catalog no. 9018, Thermo Fisher) were coated with SARS-CoV RBD, SARS-CoV-2 RBD, S1, or N protein in phosphate-buffered saline (PBS) at a concentration of 0.1 μg per well (0.025 μg per well for the nucleocapsid IgG assay) overnight at 4°C. On the next day, wells were washed 3x with PBS-0.1% Tween 20 (PBS-T) and blocked with PBS-T containing 3% non-fat milk powder for 1 hour at room temperature (RT). Wells were then incubated with plasma samples from COVID-19 patients at a dilution of 1:100 in PBS-T containing 1% non-fat milk for 1 hour at 37°C. Two negative and two positive plasma pool wells and two blank wells incubated with PBS-T containing 1% non-fat milk powder were included on each plate. After washing 3x with PBS-T, horseradish peroxidase conjugated goat anti-human IgG (γ-chain specific, catalog no. 62-8420, Thermo Fisher, 1:6,000 dilution), IgM (μ-chain specific, catalog no. A6907, Sigma, 1:6,000 dilution), or IgA (α-chain specific, catalog no. P0216, Agilent, 1:5,000 dilution) in PBS-T containing 1% non-fat milk was added and incubated for 1 hour at RT. Wells were washed 3x with PBS-T and dried by vigorous tapping of plates on paper towels. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution was added and the reaction was stopped after 12 min by addition of 0.16 M sulfuric acid. The optical density (OD) at 450 nanometers was measured with an EMax Plus microplate reader (Molecular Devices, San Jose, CA); values for blank wells were subtracted from values obtained for plasma samples. The cutoff value for seroconversion was calculated by adding 3 standard deviations to mean ELISA ODs of 94 historical negative control samples from healthy blood donors (collected before the pandemic for an unrelated seroprevalence study) obtained by testing the samples in all protein/isotype assays (table S4). Additional details for the manual and clinical lab instrument ELISA assay setup are provided” | concepts[85] |
| concept | recently class-switched B cells — not defined in this paper's own words; circulating B-cell expansion near seroconversion — “At early time points post-onset of symptoms, we saw no evidence of pre-existing antibodies that recognize SARS-CoV-2, suggesting that there is minimal cross-reactivity of SARS-CoV-2 antigens with community coronaviruses. These findings do not preclude the possibility that memory B cells stimulated by prior coronavirus exposure may be cross-reactive and form part of the response to SARS-CoV-2. IgM, IgG and IgA antibodies specific for SARS-CoV-2 antigens typically become detectable in patients’ blood at similar median times of about 2 weeks after onset of symptoms (this study and ( 32 , 33 )), although IgA timing is the most variable. As we have recently reported, large polyclonal proliferations of recently class-switched B cells expressing IgG and IgA subtypes with low antibody somatic mutation frequencies appear in the circulation around the time of seroconversion, likely including plasmablasts that contribute to the observed serological responses” | concepts[86] |
| concept | plasmablasts — not defined in this paper's own words; antibody-secreting cells likely contributing to serological response — “At early time points post-onset of symptoms, we saw no evidence of pre-existing antibodies that recognize SARS-CoV-2, suggesting that there is minimal cross-reactivity of SARS-CoV-2 antigens with community coronaviruses. These findings do not preclude the possibility that memory B cells stimulated by prior coronavirus exposure may be cross-reactive and form part of the response to SARS-CoV-2. IgM, IgG and IgA antibodies specific for SARS-CoV-2 antigens typically become detectable in patients’ blood at similar median times of about 2 weeks after onset of symptoms (this study and ( 32 , 33 )), although IgA timing is the most variable. As we have recently reported, large polyclonal proliferations of recently class-switched B cells expressing IgG and IgA subtypes with low antibody somatic mutation frequencies appear in the circulation around the time of seroconversion, likely including plasmablasts that contribute to the observed serological responses” | concepts[87] |
| concept | observational study — not defined in this paper's own words; design that cannot establish whether antibodies caused lower viral loads — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood. Outpatients and admitted non-ICU patients had the lowest viral loads in their nasopharyngeal swabs, but whether this is due to their antibody responses cannot be determined from this observational study. Associations of mortality in COVID-19 patients with SARS-CoV-2 viral load assessed by rRT-PCR applied to nasopharyngeal swab specimens have also been found in other studies ( 34 , 35 ). Our data are consistent with results reported from a panel of antibody assays applied to single time point samples from COVID-19 patients who recovered or died of their disease, which found higher values of spike-targeting responses in the convalescents ( 19 ). Patients with more severe illness in our study eventually raised higher antibody titers than those with milder disease, consistent with prior publications ( 32 , 36 ) and reports of other coronavirus infections ( 21 – 23 ). Patients with more severe disease also had somewhat higher viral loads than patients with milder illness, suggesting that larger initial amounts of viral antigen may contribute to their greater serological responses” | concepts[88] |
| concept | larger initial amounts of viral antigen — not defined in this paper's own words; proposed driver of larger responses in severe illness — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood. Outpatients and admitted non-ICU patients had the lowest viral loads in their nasopharyngeal swabs, but whether this is due to their antibody responses cannot be determined from this observational study. Associations of mortality in COVID-19 patients with SARS-CoV-2 viral load assessed by rRT-PCR applied to nasopharyngeal swab specimens have also been found in other studies ( 34 , 35 ). Our data are consistent with results reported from a panel of antibody assays applied to single time point samples from COVID-19 patients who recovered or died of their disease, which found higher values of spike-targeting responses in the convalescents ( 19 ). Patients with more severe illness in our study eventually raised higher antibody titers than those with milder disease, consistent with prior publications ( 32 , 36 ) and reports of other coronavirus infections ( 21 – 23 ). Patients with more severe disease also had somewhat higher viral loads than patients with milder illness, suggesting that larger initial amounts of viral antigen may contribute to their greater serological responses” | concepts[89] |
| concept | greater serological responses — not defined in this paper's own words; higher responses potentially driven by more antigen — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood. Outpatients and admitted non-ICU patients had the lowest viral loads in their nasopharyngeal swabs, but whether this is due to their antibody responses cannot be determined from this observational study. Associations of mortality in COVID-19 patients with SARS-CoV-2 viral load assessed by rRT-PCR applied to nasopharyngeal swab specimens have also been found in other studies ( 34 , 35 ). Our data are consistent with results reported from a panel of antibody assays applied to single time point samples from COVID-19 patients who recovered or died of their disease, which found higher values of spike-targeting responses in the convalescents ( 19 ). Patients with more severe illness in our study eventually raised higher antibody titers than those with milder disease, consistent with prior publications ( 32 , 36 ) and reports of other coronavirus infections ( 21 – 23 ). Patients with more severe disease also had somewhat higher viral loads than patients with milder illness, suggesting that larger initial amounts of viral antigen may contribute to their greater serological responses” | concepts[90] |
| concept | inflammatory mediators — not defined in this paper's own words; non-antibody contributor to outcomes — “In the detailed serological time courses of the hospitalized patients in this study it was evident that the patterns of antibody responses could not fully explain patient outcomes, including death. Substantial numbers of patients recovered from their illness and were discharged from hospital before they had formed detectable antibody responses, but minimal serological responses were also seen in patients who died of COVID-19 at early times post-onset of symptoms. Similarly, individuals with moderate antibody production were seen across the full spectrum of inpatient disease severity, and many patients who died of their disease generated high levels of antibodies, RBD-ACE2 blocking activity, and neutralizing titers. Differences between individuals in other aspects of the immune response or disease course, such as production of inflammatory mediators, T cell responses, host cell and tissue vulnerability to the damage during viral infection, coagulopathy, and secondary infections, are all likely to contribute to patient outcomes” | concepts[91] |
| concept | T cell responses — not defined in this paper's own words; non-antibody immune contributor to outcomes — “In the detailed serological time courses of the hospitalized patients in this study it was evident that the patterns of antibody responses could not fully explain patient outcomes, including death. Substantial numbers of patients recovered from their illness and were discharged from hospital before they had formed detectable antibody responses, but minimal serological responses were also seen in patients who died of COVID-19 at early times post-onset of symptoms. Similarly, individuals with moderate antibody production were seen across the full spectrum of inpatient disease severity, and many patients who died of their disease generated high levels of antibodies, RBD-ACE2 blocking activity, and neutralizing titers. Differences between individuals in other aspects of the immune response or disease course, such as production of inflammatory mediators, T cell responses, host cell and tissue vulnerability to the damage during viral infection, coagulopathy, and secondary infections, are all likely to contribute to patient outcomes” | concepts[92] |
| concept | coagulopathy — not defined in this paper's own words; disease-course contributor to patient outcome — “In the detailed serological time courses of the hospitalized patients in this study it was evident that the patterns of antibody responses could not fully explain patient outcomes, including death. Substantial numbers of patients recovered from their illness and were discharged from hospital before they had formed detectable antibody responses, but minimal serological responses were also seen in patients who died of COVID-19 at early times post-onset of symptoms. Similarly, individuals with moderate antibody production were seen across the full spectrum of inpatient disease severity, and many patients who died of their disease generated high levels of antibodies, RBD-ACE2 blocking activity, and neutralizing titers. Differences between individuals in other aspects of the immune response or disease course, such as production of inflammatory mediators, T cell responses, host cell and tissue vulnerability to the damage during viral infection, coagulopathy, and secondary infections, are all likely to contribute to patient outcomes” | concepts[93] |
| concept | secondary infections — not defined in this paper's own words; additional contributor to patient outcome — “In the detailed serological time courses of the hospitalized patients in this study it was evident that the patterns of antibody responses could not fully explain patient outcomes, including death. Substantial numbers of patients recovered from their illness and were discharged from hospital before they had formed detectable antibody responses, but minimal serological responses were also seen in patients who died of COVID-19 at early times post-onset of symptoms. Similarly, individuals with moderate antibody production were seen across the full spectrum of inpatient disease severity, and many patients who died of their disease generated high levels of antibodies, RBD-ACE2 blocking activity, and neutralizing titers. Differences between individuals in other aspects of the immune response or disease course, such as production of inflammatory mediators, T cell responses, host cell and tissue vulnerability to the damage during viral infection, coagulopathy, and secondary infections, are all likely to contribute to patient outcomes” | concepts[94] |
| concept | reinfection — not defined in this paper's own words; repeat infection whose protection remains uncertain — “There is an urgent need to understand how long antibody titers against the virus persist after infection, now that the pandemic has been underway for more than half a year in many countries, and initial case reports of proven reinfection by SARS-CoV-2 have begun to appear ( 37 , 38 ). Studies differing in their patient populations, disease severity, and serological assays, have disagreed on the duration of SARS-CoV-2 antibody responses ( 14 , 16 , 32 ). Our data derived from inpatients, outpatients and asymptomatic individuals, with an additional asymptomatic validation cohort, show clearly that not only IgM and IgA, but also IgG titers to RBD, S1 and N antigens, RBD-ACE2 blocking activity and spike pseudotyped viral neutralization titers all begin to decrease in patients after approximately the first month post-onset of symptoms. The decline in antibody titers is most evident in individuals who had asymptomatic infection or mild illness, who produce lower levels of antibodies at the peak of their responses. We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately ( 16 ). Our data do not permit us to predict what fraction of the population will be susceptible to reinfection at a given time after their initial illness, or whether individuals will maintain sustained plateaus of lower antibody levels after an initial decrease; additional time and follow-up will be required to obtain this information” | concepts[95] |
| concept | isotype — not defined in this paper's own words; IgM, IgG, or IgA class hidden by pan-Ig aggregation — “There is an urgent need to understand how long antibody titers against the virus persist after infection, now that the pandemic has been underway for more than half a year in many countries, and initial case reports of proven reinfection by SARS-CoV-2 have begun to appear ( 37 , 38 ). Studies differing in their patient populations, disease severity, and serological assays, have disagreed on the duration of SARS-CoV-2 antibody responses ( 14 , 16 , 32 ). Our data derived from inpatients, outpatients and asymptomatic individuals, with an additional asymptomatic validation cohort, show clearly that not only IgM and IgA, but also IgG titers to RBD, S1 and N antigens, RBD-ACE2 blocking activity and spike pseudotyped viral neutralization titers all begin to decrease in patients after approximately the first month post-onset of symptoms. The decline in antibody titers is most evident in individuals who had asymptomatic infection or mild illness, who produce lower levels of antibodies at the peak of their responses. We note that reported results failing to find a decrease in SARS-CoV-2 antibodies after several months post-infection have relied on “pan-Ig” assays that cannot evaluate each isotype separately ( 16 ). Our data do not permit us to predict what fraction of the population will be susceptible to reinfection at a given time after their initial illness, or whether individuals will maintain sustained plateaus of lower antibody levels after an initial decrease; additional time and follow-up will be required to obtain this information” | concepts[96] |
| concept | prospective validation cohort — not defined in this paper's own words; cohort exempt from retrospective sampling bias — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[97] |
| concept | decreasing antibody levels — not defined in this paper's own words; waning serum titers that do not necessarily equal lost immunity — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[98] |
| concept | all immunity will be lost — not defined in this paper's own words; overstrong interpretation explicitly rejected by the paper — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[99] |
| concept | local mucosal antibody production — not defined in this paper's own words; possible airway protection after serum decline — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[100] |
| concept | memory B and T cells — not defined in this paper's own words; recall cells that could accelerate later responses — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[101] |
| concept | seroprevalence studies — not defined in this paper's own words; population studies biased downward as antibodies wane — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[102] |
| concept | frequent boosting — not defined in this paper's own words; possible vaccination requirement if antibodies do not persist — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[103] |
| concept | vaccination strategies — not defined in this paper's own words; vaccine approaches differing from natural infection — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[104] |
| concept | natural infection — not defined in this paper's own words; immune exposure contrasted with vaccination — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[105] |
| concept | adjuvants — not defined in this paper's own words; vaccine components absent from natural infection — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[106] |
| concept | short- or long-lived plasma cells — not defined in this paper's own words; cell populations governing antibody persistence — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[107] |
| concept | T cell memory — not defined in this paper's own words; longer-term cellular immune memory — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[108] |
| concept | rRT-PCR-confirmed SARS-CoV-2 infection — not defined in this paper's own words; cohort inclusion criterion — “The objective of this study was to investigate correlations between humoral immune responses to SARS-CoV-2, including antibodies blocking binding of RBD to the human ACE2 receptor or neutralizing spike pseudovirus, and viral RNA loads in the nasopharynx and blood in different COVID-19 patient groups and individual patients. On March 4, 2020, the Stanford Health Care Clinical Virology Laboratory began rRT-PCR testing on nasopharyngeal specimens from suspected COVID-19 patients using a laboratory-developed SARS-CoV-2 rRT-PCR assay ( 46 , 47 ). For this study, we included specimens from patients with rRT-PCR-confirmed SARS-CoV-2 infection who reported with symptoms of COVID-19 to Stanford Healthcare-associated clinical sites between March 2020 and August 2020; and specimens from rRT-PCR positive outpatients and asymptomatic individuals identified between April 2020 and May 2020 through occupational health screening including rRT-PCR and serology testing for RBD IgM/G at Stanford Clinical Laboratories. The screening program that detected asymptomatic SARS-CoV-2 infected individuals was offered to all Stanford Healthcare employees on a voluntary basis, and screened employees with nasopharyngeal swab rRT-PCR testing and serology. In addition, we included a validation cohort of 14 asymptomatic and mildly ill individuals with prospective sample collection for up to four months post-enrollment. This study was approved by the Stanford University Institutional Review Board (Protocols IRB-48973 and IRB-55689)” | concepts[109] |
| concept | occupational health screening — not defined in this paper's own words; route identifying outpatient and asymptomatic participants — “The objective of this study was to investigate correlations between humoral immune responses to SARS-CoV-2, including antibodies blocking binding of RBD to the human ACE2 receptor or neutralizing spike pseudovirus, and viral RNA loads in the nasopharynx and blood in different COVID-19 patient groups and individual patients. On March 4, 2020, the Stanford Health Care Clinical Virology Laboratory began rRT-PCR testing on nasopharyngeal specimens from suspected COVID-19 patients using a laboratory-developed SARS-CoV-2 rRT-PCR assay ( 46 , 47 ). For this study, we included specimens from patients with rRT-PCR-confirmed SARS-CoV-2 infection who reported with symptoms of COVID-19 to Stanford Healthcare-associated clinical sites between March 2020 and August 2020; and specimens from rRT-PCR positive outpatients and asymptomatic individuals identified between April 2020 and May 2020 through occupational health screening including rRT-PCR and serology testing for RBD IgM/G at Stanford Clinical Laboratories. The screening program that detected asymptomatic SARS-CoV-2 infected individuals was offered to all Stanford Healthcare employees on a voluntary basis, and screened employees with nasopharyngeal swab rRT-PCR testing and serology. In addition, we included a validation cohort of 14 asymptomatic and mildly ill individuals with prospective sample collection for up to four months post-enrollment. This study was approved by the Stanford University Institutional Review Board (Protocols IRB-48973 and IRB-55689)” | concepts[110] |
| concept | historical negative control samples — not defined in this paper's own words; pre-pandemic samples setting the ELISA seroconversion cutoff — “The ELISA procedure in this study was modified from a protocol published by Stadlbauer ( 48 ). 96-well Corning Costar high binding plates (catalog no. 9018, Thermo Fisher) were coated with SARS-CoV RBD, SARS-CoV-2 RBD, S1, or N protein in phosphate-buffered saline (PBS) at a concentration of 0.1 μg per well (0.025 μg per well for the nucleocapsid IgG assay) overnight at 4°C. On the next day, wells were washed 3x with PBS-0.1% Tween 20 (PBS-T) and blocked with PBS-T containing 3% non-fat milk powder for 1 hour at room temperature (RT). Wells were then incubated with plasma samples from COVID-19 patients at a dilution of 1:100 in PBS-T containing 1% non-fat milk for 1 hour at 37°C. Two negative and two positive plasma pool wells and two blank wells incubated with PBS-T containing 1% non-fat milk powder were included on each plate. After washing 3x with PBS-T, horseradish peroxidase conjugated goat anti-human IgG (γ-chain specific, catalog no. 62-8420, Thermo Fisher, 1:6,000 dilution), IgM (μ-chain specific, catalog no. A6907, Sigma, 1:6,000 dilution), or IgA (α-chain specific, catalog no. P0216, Agilent, 1:5,000 dilution) in PBS-T containing 1% non-fat milk was added and incubated for 1 hour at RT. Wells were washed 3x with PBS-T and dried by vigorous tapping of plates on paper towels. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution was added and the reaction was stopped after 12 min by addition of 0.16 M sulfuric acid. The optical density (OD) at 450 nanometers was measured with an EMax Plus microplate reader (Molecular Devices, San Jose, CA); values for blank wells were subtracted from values obtained for plasma samples. The cutoff value for seroconversion was calculated by adding 3 standard deviations to mean ELISA ODs of 94 historical negative control samples from healthy blood donors (collected before the pandemic for an unrelated seroprevalence study) obtained by testing the samples in all protein/isotype assays (table S4). Additional details for the manual and clinical lab instrument ELISA assay setup are provided” | concepts[111] |
| concept | cutoff value for seroconversion — not defined in this paper's own words; mean negative OD plus three standard deviations — “The ELISA procedure in this study was modified from a protocol published by Stadlbauer ( 48 ). 96-well Corning Costar high binding plates (catalog no. 9018, Thermo Fisher) were coated with SARS-CoV RBD, SARS-CoV-2 RBD, S1, or N protein in phosphate-buffered saline (PBS) at a concentration of 0.1 μg per well (0.025 μg per well for the nucleocapsid IgG assay) overnight at 4°C. On the next day, wells were washed 3x with PBS-0.1% Tween 20 (PBS-T) and blocked with PBS-T containing 3% non-fat milk powder for 1 hour at room temperature (RT). Wells were then incubated with plasma samples from COVID-19 patients at a dilution of 1:100 in PBS-T containing 1% non-fat milk for 1 hour at 37°C. Two negative and two positive plasma pool wells and two blank wells incubated with PBS-T containing 1% non-fat milk powder were included on each plate. After washing 3x with PBS-T, horseradish peroxidase conjugated goat anti-human IgG (γ-chain specific, catalog no. 62-8420, Thermo Fisher, 1:6,000 dilution), IgM (μ-chain specific, catalog no. A6907, Sigma, 1:6,000 dilution), or IgA (α-chain specific, catalog no. P0216, Agilent, 1:5,000 dilution) in PBS-T containing 1% non-fat milk was added and incubated for 1 hour at RT. Wells were washed 3x with PBS-T and dried by vigorous tapping of plates on paper towels. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution was added and the reaction was stopped after 12 min by addition of 0.16 M sulfuric acid. The optical density (OD) at 450 nanometers was measured with an EMax Plus microplate reader (Molecular Devices, San Jose, CA); values for blank wells were subtracted from values obtained for plasma samples. The cutoff value for seroconversion was calculated by adding 3 standard deviations to mean ELISA ODs of 94 historical negative control samples from healthy blood donors (collected before the pandemic for an unrelated seroprevalence study) obtained by testing the samples in all protein/isotype assays (table S4). Additional details for the manual and clinical lab instrument ELISA assay setup are provided” | concepts[112] |
| concept | percentage of blocking — not defined in this paper's own words; normalized RBD-ACE2 competition output — “96-well Corning Costar high binding plates (Thermo Fisher: cat. 9018) were coated with SARS-CoV-2 spike RBD protein in PBS at a concentration of 0.1 μg per well overnight at 4°C. All competition ELISA steps were carried out on the next day at RT. Wells were washed 3x with PBS-T and blocked with PBS-T containing 3% non-fat milk powder for 1 hour. Wells were then incubated with plasma samples from COVID-19 patients at a dilution of 1:10 in PBS-T containing 1% non-fat milk for 1 hour. Two quality controls (Access SARS-CoV-2 IgG QC, QC1-QC2, 2 levels, catalog no. C58964 , Beckman Coulter) and two blank wells incubated with PBS-T containing 1% non-fat milk were included on each plate. ACE2-mFc diluted to 0.5 μg/ml in 1% non-fat milk powder was added without washing steps and incubated for an additional 45 min. After washing 3x with PBS-T, horseradish peroxidase conjugated goat anti-mouse IgG (Fc specific, catalog no. 31439, Invitrogen, 1:10’000 dilution) in PBS-T containing 1% non-fat milk was added and incubated for 45 min. Wells were washed 3x with PBS-T and dried by vigorous tapping of plates on paper towels. TMB substrate solution was added and the reaction was stopped after 12 min by addition of 0.16 M sulfuric acid. The OD at 450 nanometers was measured with an EMax Plus microplate reader (Molecular Devices, San Jose, CA). OD values were converted to percentage of blocking using the following formula: 100*(1-(sample OD - 0.2)/(QC1 OD – 0.2)), taking into account the background noise of the assay of 0.2 as determined after testing pre-pandemic control plasma samples. Additional details for competition ELISA assay setup and optimization are provided” | concepts[113] |
| concept | technical duplicate — not defined in this paper's own words; replicate structure used in neutralization testing — “SARS-CoV-2 pseudotyped lentivirus assays were performed as described previously. Briefly, spike-pseudotyped lentivirus was produced in HEK293T cells as described in ( 49 ) using a 5-plasmid system described in ( 27 ). For viral neutralization assays, ACE2/HeLa cells ( 28 ) were plated in 96-well tissue culture plates one day prior to infection. Prior to neutralization assays, patient plasma samples were heat inactivated for 1 hour at 56°C. Plasma samples and virus were incubated with the cells at 37°C for ~48 hours. After incubation, cells were lysed with BriteLite assay readout solution (Perkin Elmer) and luminescence values were obtained with a BioTek plate reader. Single-dilution point neutralization assays were performed in technical duplicate in two different experimental replicates. Percent neutralization was determined by normalizing raw luciferase values to 0% infectivity (average from cells only wells) and 100% infectivity (average from virus only wells) in GraphPad Prism 8.4.1. Plasma dilution series shown in Figure S9 were performed in technical duplicate; normalized % infectivity values were fit with a three-parameter non-linear regression inhibitor curve in GraphPad Prism 8.4.1 and fits were constrained to have a value of 0% at the bottom of the fit” | concepts[114] |
| concept | three-parameter non-linear regression inhibitor curve — not defined in this paper's own words; curve fitted to plasma dilution series — “SARS-CoV-2 pseudotyped lentivirus assays were performed as described previously. Briefly, spike-pseudotyped lentivirus was produced in HEK293T cells as described in ( 49 ) using a 5-plasmid system described in ( 27 ). For viral neutralization assays, ACE2/HeLa cells ( 28 ) were plated in 96-well tissue culture plates one day prior to infection. Prior to neutralization assays, patient plasma samples were heat inactivated for 1 hour at 56°C. Plasma samples and virus were incubated with the cells at 37°C for ~48 hours. After incubation, cells were lysed with BriteLite assay readout solution (Perkin Elmer) and luminescence values were obtained with a BioTek plate reader. Single-dilution point neutralization assays were performed in technical duplicate in two different experimental replicates. Percent neutralization was determined by normalizing raw luciferase values to 0% infectivity (average from cells only wells) and 100% infectivity (average from virus only wells) in GraphPad Prism 8.4.1. Plasma dilution series shown in Figure S9 were performed in technical duplicate; normalized % infectivity values were fit with a three-parameter non-linear regression inhibitor curve in GraphPad Prism 8.4.1 and fits were constrained to have a value of 0% at the bottom of the fit” | concepts[115] |
| concept | Wilcoxon rank sum testing — not defined in this paper's own words; between-severity-category comparison — “A volume of 400 μL of EDTA-anticoagulated plasma was extracted by Qiagen EZ1 Virus Mini Kit v2.0 (Qiagen Germantown, MD). Molecular testing for the presence of SARS-CoV-2 RNA in plasma was performed with a modification of a published rRT-PCR assay targeting the envelope ( E ) gene ( 46 , 47 ). The standard Ct values of positive tests with this assay range from Ct <20 to 45 cycles. Testing of plasma samples with a Ct value of 40 or greater was repeated to ensure reproducibility of the positive result. As viral culture was not performed as part of this study, presence of SARS-CoV-2 in tested plasma was defined as RNAemia. Statistics GraphPad Prism version 8.0 (GraphPad Software, San Diego, California, USA) software was used to visualize ELISA data, analyze for differences in antibody responses between disease categories by Wilcoxon rank sum testing, and carry out linear regression of pseudotyped virus neutralization, RBD-ACE2 blocking and antibody ELISA results. The goodness of fit for linear regression analyses was reported as the coefficient of determination, R 2 . Locally estimated scatterplot smoothing for the development and decrease of antibody responses over time was performed using the loess method in the R statistical package version 3.6.1 ( 50 ). Correlation between antibody OD 450 values, RNAemia, and ACE2-RBD blocking assay OD 450 values were calculated as Spearman correlations with the R cor function” | concepts[116] |
| concept | linear regression — not defined in this paper's own words; analysis relating functional and binding assay results — “A volume of 400 μL of EDTA-anticoagulated plasma was extracted by Qiagen EZ1 Virus Mini Kit v2.0 (Qiagen Germantown, MD). Molecular testing for the presence of SARS-CoV-2 RNA in plasma was performed with a modification of a published rRT-PCR assay targeting the envelope ( E ) gene ( 46 , 47 ). The standard Ct values of positive tests with this assay range from Ct <20 to 45 cycles. Testing of plasma samples with a Ct value of 40 or greater was repeated to ensure reproducibility of the positive result. As viral culture was not performed as part of this study, presence of SARS-CoV-2 in tested plasma was defined as RNAemia. Statistics GraphPad Prism version 8.0 (GraphPad Software, San Diego, California, USA) software was used to visualize ELISA data, analyze for differences in antibody responses between disease categories by Wilcoxon rank sum testing, and carry out linear regression of pseudotyped virus neutralization, RBD-ACE2 blocking and antibody ELISA results. The goodness of fit for linear regression analyses was reported as the coefficient of determination, R 2 . Locally estimated scatterplot smoothing for the development and decrease of antibody responses over time was performed using the loess method in the R statistical package version 3.6.1 ( 50 ). Correlation between antibody OD 450 values, RNAemia, and ACE2-RBD blocking assay OD 450 values were calculated as Spearman correlations with the R cor function” | concepts[117] |
| concept | coefficient of determination, R 2 — not defined in this paper's own words; goodness-of-fit statistic for linear regressions — “A volume of 400 μL of EDTA-anticoagulated plasma was extracted by Qiagen EZ1 Virus Mini Kit v2.0 (Qiagen Germantown, MD). Molecular testing for the presence of SARS-CoV-2 RNA in plasma was performed with a modification of a published rRT-PCR assay targeting the envelope ( E ) gene ( 46 , 47 ). The standard Ct values of positive tests with this assay range from Ct <20 to 45 cycles. Testing of plasma samples with a Ct value of 40 or greater was repeated to ensure reproducibility of the positive result. As viral culture was not performed as part of this study, presence of SARS-CoV-2 in tested plasma was defined as RNAemia. Statistics GraphPad Prism version 8.0 (GraphPad Software, San Diego, California, USA) software was used to visualize ELISA data, analyze for differences in antibody responses between disease categories by Wilcoxon rank sum testing, and carry out linear regression of pseudotyped virus neutralization, RBD-ACE2 blocking and antibody ELISA results. The goodness of fit for linear regression analyses was reported as the coefficient of determination, R 2 . Locally estimated scatterplot smoothing for the development and decrease of antibody responses over time was performed using the loess method in the R statistical package version 3.6.1 ( 50 ). Correlation between antibody OD 450 values, RNAemia, and ACE2-RBD blocking assay OD 450 values were calculated as Spearman correlations with the R cor function” | concepts[118] |
| concept | loess method — not defined in this paper's own words; locally smoothed antibody trajectory analysis — “A volume of 400 μL of EDTA-anticoagulated plasma was extracted by Qiagen EZ1 Virus Mini Kit v2.0 (Qiagen Germantown, MD). Molecular testing for the presence of SARS-CoV-2 RNA in plasma was performed with a modification of a published rRT-PCR assay targeting the envelope ( E ) gene ( 46 , 47 ). The standard Ct values of positive tests with this assay range from Ct <20 to 45 cycles. Testing of plasma samples with a Ct value of 40 or greater was repeated to ensure reproducibility of the positive result. As viral culture was not performed as part of this study, presence of SARS-CoV-2 in tested plasma was defined as RNAemia. Statistics GraphPad Prism version 8.0 (GraphPad Software, San Diego, California, USA) software was used to visualize ELISA data, analyze for differences in antibody responses between disease categories by Wilcoxon rank sum testing, and carry out linear regression of pseudotyped virus neutralization, RBD-ACE2 blocking and antibody ELISA results. The goodness of fit for linear regression analyses was reported as the coefficient of determination, R 2 . Locally estimated scatterplot smoothing for the development and decrease of antibody responses over time was performed using the loess method in the R statistical package version 3.6.1 ( 50 ). Correlation between antibody OD 450 values, RNAemia, and ACE2-RBD blocking assay OD 450 values were calculated as Spearman correlations with the R cor function” | concepts[119] |
| concept | Spearman correlations — not defined in this paper's own words; rank correlations among antibody, RNAemia, and blocking values — “A volume of 400 μL of EDTA-anticoagulated plasma was extracted by Qiagen EZ1 Virus Mini Kit v2.0 (Qiagen Germantown, MD). Molecular testing for the presence of SARS-CoV-2 RNA in plasma was performed with a modification of a published rRT-PCR assay targeting the envelope ( E ) gene ( 46 , 47 ). The standard Ct values of positive tests with this assay range from Ct <20 to 45 cycles. Testing of plasma samples with a Ct value of 40 or greater was repeated to ensure reproducibility of the positive result. As viral culture was not performed as part of this study, presence of SARS-CoV-2 in tested plasma was defined as RNAemia. Statistics GraphPad Prism version 8.0 (GraphPad Software, San Diego, California, USA) software was used to visualize ELISA data, analyze for differences in antibody responses between disease categories by Wilcoxon rank sum testing, and carry out linear regression of pseudotyped virus neutralization, RBD-ACE2 blocking and antibody ELISA results. The goodness of fit for linear regression analyses was reported as the coefficient of determination, R 2 . Locally estimated scatterplot smoothing for the development and decrease of antibody responses over time was performed using the loess method in the R statistical package version 3.6.1 ( 50 ). Correlation between antibody OD 450 values, RNAemia, and ACE2-RBD blocking assay OD 450 values were calculated as Spearman correlations with the R cor function” | concepts[120] |
| concept | SARS-CoV-2 — not defined in this paper's own words; designation for the virus causing COVID-19 — “The virus causing COVID-19 belongs to the Sarbecovirus subgenus (genus Betacoronavirus ) together with the severe acute respiratory syndrome coronavirus (SARS-CoV) and has been designated SARS-CoV-2 ( 9 ). Coronaviruses contain four structural proteins, including spike, envelope, membrane, and nucleocapsid (N) proteins. The spike surface glycoprotein contains the receptor binding domain (RBD), which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachment, fusion of viral and host membranes, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2 develop antibodies to the spike and N proteins, which are therefore used as antigens in clinical serology assays. The spike protein is an important target for neutralizing antibodies, as they can prevent viral entry into host cells” | concepts[121] |
| concept | SARS-CoV RBD — not defined in this paper's own words; RBD of the earlier SARS coronavirus — “S1-specific IgM, IgG, and IgA showed antibody kinetics that were very similar to those seen for RBD (figs. S3 to S5 and table S2). While N-specific IgG responses showed high positivity rates with antibody kinetics similar to those for RBD and S1, IgM antibody responses to N were strikingly low in most patients (figs. S4 and S6 and table S2). There was no consistent difference in RBD, S1, and N antibody titers or RBD-ACE2 blocking antibodies between ICU patients who survived compared to patients who died (fig. S7). We further evaluated the breadth of antibody responses in different disease severity categories by testing for SARS-CoV RBD binding. Most monoclonal antibodies targeting SARS-CoV RBD fail to bind SARS-CoV-2 RBD, indicating distinct antigenicity despite sequence and structural similarity of the two proteins ( 25 , 26 ). Nine of 13 ICU patients, three of 25 admitted non-ICU patients (fig. S8A) and five of 82 outpatients (fig. S8B), developed SARS-CoV RBD IgG titers during the course of their infection. The time course of anti-SARS-CoV RBD positivity in serial samples from individual patients did not always mirror anti-SARS-CoV-2 RBD IgG responses, suggesting limited clonal or oligoclonal B cell responses with this broader reactivity within the overall polyclonal anti-SARS-CoV-2 serological response” | concepts[122] |
| concept | SARS-CoV-2 RBD — not defined in this paper's own words; RBD of SARS-CoV-2 — “S1-specific IgM, IgG, and IgA showed antibody kinetics that were very similar to those seen for RBD (figs. S3 to S5 and table S2). While N-specific IgG responses showed high positivity rates with antibody kinetics similar to those for RBD and S1, IgM antibody responses to N were strikingly low in most patients (figs. S4 and S6 and table S2). There was no consistent difference in RBD, S1, and N antibody titers or RBD-ACE2 blocking antibodies between ICU patients who survived compared to patients who died (fig. S7). We further evaluated the breadth of antibody responses in different disease severity categories by testing for SARS-CoV RBD binding. Most monoclonal antibodies targeting SARS-CoV RBD fail to bind SARS-CoV-2 RBD, indicating distinct antigenicity despite sequence and structural similarity of the two proteins ( 25 , 26 ). Nine of 13 ICU patients, three of 25 admitted non-ICU patients (fig. S8A) and five of 82 outpatients (fig. S8B), developed SARS-CoV RBD IgG titers during the course of their infection. The time course of anti-SARS-CoV RBD positivity in serial samples from individual patients did not always mirror anti-SARS-CoV-2 RBD IgG responses, suggesting limited clonal or oligoclonal B cell responses with this broader reactivity within the overall polyclonal anti-SARS-CoV-2 serological response” | concepts[123] |
| concept | RBD IgM and IgA — not defined in this paper's own words; isotypes more variable and less correlated with functional assays — “Neutralizing antibodies are increased in inpatients compared to outpatients, and correlate with RBD-ACE2 blocking and RBD IgG titers Antibody neutralization of live SARS-CoV-2, or of pseudotyped viruses such as lentiviruses expressing the SARS-CoV-2 spike may represent the most physiologically relevant surrogates for humoral immunity in vivo, but are poorly scalable due to restrictive biosafety requirements for SARS-CoV-2 and less easily standardized assay components and protocols, compared to tests using purified proteins. We tested SARS-CoV-2 spike-pseudotyped lentivirus neutralization ( 27 ) in HeLa cells overexpressing ACE2 ( 28 ) by inpatient and outpatient samples, and evaluated correlations with the more scalable ELISAs for RBD, S1 and RBD-ACE2 blocking ( Fig. 3A, B ). As with RBD-ACE2 blocking, neutralizing antibody activity was higher in inpatients compared to outpatients, and began to decrease after about one month after symptom onset. Neutralization was well-correlated with RBD IgG titers and RBD-ACE2 blocking (linear regression coefficient of determination R 2 of 0.6995 and 0.6824 for inpatients; 0.7338 and 0.6839 for outpatients). The RBD-ACE2 blocking assay was less sensitive than neutralization or RBD IgG ELISA. RBD IgM and IgA ELISA results were much more variable and did not correlate as well with neutralization or RBD-ACE2 blocking compared to RBD IgG” | concepts[124] |
| concept | presence of SARS-CoV-2 in tested plasma — not defined in this paper's own words; study definition of RNAemia — “A volume of 400 μL of EDTA-anticoagulated plasma was extracted by Qiagen EZ1 Virus Mini Kit v2.0 (Qiagen Germantown, MD). Molecular testing for the presence of SARS-CoV-2 RNA in plasma was performed with a modification of a published rRT-PCR assay targeting the envelope ( E ) gene ( 46 , 47 ). The standard Ct values of positive tests with this assay range from Ct <20 to 45 cycles. Testing of plasma samples with a Ct value of 40 or greater was repeated to ensure reproducibility of the positive result. As viral culture was not performed as part of this study, presence of SARS-CoV-2 in tested plasma was defined as RNAemia. Statistics GraphPad Prism version 8.0 (GraphPad Software, San Diego, California, USA) software was used to visualize ELISA data, analyze for differences in antibody responses between disease categories by Wilcoxon rank sum testing, and carry out linear regression of pseudotyped virus neutralization, RBD-ACE2 blocking and antibody ELISA results. The goodness of fit for linear regression analyses was reported as the coefficient of determination, R 2 . Locally estimated scatterplot smoothing for the development and decrease of antibody responses over time was performed using the loess method in the R statistical package version 3.6.1 ( 50 ). Correlation between antibody OD 450 values, RNAemia, and ACE2-RBD blocking assay OD 450 values were calculated as Spearman correlations with the R cor function” | concepts[125] |
| concept | waning antibody levels — not defined in this paper's own words; declining levels that bias seroprevalence — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[126] |
| concept | Reduction in RNAemia — not defined in this paper's own words; decline in plasma viral RNA — “Viral RNAemia is detected in up to a third of COVID-19 patients, most often in patients with severe disease ( 3 , 31 ). RNAemia was evaluated in a subset of patients, and detected in 15 of 25 admitted patients, 13 of 15 ICU patients who recovered, and 2 of 2 patients who died of their disease. Reduction in RNAemia was strongly correlated with the appearance of plasma antibodies (Spearman’s correlation coefficients of -0.47 for IgM, -0.43 for IgG, and -0.44 for IgA, p<0.001 for each)” | concepts[127] |
| concept | appearance of plasma antibodies — not defined in this paper's own words; serological appearance correlated with RNAemia reduction — “Viral RNAemia is detected in up to a third of COVID-19 patients, most often in patients with severe disease ( 3 , 31 ). RNAemia was evaluated in a subset of patients, and detected in 15 of 25 admitted patients, 13 of 15 ICU patients who recovered, and 2 of 2 patients who died of their disease. Reduction in RNAemia was strongly correlated with the appearance of plasma antibodies (Spearman’s correlation coefficients of -0.47 for IgM, -0.43 for IgG, and -0.44 for IgA, p<0.001 for each)” | concepts[128] |
| concept | higher ratios of spike RBD and S1 domains compared to N antigen — not defined in this paper's own words; relative specificity associated with mild illness — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood. Outpatients and admitted non-ICU patients had the lowest viral loads in their nasopharyngeal swabs, but whether this is due to their antibody responses cannot be determined from this observational study. Associations of mortality in COVID-19 patients with SARS-CoV-2 viral load assessed by rRT-PCR applied to nasopharyngeal swab specimens have also been found in other studies ( 34 , 35 ). Our data are consistent with results reported from a panel of antibody assays applied to single time point samples from COVID-19 patients who recovered or died of their disease, which found higher values of spike-targeting responses in the convalescents ( 19 ). Patients with more severe illness in our study eventually raised higher antibody titers than those with milder disease, consistent with prior publications ( 32 , 36 ) and reports of other coronavirus infections ( 21 – 23 ). Patients with more severe disease also had somewhat higher viral loads than patients with milder illness, suggesting that larger initial amounts of viral antigen may contribute to their greater serological responses” | concepts[129] |
| concept | mildest illness — not defined in this paper's own words; outpatient end of the severity spectrum — “Which features of patient antibody responses were associated with disease severity? To our surprise, we found that outpatients with the mildest illness showed higher ratios of spike RBD and S1 domains compared to N antigen, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spike antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood. Outpatients and admitted non-ICU patients had the lowest viral loads in their nasopharyngeal swabs, but whether this is due to their antibody responses cannot be determined from this observational study. Associations of mortality in COVID-19 patients with SARS-CoV-2 viral load assessed by rRT-PCR applied to nasopharyngeal swab specimens have also been found in other studies ( 34 , 35 ). Our data are consistent with results reported from a panel of antibody assays applied to single time point samples from COVID-19 patients who recovered or died of their disease, which found higher values of spike-targeting responses in the convalescents ( 19 ). Patients with more severe illness in our study eventually raised higher antibody titers than those with milder disease, consistent with prior publications ( 32 , 36 ) and reports of other coronavirus infections ( 21 – 23 ). Patients with more severe disease also had somewhat higher viral loads than patients with milder illness, suggesting that larger initial amounts of viral antigen may contribute to their greater serological responses” | concepts[130] |
| concept | Neutralization — not defined in this paper's own words; functional virus-blocking activity — “Neutralizing antibodies are increased in inpatients compared to outpatients, and correlate with RBD-ACE2 blocking and RBD IgG titers Antibody neutralization of live SARS-CoV-2, or of pseudotyped viruses such as lentiviruses expressing the SARS-CoV-2 spike may represent the most physiologically relevant surrogates for humoral immunity in vivo, but are poorly scalable due to restrictive biosafety requirements for SARS-CoV-2 and less easily standardized assay components and protocols, compared to tests using purified proteins. We tested SARS-CoV-2 spike-pseudotyped lentivirus neutralization ( 27 ) in HeLa cells overexpressing ACE2 ( 28 ) by inpatient and outpatient samples, and evaluated correlations with the more scalable ELISAs for RBD, S1 and RBD-ACE2 blocking ( Fig. 3A, B ). As with RBD-ACE2 blocking, neutralizing antibody activity was higher in inpatients compared to outpatients, and began to decrease after about one month after symptom onset. Neutralization was well-correlated with RBD IgG titers and RBD-ACE2 blocking (linear regression coefficient of determination R 2 of 0.6995 and 0.6824 for inpatients; 0.7338 and 0.6839 for outpatients). The RBD-ACE2 blocking assay was less sensitive than neutralization or RBD IgG ELISA. RBD IgM and IgA ELISA results were much more variable and did not correlate as well with neutralization or RBD-ACE2 blocking compared to RBD IgG” | concepts[131] |
| concept | sampling time points — not defined in this paper's own words; patient observation times in the retrospective cohorts — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[132] |
| concept | length of hospital stay and subsequent healthcare visits — not defined in this paper's own words; determinants of retrospective sampling opportunity — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[133] |
| concept | disease categories — not defined in this paper's own words; admitted, ICU, and deceased clinical groups — “Inpatient SARS-CoV-2 antibody time course patterns are associated with decreases in RNAemia, but not disease outcome We hypothesized that detailed examination of the time course of SARS-CoV-2 antibody responses could identify distinct patterns associated with admitted non-ICU patients ( Fig. 6 , fig. S10), ICU patients who recovered from their illness ( Fig. 7 , fig. S11A), or those who died ( Fig. 8 , fig. S11B). We identified three main patterns of response: at the time point closest to their discharge from hospital, or death, Group 1 individuals had SARS-CoV-2 antibody responses with no RBD IgG or RBD-ACE2 blocking activity; Group 2 individuals had up to 25% RBD-ACE2 blocking activity; and Group 3 individuals developed high levels of antibodies and over 25% RBD-ACE2 blocking activity. These patterns of response were not strongly associated with disease severity, but instead were shared across disease categories. Group 1 patients included admitted patients who recovered from their illness rapidly and were discharged before they had developed detectable antibody responses, but also included patients who recovered from their illness without antibody production after prolonged ICU stays, and patients who died of COVID-19 prior to antibody development. Similarly, Group 2 patterns with low levels of RBD-ACE2 blocking were shared across all three patient categories. In Group 3 (high levels of antibody production and >25% RBD-ACE2 blocking activity) admitted non-ICU patients differed from ICU or deceased patients in that they developed their robust antibody responses during short hospital stays prior to discharge, whereas ICU or deceased patients typically had prolonged hospital courses. In patients tested for neutralizing antibodies (13 admitted non-ICU patients, 16 ICU surviving patients, and 16 deceased patients), the results were closely correlated with IgG titers to RBD, or RBD-ACE2 blocking” | concepts[134] |
| concept | SARS-CoV-2-infection upon re-exposure — not defined in this paper's own words; repeat exposure outcome potentially impeded by mucosal antibodies — “A limitation of our study is that, apart from the prospective validation cohort, the sampling time points for each patient were determined by their length of hospital stay and subsequent healthcare visits, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levels do not necessarily indicate that all immunity will be lost. It is possible that local mucosal antibody production in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposure ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cells stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfection reports offer some hope that SARS-CoV-2 may behave similarly to other community coronaviruses, with reinfection generally producing milder illness than the initial infection ( 40 , 41 ). One implication of our finding of waning antibody levels is that seroprevalence studies may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccination will last, and whether frequent boosting will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategies undergoing clinical trials differ from natural infection in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvants ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infection, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populations, short- or long-lived plasma cells, and T cell memory to SARS-CoV-2 as well as other coronaviruses should begin to clarify some of these key mechanistic points” | concepts[135] |
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Sci Immunol Sci Immunol 3837 pheaaas 101688624 SciImmunol Science Immunology 2470-9468 pmc-is-collection-domain yes pmc-collection-title AAAS - PMC COVID-19 Collection PMC7857392 PMC7857392.1 7857392 7857392 33288645 10.1126/sciimmunol.abe0240 abe0240 1 Research Articles Research Articles R-Articles Coronavirus Defining the features and duration of antibody responses to SARS-CoV-2term infection associated with disease severityterm and outcome http://orcid.org/0000-0002-1582-1720 RöltgenpersName Katharina 1 † http://orcid.org/0000-0001-6408-9495 PowellpersName Abigail E. 2 † http://orcid.org/0000-0003-0377-6464 WirzpersName Oliver F. 1 † http://orcid.org/0000-0002-1713-3163 StevenspersName Bryan A. 1 † http://orcid.org/0000-0003-1977-253X HoganpersName Catherine A. 1 NajeebpersName Javaria 3 HunterpersName Molly 4 http://orcid.org/0000-0002-3712-9188 WangpersName Hannah 1 http://orcid.org/0000-0001-9837-4032 SahoopersName Malaya K. 1 HuangpersName ChunHong 1 YamamotopersName Fumiko 1 ManoharpersName Monali 5 6 ManalacpersName Justin 1 http://orcid.org/0000-0003-2606-9367 Otrelo-CardosopersName Ana R. 3 PhampersName Tho D. 1 7 http://orcid.org/0000-0002-6921-1012 RustagipersName Arjun 8 http://orcid.org/0000-0001-6969-6200 RogerspersName Angela J. 5 http://orcid.org/0000-0001-9385-7158 ShahpersName Nigam H. 9 http://orcid.org/0000-0001-6946-7627 BlishpersName Catherine A. 8 10 http://orcid.org/0000-0002-6532-9873 CochranpersName Jennifer R. 11 http://orcid.org/0000-0002-3664-0072 JardetzkypersName Theodore S. 3 ZehnderpersName James L. 1 http://orcid.org/0000-0002-3894-685X WangpersName Taia T. 8 10 12 http://orcid.org/0000-0001-5852-7639 NarasimhanpersName Balasubramanian 13 14 GombarpersName Saurabh 1 http://orcid.org/0000-0003-0553-5090 TibshiranipersName Robert 13 14 http://orcid.org/0000-0002-2146-2955 NadeaupersName Kari C. 5 6 http://orcid.org/0000-0001-6503-4541 KimpersName Peter S. 2 10 ‡ http://orcid.org/0000-0001-8751-4810 PinskypersName Benjamin A. 1 8 ‡ http://orcid.org/0000-0003-0963-044X BoydpersName Scott D. 1 6 ‡ * 1 Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. 2 Stanford ChEM-H and Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA. 3 Department of Structural Biology, Stanford University, Stanford, USA. 4 ATUM, Newark, CA, USA 5 Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, Stanford University, Stanford, CA, USA. 6 Sean N. Parker Center for Allergy and Asthma Research, Stanford, CA, USA. 7 Stanford Blood Center, Palo Alto, CA, USA. 8 Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford, CA, USA. 9 Stanford Center for Biomedical Informatics Research, Stanford University, Stanford, California, USA 10 Chan Zuckerberg Biohub, San Francisco, CA, USA. 11 Department of Bioengineering, Stanford University, Stanford, CA, USA. 12 Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA. 13 Department of Statistics, Stanford University, Stanford, CA, USA. 14 Department of Biomedical Data Sciences, Stanford University, Stanford, CA, USA. * Corresponding author: sboyd1@stanford.edu † these authors contributed equally to this work.
‡ these authors contributed equally to this work.
07 12 2020 5 54 374625 eabe0240 27 7 2020 05 10 2020 03 12 2020 03 02 2021 05 02 2021 05 10 2025 Copyright © 2020, American Association for the Advancement of Science 2020 American Association for the Advancement of Science https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution license , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Illness severity in COVID-19 correlates with specificity of serological responses, but antibody levels decrease in most patients.
SARS-CoV-2-specific antibodiesterm, particularly those preventing viral spike receptor binding domain (RBD)term interaction with host angiotensin-converting enzyme 2 (ACE2) receptorterm, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samplesterm from 79 hospitalized COVID-19 patientsterm and 175 SARS-CoV-2-infected outpatients and asymptomatic individualsterm. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1term or RBD domainsterm of spiketerm compared to nucleocapsid antigenterm were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemiaterm, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assaysterm and a scalable ELISAterm measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals’ SARS-CoV-2term antibodies, including IgG, progressively decreased during observation up to five months post-infection.
pmc-status-qastatus 0 pmc-status-live yes pmc-status-embargo no pmc-status-released yes pmc-prop-open-access yes pmc-prop-olf no pmc-prop-manuscript no pmc-prop-legally-suppressed no pmc-prop-has-pdf yes pmc-prop-has-supplement yes pmc-prop-pdf-only no pmc-prop-suppress-copyright no pmc-prop-is-real-version no pmc-prop-is-scanned-article no pmc-prop-preprint no pmc-prop-in-epmc yes pmc-license-ref CC BY INTRODUCTION A novel coronavirus first described in Wuhan, China in December 2019 ( 1 ) has led to a coronavirus disease (COVID-19) pandemic and a global economic shutdown amid unprecedented social distancing measures. The clinical spectrum of COVID-19 ranges from asymptomatic infectionterm and mild upper respiratory tract illness in the majority of patients, to severe viral pneumoniaterm with respiratory failureterm, multiorgan failureterm, and death ( 2 – 4 ). Older adults and people with serious underlying health conditions are at greatest risk for severe illness and death ( 5 – 8 ). Host immune responses may be one of the most important determinants for disease progression and outcome, but this remains to be established.
The virus causing COVID-19 belongs to the Sarbecovirus subgenusterm (genus Betacoronavirusterm ) together with the severe acute respiratory syndrome coronavirus (SARS-CoVterm) and has been designated SARS-CoV-2term ( 9 ). Coronaviruses contain four structural proteinsterm, including spiketerm, envelopeterm, membraneterm, and nucleocapsid (N) proteinsterm. The spike surface glycoproteinterm contains the receptor binding domain (RBD)term, which binds strongly to human ACE2 receptors ( 1 , 10 ), and plays a major role in viral attachmentterm, fusion of viral and host membranesterm, and entry of the virus into host cells ( 11 ). Most individuals infected with SARS-CoV-2term develop antibodies to the spiketerm and N proteins, which are therefore used as antigens in clinical serology assaysterm. The spiketerm protein is an important target for neutralizing antibodiesterm, as they can prevent viral entry into host cellsterm ( 12 , 13 ). Current information on the role of antibodies in viral clearanceterm and modulation of disease severityterm as well as the durabilityterm of these responses following primary infection is limited or controversial. Improved understanding of humoral immunityterm to SARS-CoV-2term is needed to inform strategies for vaccinationterm and the use of therapeutics in the form of neutralizing antibodiesterm or convalescent plasmaterm. Reports about the longevity of antibody titersterm to SARS-CoV-2term are not in full agreement, with some finding a rapid waning of virus-specific IgG antibodiesterm by approximately 3 months after infection ( 14 , 15 ), and others emphasizing stable titers detected over several weeks or several months ( 16 – 18 ). Virus-specific antibody responses appear to be elevated in COVID-19 patients with severe disease as opposed to asymptomatic or mildly ill individuals, raising concerns about the effectiveness of antibody responses to SARS-CoV-2term. A suggestion that the quality rather than quantity of antibodies may predict the outcome of infection is provided by a recent report applying a panel of serological assays to COVID-19 patients who convalesced or died ( 19 ). Nonhuman primates challenged with SARS-CoV-2term after vaccinationterm with spike-based DNA vaccines developed neutralizing antibodiesterm and immune correlates of protection, suggesting that antibody responses may be more effective in preventing than resolving disease ( 20 ).
We performed a comprehensive analysis of SARS-CoV-2 RBDterm, S1term and N protein specific antibodies, and RBD-ACE2 blockingterm antibodies, spike-pseudotyped virus neutralizationterm, and viral RNA measurementsterm in nasopharyngeal swabterm and plasma samples of individuals infected with SARS-CoV-2term. A total of 254 individuals (79 inpatients and 175 outpatients or asymptomatic individuals) were studied. RBD-ACE2 blockingterm antibodies and spike-pseudotyped lentivirus neutralizationterm were well-correlated with IgG specific for RBD. Notably, higher ratios of IgG antibodies targeting S1term or RBD compared to N were strongly associated with clinically milder infection. Viral RNAemiaterm decreased to undetectable levels rapidly once plasma antibodies and RBD-ACE2 blockingterm activity appeared. Outpatients and asymptomatic individualsterm show substantial and progressive decreasesterm in SARS-CoV-2term specific antibodies after the first month of documented infection.
RESULTS Study design and patient demographics A total of 254 individuals with a positive SARS-CoV-2term real-time RT-PCRterm (rRT-PCR) nasopharyngeal swabterm test were included in the study (fig. S1term). Study subjects were identified either after routine serology testing or occupational health screeningterm in the Stanford Health Care Clinical Laboratories for anti-SARS-CoV-2 RBD IgMterm and IgG antibodies (136 asymptomatic individuals or outpatients), or after they reported to Stanford Health Care-associated clinical sites with symptoms of COVID-19. This included 24 outpatients, 35 hospitalized patients who were not admitted to the intensive care unit (ICU)term, and 20 ICU inpatients who survived their illness. To evaluate serological responses associated with patient mortality, we also analyzed specimens from 25 patients who died of COVID-19 (one outpatient, seven admitted non-ICU, and seventeen admitted ICU patients). Of patients who were treated in the ICU, 26 of 37 (70%) required mechanical ventilationterm, including 15 patients who died.
Demographic and clinical characteristics of patients stratified by disease status are presented in Table 1 . The percentage of males, and those with comorbidities of hypertension or diabetes mellitus increased with patient disease severityterm. Outpatients and asymptomatic individualsterm were younger and had the lowest levels of obesity compared to more severely ill patients. Notably, levels of viral RNA measured by rRT-PCR of nasopharyngeal swabsterm at diagnosis showed a progressive increase (lower rRT-PCR cycle threshold, Ctterm) with disease severityterm; patients who died had the highest viral loads (fig. S2). Plasma samples for in-depth serological testing were available from all inpatients and 86 of the 160 outpatient survivors. Demographic and clinical characteristics of outpatients and asymptomatic individualsterm with and without plasma availability are presented in table S1term. A total of 828 samples were analyzed with ELISAs measuring IgM, IgG and IgA specific for SARS-CoV-2 RBDterm, S1term or N, as well as the RBD-ACE2 blockingterm assay ( Fig. 1A-C ). Representative samples were also tested for antibody cross-reactivityterm to SARS-CoV RBDterm, viral RNA in the blood (RNAemiaterm), and neutralizationterm of spike-pseudotyped lentivirus ( Fig. 1D ). We also tested 45 plasma specimens from a validation cohortterm of 14 asymptomatic and outpatient SARS-CoV-2term rRT-PCR-positive individuals with monthly prospective sample collection for up to four months post-enrollment.
Table 1 Patient Demographic and Clinical Characteristics.
Characteristics Outpatients and asymptomatic individualsterm (n=160) Admitted, non-ICU (n=35) Admitted, ICU (n=20) Deceased* (n=25) Age, median (IQR) 41 (32-56) 55 (40-71) 44 (37-63) 76 (64-85) Sex (%) Female 101 (63) 20 (57) 10 (50) 10 (40) Male 59 (37) 15 (43) 10 (50) 15 (60) Diagnostic rRT-PCR Ct, median (IQR) 29.7 (21.8-37.3) 30.6 (21.1-35.4) 25.2 (22.5-29.9) 20.4 (16.5-28.8) Comorbidities, number of individuals (% present) Obesity 43 (27 NA) (32) 15 (1 NA) (43) 11 (55) 10 (40) Hypertension 30 (19) 8 (23) 7 (35) 19 (76) DM 16 (10) 7 (20) 7 (35) 14 (56) Symptoms on presentation, N of individuals (% present) Cough 93 (58) 27 (77) 17 (85) 15 (60) Fever 72 (45) 23 (66) 15 (75) 11 (44) SOB 30 (19) 25 (71) 15 (75) 18 (72) Myalgia 60 (38) 15 (43) 11 (55) 3 (12) GI 29 (18) 19 (54) 10 (50) 5 (20) Fatigue 56 (35) 17 (49) 6 (30) 7 (28) Chills 29 (18) 10 (29) 8 (40) 3 (12) Headache 37 (23) 6 (17) 5 (25) 1 (4) Mechanical ventilationterm (%) 0 0 11 (55) 15 (60) Length of hospital stay, median days (IQR) NA 5 (2-8) 17 (9-49) 15 (5-29) Number of plasma specimens per patient, median (IQR) 1 (1-2) 5 (4-8) 13 (8-21) 8 (3-11) ICU, intensive care unit; IQR, inter quartile range; rRT-PCR Ct, real-time reverse transcription polymerase chain reaction cycle threshold; NA, not available; DM, diabetes mellitus; SOB, shortness of breath; GI, gastrointestinal. *Of the 25 deceased patients, one, seven, and seventeen were categorized as outpatient, admitted non-ICU, and admitted ICU, respectively. The fourteen individuals that were part of the validation cohortterm were not included in this table.
Fig. 1 Serological testing of plasma from SARS-CoV-2term rRT-PCR+ individuals.
Plasma samples from SARS-CoV-2term rRT-PCR-positive individuals ( A ) were analyzed for the presence of antibodies binding to SARS-CoV-2term spiketerm RBD ( B ). *Plasma was also tested for antibodies specific for SARS-CoV-2term S1term and N proteins, and SARS-CoV RBDterm. In addition, samples were tested for antibodies blocking the interaction of ACE2 and RBD in an ACE2 competition ELISAterm ( C ). Spiketerm SARS-CoV-2term pseudotyped lentiviral neutralization assaysterm were performed on selected plasma samples ( D ). Spike-mediated virus entry into HeLa cells overexpressing the ACE2 receptorterm was measured via luciferase reporter activityterm.
Anti-RBD, S1term and N antibody responses and duration are associated with disease severityterm Lower antibody responses in patients with mild symptoms compared to those with more severe disease have been reported for other coronavirus infections, such as MERS-CoV ( 21 – 23 ) as well as for SARS-CoV-2term ( 15 , 24 ). The detection rate of RBD-binding antibodies at one-week intervals after symptom onset are shown in table S2; most individuals seroconverted by week 2term post-onset of symptoms ( Fig. 2A, B ). Positivity rates for RBD IgMterm, IgG and IgA reached their maximum at weeks 4, 6 and 5, respectively, with most patients negative for IgM and IgA after 12 weeks, while RBD IgGterm levels showed a slower, but progressive decline, or a continued negative result in those who failed to generate IgG at earlier time points ( Fig. 2A , fig. S3, and table S2). Outpatients had lower titers of RBD IgMterm, IgG, and IgA compared to inpatients, and a more rapid decline of titers ( Fig. 2B , fig. S4). Patients who required ICU care, and those who died, developed and maintained the highest levels of IgM, IgG and IgA, as well as RBD-ACE2 blockingterm antibodies, throughout the time course ( Fig. 2C ).
Fig. 2 Development of anti-SARS-CoV-2 RBD antibody responses in COVID-19 patients.
828 longitudinal plasma samples collected from 80 COVID-19 inpatients and deceased individuals (714 samples) ( A ) and 86 outpatients (114 samples) ( B ) were tested by ELISA at a dilution of 1:100 for the presence of SARS-CoV-2term RBD-specific IgM, IgG, and IgA antibodies and for antibodies blocking binding of ACE2 to RBD. ELISA data stratified by the 86 outpatients (Outpt), 35 hospitalized patients who did not require ICU care (Admit), and the 20 ICU patients and 25 patients who died, from week 1 to ≥ 7 weeks post-onset of symptoms ( C ). Boxes indicate the interquartile range and whiskers show the minimum and maximum values for each group. Dotted lines denote the assay cutoff. Mean values for duplicate measurements are shown. Statistical testing comparisons are P1 = Outpt vs Admit/ICU/Deceased, P2 = Admit vs ICU/Deceased, by two-sided Wilcoxon rank sum test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data for 14 samples from 2 patients (one admitted non-ICU and one deceased patient) are not plotted because the time of symptom onset was unknown. Mean ELISA OD 450 values of duplicate measurements are shown for each sample.
S1-specific IgM, IgG, and IgA showed antibody kineticsterm that were very similar to those seen for RBD (figs. S3 to S5 and table S2). While N-specific IgG responses showed high positivity rates with antibody kineticsterm similar to those for RBD and S1term, IgM antibody responses to N were strikingly low in most patients (figs. S4 and S6 and table S2). There was no consistent difference in RBD, S1term, and N antibody titersterm or RBD-ACE2 blockingterm antibodies between ICU patients who survived compared to patients who died (fig. S7).
We further evaluated the breadth of antibody responses in different disease severityterm categories by testing for SARS-CoV RBDterm binding. Most monoclonal antibodies targeting SARS-CoV RBDterm fail to bind SARS-CoV-2 RBDterm, indicating distinct antigenicityterm despite sequence and structural similarity of the two proteins ( 25 , 26 ). Nine of 13 ICU patients, three of 25 admitted non-ICU patients (fig. S8A) and five of 82 outpatients (fig. S8B), developed SARS-CoV RBDterm IgG titers during the course of their infection. The time course of anti-SARS-CoV RBD positivity in serial samples from individual patients did not always mirror anti-SARS-CoV-2 RBD IgGterm responses, suggesting limited clonal or oligoclonal B cell responsesterm with this broader reactivity within the overall polyclonal anti-SARS-CoV-2 serological responseterm (figs. S8C, D).
Neutralizing antibodiesterm are increased in inpatients compared to outpatients, and correlate with RBD-ACE2 blockingterm and RBD IgGterm titers Antibody neutralizationterm of live SARS-CoV-2term, or of pseudotyped virusesterm such as lentiviruses expressing the SARS-CoV-2term spiketerm may represent the most physiologically relevant surrogates for humoral immunityterm in vivo, but are poorly scalable due to restrictive biosafety requirementsterm for SARS-CoV-2term and less easily standardized assay components and protocols, compared to tests using purified proteinsterm. We tested SARS-CoV-2term spike-pseudotyped lentivirus neutralizationterm ( 27 ) in HeLa cells overexpressing ACE2 ( 28 ) by inpatient and outpatient samples, and evaluated correlations with the more scalable ELISAsterm for RBD, S1term and RBD-ACE2 blockingterm ( Fig. 3A, B ). As with RBD-ACE2 blockingterm, neutralizing antibody activity was higher in inpatients compared to outpatients, and began to decrease after about one month after symptom onset. Neutralizationterm was well-correlated with RBD IgGterm titers and RBD-ACE2 blockingterm (linear regression coefficient of determination R 2term of 0.6995 and 0.6824 for inpatients; 0.7338 and 0.6839 for outpatients). The RBD-ACE2 blockingterm assay was less sensitive than neutralizationterm or RBD IgGterm ELISA. RBD IgM and IgAterm ELISA results were much more variable and did not correlate as well with neutralizationterm or RBD-ACE2 blockingterm compared to RBD IgGterm.
Fig. 3 Correlation of spike-pseudotyped viral neutralizationterm, RBD-ACE2 blockingterm and RBD-specific serology results.
Plasma samples from inpatients (n = 188) ( A ) and outpatients (n = 96) ( B ) collected at different time points post-onset of symptoms were tested at a dilution of 1:1250 for their pseudovirus neutralizationterm activity. Correlations with RBD IgMterm, IgG, IgA (1:100 diluted plasma samples), and RBD-ACE2 blockingterm ELISA (1:10 diluted plasma samples) data were assessed with simple linear regressionterm and 95% confidence bands (grey shading) of the best-fit line. Correlations between RBD IgMterm, IgG, and IgA data and RBD-ACE2 blockingterm ELISA results were done on the full sample set from inpatients (n = 714) and outpatients (n = 114). Plots show mean ELISA OD 450 values of duplicate measurements and average percent neutralizationterm from duplicate testing in each of two replicate experiments.
Validation of decreasing antibody responses in mildly ill and asymptomatic individuals Most individuals who become infected with SARS-CoV-2term infection do not require hospitalization to recover from their illness, and a sizeable fraction (approximately 40-45%) ( 29 ) remain asymptomatic. We carried out further analysis of antibody responses in a larger set of samples from 136 outpatients and asymptomatic individualsterm who tested positive by rRT-PCR for SARS-CoV-2term RNA in nasopharyngeal swabsterm and had serological testing conducted in the Stanford Health Care Clinical Laboratory. We also tested an independent validation setterm of 45 plasma samples from an additional 14 asymptomatic or mildly ill individuals. As seen for outpatients in Fig. 2B , the asymptomatic and outpatient individuals showed relatively low titers and rapid decline of RBD IgMterm and IgG ( Fig. 4A , table S3). The timing of infection in asymptomatic individuals is less certain than for symptomatic individuals, whose symptoms usually develop within 2 to 14 days after exposure to the virus ( 30 ). Plotting of serological responses of outpatients and asymptomatic individualsterm relative to the date of their first positive rRT-PCR test for infection nonetheless showed a similar time course to that seen for outpatients plotted as days post onset of symptoms ( Fig. 2B ). Notably, the amount of viral RNA detected in diagnostic nasopharyngeal swabsterm was correlated with the antibody titersterm measured in these individuals ( Fig. 4B ).
Fig. 4 Development of anti-SARS-CoV-2 spiketerm RBD antibody responses in SARS-CoV-2term rRT-PCR+ asymptomatic individuals and outpatients over time.
176 plasma samples from 136 SARS-CoV-2term rRT-PCR+ individuals were tested for RBD IgMterm and IgG. The x-axis indicates the time following the first positive rRT-PCR test ( A ). Dotted lines denote the assay cutoff for positive results. RBD IgMterm and IgG are shown for the latest available timepoint for subjects with low (Ct >12-25), middle (Ct >25-35) and high (Ct >35-40) SARS-CoV-2term rRT-PCR Ct at diagnosis ( B ). 45 plasma samples collected from 14 rRT-PCR+ asymptomatic individuals and outpatients sampled at monthly intervals (V1 = enrollment, V2 to V5 = months 1 to 4 post-enrollment) were tested for RBD IgMterm, IgG, IgA at a dilution of 1:100, as well as RBD-ACE2 blockingterm antibodies at a dilution of 1:10 ( C ). The 45 plasma samples were further tested for pseudoviral neutralizationterm at a dilution of 1:1250 ( D ). Box-whisker ELISA OD 450 and blocking/neutralizationterm percent plots show the interquartile range as the box and the minimum and maximum values as the ends of the whiskers. Correlations between virus neutralizationterm and RBD IgMterm, IgG, IgA, and RBD-ACE2 blockingterm are shown with superimposed simple linear regressionterm and 95% confidence bands (grey shading) of the best-fit line ( E ). Plots show mean ELISA OD 450 values of duplicate measurements and average percent neutralizationterm from duplicate testing in each of two replicate experiments.
Plasma specimens from the independent validation cohortterm of asymptomatic or outpatient individuals were collected prospectively during monthly visits up to four months following recruitment ( Fig. 4C, D , fig. S9). RBD IgMterm, IgG and IgA, RBD-ACE2 blockingterm and neutralization assaysterm all showed a progressive decreaseterm during the months sampled. Neutralizing antibodiesterm were best correlated with RBD IgGterm titers, and somewhat less well with RBD-ACE2 blockingterm ELISA in these individuals (R 2 = 0.751 and 0.5221, respectively) ( Fig. 4E ).
Outpatients and non-ICU inpatients show preferential antibody targetingterm of RBD and S1term compared to N It is an open question whether antibody responses in the initial weeks of SARS-CoV-2term infection have a role in modulating disease severityterm. Having found that patients with milder illness or asymptomatic infectionterm had lower levels of SARS-CoV-2term antibodies to RBD, S1term and N compared to severely ill patients, we hypothesized that the relative targeting of the antibody response between antigens might be associated with different disease severityterm. In a recent study, serological analysis of samples from patients who died of COVID-19 compared to individuals who convalesced suggested an enrichment of an aggregate measure of spiketerm antibodies or antibody functional activities in the convalescent ( 19 ). We calculated the ratios of RBD to N ( Fig. 5A ) and S1term to N ( Fig. 5B ) ELISA results for IgM, IgG and IgA for all specimens that had detectable antibodies. Notably, in weeks 1 and 2 post-onset of symptoms, outpatient IgG RBD/Nterm and S1/Nterm ratios were higher than those of admitted non-ICU patients, and patients who died of their illness; admitted and ICU patients also had higher RBD/Nterm ratios compared to patients who died. In weeks 3 and 4, and after 4 weeks, outpatients and admitted patients had higher S1/Nterm IgG ratios compared to ICU patients and those who died. IgA responses showed a similar pattern to IgG in weeks 3 and 4, and after week 4 post-onset of symptoms. The IgM ratios to these antigens was more variable and did not show a consistent relationship to disease severityterm.
Fig. 5 RBD/Nterm and S1/Nterm antibody response ratios in patients with different disease severityterm.
Ratios of RBD/Nterm ( A ) and anti-S1/N ( B ) IgM, IgG, and IgA ELISA OD 450 values are shown for outpatients (Outpt), hospitalized patients who did not (Admit) or did (ICU) require ICU care, and deceased patients (Death) over time. Box-whisker ELISA OD 450 ratio plots illustrate the interquartile range as the box and the minimum and maximum values as the ends of the whiskers. Comparisons between groups were by the two-sided Wilcoxon rank sum test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The dotted line denotes an equal ratio of antigen-specific antibodies.
Inpatient SARS-CoV-2term antibody time course patterns are associated with decreases in RNAemiaterm, but not disease outcome We hypothesized that detailed examination of the time course of SARS-CoV-2term antibody responses could identify distinct patterns associated with admitted non-ICU patients ( Fig. 6 , fig. S10), ICU patients who recovered from their illness ( Fig. 7 , fig. S11A), or those who died ( Fig. 8 , fig. S11B). We identified three main patterns of responseterm: at the time point closest to their discharge from hospital, or death, Group 1term individuals had SARS-CoV-2term antibody responses with no RBD IgGterm or RBD-ACE2 blockingterm activity; Group 2term individuals had up to 25% RBD-ACE2 blockingterm activity; and Group 3term individuals developed high levels of antibodies and over 25% RBD-ACE2 blockingterm activity. These patterns of response were not strongly associated with disease severityterm, but instead were shared across disease categoriesterm. Group 1term patients included admitted patients who recovered from their illness rapidly and were discharged before they had developed detectable antibody responses, but also included patients who recovered from their illness without antibody production after prolonged ICU stays, and patients who died of COVID-19 prior to antibody development. Similarly, Group 2term patterns with low levels of RBD-ACE2 blockingterm were shared across all three patient categories. In Group 3term (high levels of antibody production and >25% RBD-ACE2 blockingterm activity) admitted non-ICU patients differed from ICU or deceased patients in that they developed their robust antibody responses during short hospital stays prior to discharge, whereas ICU or deceased patients typically had prolonged hospital courses. In patients tested for neutralizing antibodiesterm (13 admitted non-ICU patients, 16 ICU surviving patients, and 16 deceased patients), the results were closely correlated with IgG titers to RBD, or RBD-ACE2 blockingterm.
Fig. 6 Correlations between anti-SARS-CoV-2 spiketerm RBD antibody responses, spiketerm pseudovirus neutralizationterm activity, and viral RNA in individual hospitalized patients who did not require ICU care.
Individual patient plots show the development of RBD IgMterm, IgG, and IgA antibody responses (upper panels for each patient) and RBD-ACE2 blockingterm antibodies (lower panels for each patient) for all available plasma sample time points. Orange shading indicates time admitted in the hospital. Representative plots for individuals with no (Group 1term), up to 25% (Group 2term) and greater than 25% (Group 3term) RBD-ACE2 blockingterm activity are shown. Plots for the remaining admitted patients are shown in fig. S10 . Viral RNAemiaterm and pseudovirus neutralizationterm activity are displayed in the lower panels of patients in which these were measured. The time point and Ct value for the diagnostic NP swab SARS-CoV-2term rRT PCR is shown as a turquoise triangle in the lower panel.
Fig. 7 Correlations between anti-SARS-CoV-2 spiketerm RBD antibody responses, spiketerm pseudovirus neutralizationterm activity, and viral RNA in individual ICU patients.
Individual patient plots show the development of RBD IgMterm, IgG, and IgA antibody responses (upper panels for each patient) and RBD-ACE2 blockingterm antibodies (lower panels for each patient) for all available plasma sample time points. Orange shading indicates time admitted in the hospital, red shading represents the timeframe patients were treated in the ICU. Representative plots for individuals with no (Group 1term), up to 25% (Group 2term), and over 25% (Group 3term) RBD-ACE2 blockingterm activity at the time point closest to discharge from hospital are shown here. Plots for the remaining ICU patients are shown in fig. S11A . The time point and Ct value for the diagnostic NP swab SARS-CoV-2term rRT PCR is shown as a turquoise triangle in the lower panel.
Fig. 8 Correlations between anti-SARS-CoV-2 spiketerm RBD antibody responses, spiketerm pseudovirus neutralizationterm activity, and viral RNA in individual deceased patients.
Individual patient plots show the development of RBD IgMterm, IgG, and IgA antibody responses (upper panels for each patient) and RBD-ACE2 blockingterm antibodies (lower panels for each patient) for all available plasma sample time points. Orange shading indicates time admitted in the hospital, while red shading represents the timeframe patients were treated in the ICU. The time of death is indicated in each plot with a star. Representative plots for individuals with no (Group 1term), up to 25% (Group 2term) and over 25% (Group 3term) RBD-ACE2 blockingterm activity are shown here. Plots for the remaining deceased patients are shown in fig. S11B . The time point and Ct value for the diagnostic NP swab SARS-CoV-2term rRT PCR is shown as a turquoise triangle in the lower panel.
Viral RNAemiaterm is detected in up to a third of COVID-19 patients, most often in patients with severe disease ( 3 , 31 ). RNAemiaterm was evaluated in a subset of patients, and detected in 15 of 25 admitted patients, 13 of 15 ICU patients who recovered, and 2 of 2 patients who died of their disease. Reduction in RNAemiaterm was strongly correlated with the appearance of plasma antibodiesterm (Spearman’s correlation coefficientsterm of -0.47 for IgM, -0.43 for IgG, and -0.44 for IgA, p<0.001 for each) ( Figs. 6 , 7 , 8 ; figs. S10, S11).
DISCUSSION Key clinical questions in the ongoing COVID-19 pandemic are the role of antibodies in modulating disease severityterm during infection, the duration of individuals’ serological responses, and the extent to which patient antibody responses may be protective against reinfectionterm. As SARS-CoV-2term vaccine candidates progress through clinical trials, comparison of vaccine-induced immune responsesterm to those stimulated by viral infection, and those of individuals who become reinfected, will help clarify immunological correlates of protectionterm. More comprehensive understanding of the role of antibodies in acute COVID-19 illness will guide effective use of therapeutic convalescent plasmaterm products and recombinant antibodiesterm targeting SARS-CoV-2term. Assuming spike-targeting vaccines are shown to be safe and effective and become widely used, monitoring N-specific antibodiesterm may be of utility in distinguishing vaccine-related antibodiesterm from those stimulated by infection.
Here, we have analyzed serological responses to SARS-CoV-2term in 254 individuals ranging from asymptomatic individuals to ICU patients, with detailed analysis of antibody responses to SARS-CoV-2 RBDterm, S1term and N antigens, and functional assays testing antibody blocking of RBD-ACE2 binding or spike-pseudotyped virus neutralizationterm. At early time points post-onset of symptoms, we saw no evidence of pre-existing antibodies that recognize SARS-CoV-2term, suggesting that there is minimal cross-reactivityterm of SARS-CoV-2term antigens with community coronaviruses. These findings do not preclude the possibility that memory B cells stimulated by prior coronavirus exposure may be cross-reactive and form part of the response to SARS-CoV-2term.
IgM, IgG and IgA antibodies specific for SARS-CoV-2term antigens typically become detectable in patients’ blood at similar median times of about 2 weeks after onset of symptoms (this study and ( 32 , 33 )), although IgA timing is the most variable. As we have recently reported, large polyclonal proliferations of recently class-switched B cellsterm expressing IgG and IgA subtypes with low antibody somatic mutation frequencies appear in the circulation around the time of seroconversionterm, likely including plasmablaststerm that contribute to the observed serological responses ( 29 ).
Which features of patient antibody responses were associated with disease severityterm? To our surprise, we found that outpatients with the mildest illnessterm showed higher ratios of spike RBD and S1 domains compared to N antigenterm, beginning in the first two weeks post-onset of symptoms. These findings could suggest that early humoral immune responses focused on spiketerm antigens help to constrain the viral infection, perhaps even at times when titers are not yet high enough to be measured in the blood. Outpatients and admitted non-ICU patients had the lowest viral loads in their nasopharyngeal swabsterm, but whether this is due to their antibody responses cannot be determined from this observational studyterm. Associations of mortality in COVID-19 patients with SARS-CoV-2term viral load assessed by rRT-PCR applied to nasopharyngeal swabterm specimens have also been found in other studies ( 34 , 35 ). Our data are consistent with results reported from a panel of antibody assays applied to single time point samples from COVID-19 patients who recovered or died of their disease, which found higher values of spike-targeting responses in the convalescents ( 19 ). Patients with more severe illness in our study eventually raised higher antibody titersterm than those with milder disease, consistent with prior publications ( 32 , 36 ) and reports of other coronavirus infections ( 21 – 23 ). Patients with more severe disease also had somewhat higher viral loads than patients with milder illness, suggesting that larger initial amounts of viral antigenterm may contribute to their greater serological responsesterm. Functional blocking of RBD-ACE2 interaction, and spike-pseudotyped virus neutralizationterm by patient antibodies appeared with a similar time course to IgM, IgG, and IgA, but were most closely correlated in magnitude with IgG titers. Neutralizationterm, RBD-ACE2 blockingterm, and RBD-specific IgGterm were all highly correlated in patients with high antibody levels, but RBD-ACE2 blockingterm was less sensitive than the neutralization assayterm, potentially because of antibodies that can neutralize by binding to non-RBD regions of the spiketerm, or lower affinity antibodies that can neutralize in the cell culture assay but do not compete as well with binding of ACE2 under the blocking assay conditions. It is currently unclear, however, which of these assays will be the best predictor of in vivo immunological protection from SARS-CoV-2term infection or reinfectionterm in vaccinated or previously infected individuals.
In the detailed serological time courses of the hospitalized patients in this study it was evident that the patterns of antibody responses could not fully explain patient outcomes, including death. Substantial numbers of patients recovered from their illness and were discharged from hospital before they had formed detectable antibody responses, but minimal serological responses were also seen in patients who died of COVID-19 at early times post-onset of symptoms. Similarly, individuals with moderate antibody production were seen across the full spectrum of inpatient disease severityterm, and many patients who died of their disease generated high levels of antibodies, RBD-ACE2 blockingterm activity, and neutralizing titers. Differences between individuals in other aspects of the immune response or disease course, such as production of inflammatory mediatorsterm, T cell responsesterm, host cell and tissue vulnerability to the damage during viral infection, coagulopathyterm, and secondary infectionsterm, are all likely to contribute to patient outcomes.
There is an urgent need to understand how long antibody titersterm against the virus persist after infection, now that the pandemic has been underway for more than half a year in many countries, and initial case reports of proven reinfectionterm by SARS-CoV-2term have begun to appear ( 37 , 38 ). Studies differing in their patient populations, disease severityterm, and serological assays, have disagreed on the duration of SARS-CoV-2term antibody responses ( 14 , 16 , 32 ). Our data derived from inpatients, outpatients and asymptomatic individualsterm, with an additional asymptomatic validation cohortterm, show clearly that not only IgM and IgA, but also IgG titers to RBD, S1term and N antigens, RBD-ACE2 blockingterm activity and spiketerm pseudotyped viral neutralizationterm titers all begin to decrease in patients after approximately the first month post-onset of symptoms. The decline in antibody titersterm is most evident in individuals who had asymptomatic infectionterm or mild illness, who produce lower levels of antibodies at the peak of their responses. We note that reported results failing to find a decrease in SARS-CoV-2term antibodies after several months post-infection have relied on “pan-Igterm” assays that cannot evaluate each isotypeterm separately ( 16 ). Our data do not permit us to predict what fraction of the population will be susceptible to reinfectionterm at a given time after their initial illness, or whether individuals will maintain sustained plateaus of lower antibody levels after an initial decrease; additional time and follow-up will be required to obtain this information. A limitation of our study is that, apart from the prospective validation cohortterm, the sampling time pointsterm for each patient were determined by their length of hospital stay and subsequent healthcare visitsterm, enabling more detailed analysis of serological responses in patients with more severe illness. It is important to note that decreasing antibody levelsterm do not necessarily indicate that all immunity will be lostterm. It is possible that local mucosal antibody productionterm in the airways could help prevent or impede SARS-CoV-2-infection upon re-exposureterm ( 39 ). Even if serum antibodies wane to undetectable levels, memory B and T cellsterm stimulated by infection could provide a faster or more effective response following future exposure. Initial reinfectionterm reports offer some hope that SARS-CoV-2term may behave similarly to other community coronaviruses, with reinfectionterm generally producing milder illness than the initial infection ( 40 , 41 ).
One implication of our finding of waning antibody levelsterm is that seroprevalence studiesterm may, over time, underestimate the proportion of the investigated population which has been previously infected with SARS-CoV-2term. The decrease in antibodies after infection also raises the question of how long antibodies elicited by vaccinationterm will last, and whether frequent boostingterm will be needed to maintain protection, assuming that safe and effective vaccines are identified. The current vaccination strategiesterm undergoing clinical trials differ from natural infectionterm in a variety of ways, including the method for generating or introducing viral antigens into the body, the site of exposure, and the presence of adjuvantsterm ( 42 – 45 ). It is possible that some of the vaccine approaches may generate more potent and long-lasting antibodies than natural infectionterm, in which the virus may have currently unknown mechanisms for subverting humoral immune responses. Further detailed study of the generation of memory B cell populationsterm, short- or long-lived plasma cellsterm, and T cell memoryterm to SARS-CoV-2term as well as other coronaviruses should begin to clarify some of these key mechanistic points.
MATERIALS AND METHODS Study design and participants The objective of this study was to investigate correlations between humoral immune responses to SARS-CoV-2term, including antibodies blocking binding of RBD to the human ACE2 receptor or neutralizing spiketerm pseudovirus, and viral RNA loads in the nasopharynx and blood in different COVID-19 patient groups and individual patients. On March 4, 2020, the Stanford Health Care Clinical Virology Laboratory began rRT-PCR testing on nasopharyngeal specimens from suspected COVID-19 patients using a laboratory-developed SARS-CoV-2term rRT-PCR assay ( 46 , 47 ). For this study, we included specimens from patients with rRT-PCR-confirmed SARS-CoV-2 infectionterm who reported with symptoms of COVID-19 to Stanford Healthcare-associated clinical sites between March 2020 and August 2020; and specimens from rRT-PCR positive outpatients and asymptomatic individualsterm identified between April 2020 and May 2020 through occupational health screeningterm including rRT-PCR and serology testing for RBD IgMterm/G at Stanford Clinical Laboratories. The screening program that detected asymptomatic SARS-CoV-2term infected individuals was offered to all Stanford Healthcare employees on a voluntary basis, and screened employees with nasopharyngeal swabterm rRT-PCR testing and serology. In addition, we included a validation cohortterm of 14 asymptomatic and mildly ill individuals with prospective sample collection for up to four months post-enrollment. This study was approved by the Stanford University Institutional Review Board (Protocols IRB-48973 and IRB-55689).
Sample and data collection Venipuncture blood samples collected in sodium heparin- or K 2 EDTA-coated vacutainers were used for serology testing and rRT-PCR detection of RNAemiaterm, respectively. After centrifugation for collection of plasma, samples were stored at -80°C.
Retrospective chart review was performed on all study participants. Collected data included age, gender, date of symptom onset, length of hospital stay, length of time admitted in the ICU, date and Ct value for the diagnostic nasopharyngeal swabterm rRT-PCR test result, the presence of underlying comorbidities, clinical symptoms, and mortality.
Production of SARS-CoVterm and SARS-CoV-2term proteins and ACE2-mFc The SARS-CoVterm and SARS-CoV-2 RBDterm proteins were expressed in Expi293F cells and purified using Nickel-NTA resin and size exclusion chromatography. The SARS-CoVterm construct (RBD-His_pTT5, GenBank AAP13441.1 ) was synthesized commercially by Twist Bioscience (San Francisco, CA); the SARS-CoV-2term construct (RBD-His_pCAGGS, GenBank MN908947.3 ) was kindly provided by Dr. Florian Krammer ( 48 ). SARS-CoV-2term S1term (spiketerm residues 1-682) and ACE2-mFc, expressed in HEK293 cells, and the N protein, expressed in E. coli were produced by the ATUM contract research organization. Soluble human ACE2 fused to a mouse Fc tag was constructed by synthesizing a gene encoding ACE2 (residues 1-615) joined by a (G 4 S)x2 linker to mouse IgG2a Fc, and placed under control of a CMV promoter by cloning into a mammalian expression plasmid.
ELISA to detect anti-SARS-CoV-2 Spiketerm RBD antibodies in plasma samples The ELISA procedure in this study was modified from a protocol published by Stadlbauer ( 48 ).
96-well Corning Costar high binding plates (catalog no. 9018, Thermo Fisher) were coated with SARS-CoV RBDterm, SARS-CoV-2 RBDterm, S1term, or N protein in phosphate-buffered saline (PBS) at a concentration of 0.1 μg per well (0.025 μg per well for the nucleocapsid IgG assay) overnight at 4°C. On the next day, wells were washed 3x with PBS-0.1% Tween 20 (PBS-T) and blocked with PBS-T containing 3% non-fat milk powder for 1 hour at room temperature (RT). Wells were then incubated with plasma samples from COVID-19 patients at a dilution of 1:100 in PBS-T containing 1% non-fat milk for 1 hour at 37°C. Two negative and two positive plasma pool wells and two blank wells incubated with PBS-T containing 1% non-fat milk powder were included on each plate. After washing 3x with PBS-T, horseradish peroxidase conjugated goat anti-human IgG (γ-chain specific, catalog no. 62-8420, Thermo Fisher, 1:6,000 dilution), IgM (μ-chain specific, catalog no. A6907, Sigma, 1:6,000 dilution), or IgA (α-chain specific, catalog no. P0216, Agilent, 1:5,000 dilution) in PBS-T containing 1% non-fat milk was added and incubated for 1 hour at RT. Wells were washed 3x with PBS-T and dried by vigorous tapping of plates on paper towels. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution was added and the reaction was stopped after 12 min by addition of 0.16 M sulfuric acid. The optical density (OD) at 450 nanometers was measured with an EMax Plus microplate reader (Molecular Devices, San Jose, CA); values for blank wells were subtracted from values obtained for plasma samples. The cutoff value for seroconversionterm was calculated by adding 3 standard deviations to mean ELISA ODs of 94 historical negative control samplesterm from healthy blood donors (collected before the pandemic for an unrelated seroprevalence study) obtained by testing the samples in all protein/isotypeterm assays (table S4). Additional details for the manual and clinical lab instrument ELISA assay setup are provided in figs. S12 and S13.
Competition ELISA to detect antibodies blocking binding of ACE2 to RBD 96-well Corning Costar high binding plates (Thermo Fisher: cat. 9018) were coated with SARS-CoV-2term spiketerm RBD protein in PBS at a concentration of 0.1 μg per well overnight at 4°C. All competition ELISA steps were carried out on the next day at RT. Wells were washed 3x with PBS-T and blocked with PBS-T containing 3% non-fat milk powder for 1 hour. Wells were then incubated with plasma samples from COVID-19 patients at a dilution of 1:10 in PBS-T containing 1% non-fat milk for 1 hour. Two quality controls (Access SARS-CoV-2term IgG QC, QC1-QC2, 2 levels, catalog no. C58964 , Beckman Coulter) and two blank wells incubated with PBS-T containing 1% non-fat milk were included on each plate. ACE2-mFc diluted to 0.5 μg/ml in 1% non-fat milk powder was added without washing steps and incubated for an additional 45 min. After washing 3x with PBS-T, horseradish peroxidase conjugated goat anti-mouse IgG (Fc specific, catalog no. 31439, Invitrogen, 1:10’000 dilution) in PBS-T containing 1% non-fat milk was added and incubated for 45 min. Wells were washed 3x with PBS-T and dried by vigorous tapping of plates on paper towels. TMB substrate solution was added and the reaction was stopped after 12 min by addition of 0.16 M sulfuric acid. The OD at 450 nanometers was measured with an EMax Plus microplate reader (Molecular Devices, San Jose, CA). OD values were converted to percentage of blockingterm using the following formula: 100*(1-(sample OD - 0.2)/(QC1 OD – 0.2)), taking into account the background noise of the assay of 0.2 as determined after testing pre-pandemic control plasma samples. Additional details for competition ELISA assay setup and optimization are provided in figs. S14 and S15.
Pseudotyped lentiviral neutralization assayterm SARS-CoV-2term pseudotyped lentivirus assays were performed as described previously. Briefly, spike-pseudotyped lentivirus was produced in HEK293T cells as described in ( 49 ) using a 5-plasmid system described in ( 27 ). For viral neutralization assaysterm, ACE2/HeLa cells ( 28 ) were plated in 96-well tissue culture plates one day prior to infection. Prior to neutralization assaysterm, patient plasma samples were heat inactivated for 1 hour at 56°C. Plasma samples and virus were incubated with the cells at 37°C for ~48 hours. After incubation, cells were lysed with BriteLite assay readout solution (Perkin Elmer) and luminescence values were obtained with a BioTek plate reader. Single-dilution point neutralization assaysterm were performed in technical duplicateterm in two different experimental replicates. Percent neutralizationterm was determined by normalizing raw luciferase values to 0% infectivity (average from cells only wells) and 100% infectivity (average from virus only wells) in GraphPad Prism 8.4.1. Plasma dilution series shown in Figure S9 were performed in technical duplicateterm; normalized % infectivity values were fit with a three-parameter non-linear regression inhibitor curveterm in GraphPad Prism 8.4.1 and fits were constrained to have a value of 0% at the bottom of the fit.
Real-time PCR to detect SARS-CoV-2term RNA in plasma A volume of 400 μL of EDTA-anticoagulated plasma was extracted by Qiagen EZ1 Virus Mini Kit v2.0 (Qiagen Germantown, MD). Molecular testing for the presence of SARS-CoV-2term RNA in plasma was performed with a modification of a published rRT-PCR assay targeting the envelopeterm ( E ) gene ( 46 , 47 ). The standard Ct values of positive tests with this assay range from Ct <20 to 45 cycles. Testing of plasma samples with a Ct value of 40 or greater was repeated to ensure reproducibility of the positive result. As viral culture was not performed as part of this study, presence of SARS-CoV-2 in tested plasmaterm was defined as RNAemiaterm.
Statistics GraphPad Prism version 8.0 (GraphPad Software, San Diego, California, USA) software was used to visualize ELISA data, analyze for differences in antibody responses between disease categoriesterm by Wilcoxon rank sum testingterm, and carry out linear regressionterm of pseudotyped virusterm neutralizationterm, RBD-ACE2 blockingterm and antibody ELISA results. The goodness of fit for linear regressionterm analyses was reported as the coefficient of determination, R 2term . Locally estimated scatterplot smoothing for the development and decrease of antibody responses over time was performed using the loess methodterm in the R statistical package version 3.6.1 ( 50 ). Correlation between antibody OD 450 values, RNAemiaterm, and ACE2-RBD blocking assay OD 450 values were calculated as Spearman correlationsterm with the R cor function.
Acknowledgments The authors thank Dana Anderson for helpful advice about protein production, and the ATUM Bio team for optimization and production of antigens. We thank Dr. Jesse Bloom, Kate Crawford, Dr. Dennis Burton, and Dr. Deli HuangpersName for sharing the plasmids, cells, and invaluable advice for implementation of the spike-pseudotyped lentiviral neutralization assayterm. Funding : This work was supported by NIH/NIAID R01AI127877 (S.D.B.), NIH/NIAID R01AI130398 (S.D.B.), NIH 1U54CA260517 (S.D.B.), NIH/NIAID T32AI007502-23 (A.R.), NIH/NIAID U19AI111825 (T.T.W.), NIH/NCATS UL1TR003142 (B.N.), an endowment to S.D.B. from the Crown Family Foundation, a postdoctoral fellowship to A.E.P from the Stanford Maternal and Child Health Research Institute, an Early Postdoc.Mobility Fellowship Stipend to O.F.W. from the Swiss National Science Foundation (SNSF), and a Coulter COVID-19 Rapid Response Award to J.R.C. and S.D.B. Author contributions : K.R, A.E.P., O.F.W., B.A.S., B.A.P., S.D.B. conceptualized and designed the study. K.R., A.E.P., O.F.W., C.A.H., M.K.S., C.H.H., F.Y., J.M. performed the experiments. B.A.S., J.N., M.H., H.W., M.K.S., M.M., A.R.O, T.D.P., A.R., A.J.R., C.A.B., J.R.C., K.C.N., B.A.P., S.G., N.H.S. collected data and/or contributed samples/reagents or EHR processing methods. C.A.H., J.M., T.S.J., J.L.Z., T.T.W., K.C.N., P.S.K., provided intellectual contributions throughout the study. K.R., B.N., R.T, performed statistical analyses. K.R., A.E.P., O.F.W., B.A.P., C.A.H., S.G., R.T., S.D.B. analyzed the data. K.R., S.D.B. wrote the manuscript. All authors edited and approved the manuscript. Competing interests : M.H. is an employee of ATUM. S.D.B. has consulted for Regeneron, Sanofi, and Novartis on topics unrelated to this study. S.D.B., K.R., P.S.K and A.E.P. have filed provisional patent applications related to serological tests for SARS-CoV-2term antibodies. K.C.N. reports grants from National Institute of Allergy and Infectious Diseases (NIAID), Food Allergy Research & Education (FARE), End Allergies Together (EAT); National Heart, Lung, and Blood Institute (NHLBI), and National Institute of Environmental Health Sciences (NIEHS). K.C.N. is Director of the FARE and World Allergy Organization (WAO) Center of Excellence at Stanford University; Advisor at Cour Pharmaceuticals; Cofounder of Before Brands, Alladapt, Latitude, and IgGenix; National Scientific Committee member for the Immune Tolerance Network (ITN) of NIAID; recipient of a Research Sponsorship from Nestle; Consultant and Advisory Board Member at Before Brands, Alladapt, IgGenix, NHLBI, and ProBio; and Data and Safety Monitoring Board member at NHLBI. C.A.B. is on the board of Catamaran Bio. The remaining authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/ . This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.
Supplementary Materials immunology.sciencemag.org/cgi/content/full/5/54/eabe0240/DC1 Fig. S1term. Study design and participant overview.
Fig. S2. SARS-CoV-2term rRT-PCR cycle threshold (Ct) values for diagnostic nasopharyngeal (NP) swabs.
Fig. S3. Distribution and trajectories of antibody responses to SARS-CoV-2term in inpatients over time.
Fig. S4. Distribution and trajectories of antibody responses to SARS-CoV-2term in outpatients over time.
Fig. S5. Development of anti-SARS-CoV-2 spiketerm S1term antibody responses in COVID-19 patients.
Fig. S6. Development of anti-SARS-CoV-2 N antibody responses in COVID-19 patients.
Fig. S7. Comparison of antibody responses against SARS-CoV-2term antigens among ICU patients who survived or died.
Fig. S8. COVID-19 inpatients develop anti-SARS-CoV/SARS-CoV-2term spiketerm RBD cross-reactive IgG responses.
Fig. S9. Pseudoviral neutralizationterm activity in longitudinal samples from the validation cohortterm.
Fig. S10. Correlations between anti-SARS-CoV-2 spiketerm RBD antibody responses, pseudoviral neutralizationterm activity, and viral RNA in individual hospitalized patients.
Fig. S11. Correlations between anti-SARS-CoV-2 spiketerm RBD antibody responses, pseudoviral neutralizationterm activity, and viral RNA in individual ICU and deceased patients.
Fig. S12. Checkerboard titration for serological RBD ELISA.
Fig. S13. Validation of the Stanford Health Care Clinical Laboratory anti-RBD IgM/G ELISA.
Fig. S14. Checkerboard titration for receptor blocking ELISA.
Fig. S15. RBD-ACE2 blockingterm ELISA optimization.
Table S1term. Demographic and clinical characteristics of outpatients and asymptomatic individualsterm with and without plasma availability.
Table S2. Inpatient seropositivity.
Table S3. Outpatient and asymptomatic individuals’ seropositivity.
Table S4. Determination of SARS-CoV-2term assay cutoff values and specificity.
Table S5. Raw data file (Excel spreadsheet).