Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity

Development of a VirScan library targeting human coronaviruses

Our existing VirScan phage-display platform is based on an oligonucleotide library encoding 56–amino acid (56-mer) peptides tiling every 28 amino acids across the proteomes of all known pathogenic human viruses (~400 species and strains) plus many bacterial proteins (8). To investigate the serological response to SARS-CoV-2 and other human coronaviruses (HCoVs), we supplemented this library with three additional sublibraries: Sublibrary 1 encodes a 56-mer peptide library tiling every 28 amino acids through each of the open reading frames (ORFs) expressed by the six HCoVs and three bat coronaviruses closely related to SARS-CoV-2; sublibrary 2 encodes 20-mer peptides tiling every 5 amino acids across the SARS-CoV-2 proteome, enabling more precise localization of epitopes; and sublibrary 3 encodes triple-alanine scanning mutants of the 56-mer peptides tiling across the SARS-CoV-2 proteome, enabling the mapping of epitope boundaries at amino acid resolution (Fig. 1, A to C, and table S1) (9, 10).

We used VirScan (Fig. 1C) to profile the antibody repertoires of nine cohorts of individuals from multiple locations, including Baltimore, MD, Boston, MA, and Seattle, WA (tables S2 to S8). These cohorts comprised longitudinal samples from individuals enrolled in prospective studies of COVID-19 infection, cross-sectional samples from patients with active COVID-19 who were receiving treatment in either hospital or outpatient settings, and cross-sectional samples from convalescent individuals with a past history of COVID-19. Our cohorts also included a diverse set of control sera collected before the COVID-19 outbreak. We profiled the targets of IgG and IgA (immunoglobulins G and A) antibodies separately: IgG and IgA are the most abundant isotypes in blood, whereas IgA is the principal isotype secreted on mucosal surfaces, including the respiratory tract. Collectively, we analyzed ~550 samples in duplicate, in total assessing ~100 million potential antibody repertoire–peptide interactions.

Detection of SARS-CoV-2 seropositivity with VirScan

To measure immune responses to SARS-CoV-2, we compared VirScan profiles of serum samples from COVID-19 patients to those of controls obtained before the emergence of SARS-CoV-2 in 2019. These pre–COVID-19 era controls facilitate identification of (i) SARS-CoV-2 peptides encoding epitopes specific to COVID-19 patients and (ii) SARS-CoV-2 peptides encoding epitopes that are cross-reactive with antibodies developed in response to the ubiquitous common-cold HCoVs. Sera from COVID-19 patients exhibited much more SARS-CoV-2 reactivity than did sera from pre–COVID-19 era controls (Fig. 1, D and E). Some cross-reactivity toward SARS-CoV-2 peptides was observed in the pre–COVID-19 era samples, but this was expected because nearly all people have been exposed to one or more HCoVs (11).

COVID-19 patient sera also showed significant levels of cross-reactivity with the other highly pathogenic HCoVs, SARS-CoV and MERS-CoV, although less was observed against the more distantly related MERS-CoV. Extensive cross-reactivity was also observed against peptides derived from the three bat coronaviruses that share the greatest proportion of sequence identity with SARS-CoV-2 (Fig. 1, A, D, and E) (9). We know that these represent cross-reactivities because, given the low prevalence and circumscribed geographical location of SARS-CoV and MERS-CoV, none of the individuals in this study are likely to have encountered these viruses.

COVID-19 patient sera also exhibited a significantly higher level of reactivity to seasonal HCoV peptides than did sera from pre–COVID-19 era controls (Fig. 1, D and E). This could be due to the elicitation of novel antibodies that cross-react or to an anamnestic response that boosts B cell memory against HCoVs. The converse is not always true: Many pre–COVID-19 era samples exhibit strong recognition of seasonal HCoV peptides but little or no recognition of SARS-CoV-2 peptides (Fig. 1D). In some cases, the concentrations of antibodies against seasonal HCoVs may be below the threshold of detection in the pre–COVID-19 era samples.

Coronavirus proteins targeted by antibodies in COVID-19 patients

Analysis of SARS-CoV-2 proteins targeted by COVID-19 patient antibodies revealed that the primary responses to SARS-CoV-2 are reactive with peptides derived from spike protein (S) and nucleoprotein (N) (Fig. 2, A and B). Compared with sera from pre–COVID-19 era controls, COVID-19 patient sera exhibit significant differential recognition of these two proteins, indicating that this recognition is a result of antibody responses to SARS-CoV-2. Third-most frequently recognized is the replicase polyprotein ORF1, but unlike S and N, ORF1 is recognized to a similar extent by sera from COVID-19 patients and pre–COVID-19 era controls. This suggests that recognition of SARS-CoV-2 ORF1 is a result of cross-reactions from antibodies elicited by exposure to other pathogens, possibly HCoVs. Antibody responses to peptides from membrane glycoprotein (M), ORF3, and ORF9b were occasionally detected in COVID-19 patients.

We also analyzed longitudinal samples from 23 COVID-19 patients. Most patients displayed an antibody response to peptides derived from S or N in the second week after symptom onset, with many displaying an antibody response by the end of the first week (Fig. 2C). The relative strength and onset of the antibody response to S and N differed markedly between individuals, and the initial immune response showed no preference for S or N. The signal intensity of antibodies recognizing SARS-CoV-2 ORF1 epitopes did not increase over time, further suggesting that ORF1 antibodies likely represent a preexisting cross-reactive response.

Identification of immunogenic regions of SARS-CoV-2 proteins

To more precisely define the immunogenic regions of the SARS-CoV-2 proteome, we examined the specific 56-mer and 20-mer peptides detected by VirScan in COVID-19 patients compared with those in pre–COVID-19 era controls. An example IgG response from a single patient to SARS-CoV-2 S and N is shown in Fig. 3A. We observed strong concordance between the viral regions enriched by the 56-mer and 20-mer fragments, demonstrating the robustness of VirScan. In many cases, we observed recognition of overlapping 56-mer peptides, indicating an epitope in the common region.

Next, we compared the protein regions recognized by IgG and IgA across COVID-19 patients (Fig. 3B). We identified four regions each in S and N that are recurrently targeted by antibodies from >15% of COVID-19 patients, with additional regions recognized less frequently. Overall, IgG and IgA recognize the same protein regions with similar frequencies across the population. However, when IgG and IgA responses were compared within individuals, we observed considerable divergence (Fig. 3C): Many epitopes were recognized by only IgG, only IgA, or both IgG and IgA within an individual patient. Together, these data suggest that patients generate distinct IgG and IgA antibody responses to SARS-CoV-2, but the targeted regions are largely shared at the population level.

Machine learning guides the design of a Luminex assay for rapid COVID-19 diagnosis

To predict SARS-CoV-2 exposure history from VirScan data, we developed a gradient-boosting algorithm (XGBoost) that integrates both IgG and IgA data and predicts current or past COVID-19 with 99.1% sensitivity and 98.4% specificity (Fig. 4, A and B). We used Shapley Additive exPlanations (SHAP)—a method to compute the contribution of each feature of the data to the predictive model (12)—to identify peptides from SARS-CoV-2 S and N plus homologous peptides from SARS-CoV and BatCoV-HKU-3 and BatCoV-279 that were highly predictive of SARS-CoV-2 exposure (Fig. 4, C and D).

We leveraged these insights to develop a simple, rapid Luminex-based diagnostic for COVID-19. We chose 12 SARS-CoV-2 peptides predicted by VirScan data and the machine learning model to be highly indicative of SARS-CoV-2 exposure history (table S9). These SARS-CoV-2 peptides, two positive control peptides from rhinovirus A and Epstein-Barr virus (EBV) that are recognized in >80% of seropositive individuals by VirScan (7), and a negative control peptide from HIV-1 were coupled to Luminex beads (13). We tested 163 COVID-19 patient samples and 165 pre–COVID-19 era controls for IgG reactivity to the Luminex panel. We detected clear responses to SARS-CoV-2 peptides in COVID-19 patient samples but rarely in the pre–COVID-19 era controls (Fig. 4E). Using the Luminex data, we developed a logistic regression model that predicts COVID-19 infection history with 89.6% sensitivity and 95.2% specificity [area under the curve (AUC) = 0.97] (Fig. 4, F and G). A subset of COVID-19–positive samples (n = 107) was also examined with an in-house enzyme-linked immunosorbent assay (ELISA) using three SARS-CoV-2 antigens: N, S, and the S receptor-binding domain (RBD). Considering a sample to be positive if it scored above the 99% specificity threshold on any one of the three ELISA antigens, we determined that the sensitivity of the Luminex assay for this subset (88.8%) was similar to that of the ELISA (90.7%) (fig. S1). Among samples run on all three assays, VirScan significantly outperformed both the Luminex and ELISAs (fig. S1, A and C). Notably, our optimal model integrated only three SARS-CoV-2 peptides—residues 386 to 406 of N (N 386-406), residues 810 to 830 of S (S 810-830), and residues 1146 to 1166 of S (S 1146-1166)—which were also the most discriminatory 20-mers in the VirScan data. IgG responses in COVID-19 patients were highly correlated between the Luminex and VirScan assays, providing orthogonal validation of the VirScan data and supporting the prevalence of SARS-CoV-2–induced humoral responses to these regions of S and N (fig. S1D).

Differential antibody responses to common viruses in hospitalized versus nonhospitalized COVID-19 patients

We next considered whether differences in the antibody response to SARS-CoV-2 or to other viruses might be associated with the severity of COVID-19. We grouped the COVID-19 patients into two subsets: those who required hospitalization (n = 101) and those who did not (n = 131). We compared the responses to peptides derived from the SARS-CoV-2 S and N proteins between the hospitalized (H) and nonhospitalized (NH) groups and found that the H group exhibited stronger and broader antibody responses to S and N peptides that might be due to epitope spreading (Fig. 5A). We then analyzed 32 NH COVID-19 samples, 32 H COVID-19 samples, and 32 pre–COVID-19 era negative controls with the Luminex assay and similarly observed that the H group had stronger and broader antibody responses to SARS-CoV-2–specific peptides than did the NH group (Fig. 5B).

VirScan also offers the opportunity to examine the history of previous viral infections and to determine correlates of COVID-19 outcomes. For example, prior viral exposure could provide some protection if cross-reactive neutralizing antibodies or T cell responses are stimulated upon exposure to SARS-CoV-2 (14, 15). Alternatively, cross-reactive antibodies to viral surface proteins could increase the risk of severe disease due to antibody-dependent enhancement (ADE), as has been observed for SARS-CoV (16, 17). Furthermore, exposure to certain viruses could affect the response to SARS-CoV-2 by altering the immune system. To examine these possibilities, we analyzed the virome-wide VirScan data and found that overall, the NH patients exhibited greater responses to individual peptides from common viruses such as rhinoviruses, influenza viruses, and enteroviruses, whereas the H patients displayed more robust responses to peptides from cytomegalovirus (CMV) and herpes simplex virus 1 (HSV-1) (Fig. 5C). These observations may be influenced by demographic differences in the NH and H cohorts, as described below.

We sought to understand whether the differential reactivity to CMV and HSV-1 between the H and NH patients was due to differences in the strength of antibody responses or the prevalence of infection (these viruses are common, but not ubiquitous like rhinoviruses, enteroviruses, and influenza viruses). Using VirScan data, we found that the H group had a higher incidence of both CMV and HSV-1 infection: 82.2% (83 of 101) of the H group were positive for CMV versus 37.4% (49 of 131) of the NH group, whereas 92.1% (93 of 101) of the H group were positive for HSV-1 versus 45.8% (60/131) of the NH group. To examine the relative strength of the antibody responses, we considered only CMV- or HSV-1–seropositive individuals from the NH and H groups: The antibody response to both CMV (Fig. 5D) and HSV-1 (fig. S2) was stronger among the NH individuals. Thus, the differing seroprevalence of CMV and HSV-1 in the NH versus H groups likely explains the results shown in Fig. 5C. We conclude that antibody responses to nearly all viruses, except SARS-CoV-2, were weaker in the H patients than in the NH patients.

These notable differences led us to examine potential demographic covariates between the NH and H groups. We found that age, sex, and race were all significantly associated with COVID-19 severity (fig. S3), as has been reported (18, 19). Older age, male sex, and non-white racial groups were significantly overrepresented in the H group compared with the NH group (fig. S3 and table S3). Furthermore, hospitalized males exhibited stronger responses to N than hospitalized females, whereas nonhospitalized males and females did not exhibit differential responses to any SARS-CoV-2 proteins (fig. S3E). Advanced age is a dominant risk factor for severe COVID-19 and is correlated with reduced immune function (20). In light of the age difference between the H (median age: 58) and NH (median age: 42) patients in our cohort, we reasoned that the antigens recognized more strongly in the NH group might reflect more general age-associated changes in humoral immunity. To test this hypothesis, we examined VirScan data for a cohort of 648 healthy, pre-pandemic donors. We characterized the recognition of each NH-associated peptide in subsets of healthy donors representing different age groups and observed a general decline in recognition with age, including a median 19% reduction in recognition from age 42 to 58 (Fig. 5E). These data suggest that age-related changes to the immune system may partially explain the observation of weaker antibody responses to most viruses in the H group. Although it is correlative and potentially influenced by other demographic differences between the NH and H cohorts, the broad age-related diminution in immune system activity that we observed could be an important aspect of the increased severity in the H group.

Cross reactivity of SARS-CoV-2 epitopes

We returned to the question of epitope cross-reactivity, this time examining antibody responses to the triple-alanine scanning library. For each 56-mer peptide spanning the SARS-CoV-2 proteome, this library contained a collection of scanning mutants: The first mutant peptide encoded three alanines instead of the first three residues, the second mutant peptide contained the three alanines moved one residue downstream, and so on (fig. S4). Antibodies that recognize the wild-type 56-mer peptide will not recognize mutant versions of the peptide containing alanine substitutions at critical residues. Thus, the location of the linear epitope can be deduced by looking for “antibody footprints,” indicated by stretches of alanine mutants missing from the pool of immunoprecipitated phage. The first and last triple-alanine mutations to interfere with binding are expected to start two amino acids before the first residue that is essential for antibody binding and end two amino acids after the last.

With respect to cross-reactivity, IgG from COVID-19 patients recognized more 56-mer peptides from the common HCoVs HKU1, OC43, 299E, and NL63 than IgG from pre–COVID-19 era controls. This difference is primarily driven by a pronounced increase in recognition of S peptides from the HCoVs and is likely a result of cross-reactivity of antibodies developed during SARS-CoV-2 infection (Fig. 6A).

We mapped the position of all HCoV S peptides that display increased recognition in COVID-19 patient samples onto the SARS-CoV-2 S protein. This revealed four immunodominant regions recognized by >25% of COVID-19 patients (Fig. 6B). Comparing the frequency of peptide recognition between the COVID-19 patients and pre–COVID-19 era controls showed that two of these immunogenic regions in SARS-CoV-2 S are likely to cross-react strongly with homologous regions of other HCoVs, as the frequency of recognition of the HCoV peptides at these regions rises in COVID-19 patients. For instance, peptides from all four seasonal HCoVs that span the region corresponding to residues 811 to 830 of SARS-CoV-2 S are frequently recognized by COVID-19 patients but much less so by pre–COVID-19 era controls, suggesting that this recognition is a result of antibodies developed or boosted in response to SARS-CoV-2 infection. Using triple-alanine scanning mutagenesis (fig. S4), we mapped the antibody footprints in this region to an 11–amino acid stretch that is highly conserved between SARS-CoV-2 and all four common HCoVs, which explains the cross-reactivity (Fig. 6, C and D). Similarly, both SARS-CoV-2 and HCoV-OC43 peptides corresponding to S 1144-1163 are recognized much more frequently by COVID-19 patients than pre–COVID-19 era controls, and triple-alanine-scanning mutagenesis confirmed that the antibody footprints are located within a 10–amino acid stretch conserved between SARS-CoV-2 and HCoV-OC43 but not the other HCoVs. By contrast, the epitope sequences around S 551-570 and S 766-785 are not conserved between SARS-CoV-2 and the seasonal HCoVs, and indeed these epitopes are not cross-reactive. One HCoV-HKU1 peptide spanning S 551-570 scores in both COVID-19 patients and pre–COVID-19 era control samples; however, its frequency of detection is not further boosted in COVID-19 patients, suggesting that the antibody that recognizes the SARS-CoV-2 S 551-570 peptide is distinct from the antibody recognizing the HCoV-HKU1 peptide, consistent with sequence differences at this location (Fig. 6C).

Notably, we detect antibody responses to SARS-CoV-2 S 811-830 in 79.9% of COVID-19 patients. However, we also see responses to the corresponding peptides from OC43 and 229E in ~20% of the pre–COVID-19 era controls, and these responses seem to cross-react with SARS-CoV-2. It is possible that some patients have preexisting antibodies to this region that cross-react and are expanded during SARS-CoV-2 infection. This might explain the very high prevalence of antibody responses to this epitope and suggests that anamnestic responses to seasonal coronaviruses may influence the antibody response to SARS-CoV-2. Of note, this region is located directly after the predicted S2′ cleavage site for SARS-CoV-2 and overlaps the fusion peptide. A recent study showed that adding an excess of the fusion peptide reduced neutralization, implying that an antibody that binds the fusion peptide might contribute to neutralization by interfering with membrane fusion (21, 22). Given the frequency of seroreactivity toward this epitope in COVID-19 patients, it will be important to determine whether the antibodies that recognize this epitope are neutralizing in future studies. If so, the prior presence of antibodies recognizing this epitope may affect the course of COVID-19 and mitigate severity.

Epitope mapping reveals hundreds of distinct SARS-CoV-2 epitopes, including likely epitopes of neutralizing antibodies

We also used the triple-alanine scanning mutagenesis library to map antibody footprints across the entire SARS-CoV-2 proteome (Fig. 7, fig. S5, and tables S10 to S19). We used a hidden Markov model (HMM) to analyze the mutagenesis data and detect antibody footprints. By integrating signals across stretches of consecutive residues, the HMM successfully distinguished antibody footprints from random noise and thus detected regions containing epitopes with improved sensitivity and far greater resolution than was possible with the 56-mer peptide data alone (see Materials and methods) (figs. S6 and S7 and tables S15 to S18). We performed hierarchical clustering on the antibody footprints identified by the HMM to determine the number of distinct epitopes (here defined as distinct antibody footprints) that we detected across the SARS-CoV-2 proteome (fig. S8 and table S10). Overall, we identified 3103 antibody footprints across 169 COVID-19 patient samples and mapped 823 distinct epitopes (table S19). These epitopes are not evenly distributed along the proteins but rather fall into 303 epitope clusters, each of which contains multiple overlapping epitopes (fig. S8). For example, across the 89 IgA samples that recognized the epitope cluster from S 1135-1165, we identified nine epitopes that overlap but have distinct triple-alanine scanning profiles that indicate distinct antibody-epitope interactions (fig. S8C). Individual epitopes are recognized at a wide range of frequencies in the COVID-19 patients. The average COVID-19 patient sample contained antibodies to ~18 distinct linear epitopes (fig. S9), although this is likely an underestimate of the total epitope count per person, as VirScan does not efficiently detect antibodies recognizing discontinuous (conformational) epitopes (although such antibodies may retain some affinity to linear peptides that constitute the epitope).

The SARS-CoV-2 epitope landscape includes regions recognized by antibodies in a large fraction of COVID-19 patients (“public” epitopes) and regions recognized by antibodies in only one or a few individuals (“private” epitopes). For example, we mapped six distinct epitopes in the region spanning N 151-175 (fig. S5C). One of these epitopes was recognized by nearly one-third of the COVID-19 patients, whereas the rest were detected by <2% of the COVID-19 patients. Similarly, the region spanning S 766-835 contained more than 20 distinct epitopes, including the highly public epitope cluster near S815 and the public epitope cluster near S770 that is preferentially recognized by IgA (Fig. 7B). The public epitope cluster near S770 was recognized in 43% of COVID-19 patient IgA samples but only 4% of COVID-19 patient IgG samples. In another example, we detected at least 20 distinct epitopes within a stretch of just 46 residues in N 363-408, 10 of which were specific to IgA and 2 of which were specific to IgG (fig. S5D). The positions of several public epitope clusters are shown mapped onto the structure of SARS-CoV-2 in fig. S10.

We also mapped at least 12 distinct epitopes in the SARS-CoV-2 RBD, including 5 in the receptor binding motif that binds ACE2, the human receptor for SARS-CoV-2, and 6 that overlap ACE2 binding sites (Fig. 7, C and D, and fig. S6A). For example, S 414-427 (labeled E2 in Fig. 7) spans residue K417 in the RBD; K417 makes a direct contact with the human ACE2 protein in structures of ACE2 bound to the RBD. Thus, antibodies that recognize E2 are likely to block ACE2 binding and have neutralizing activity (Fig. 7E). Epitope S 454-463 (labeled E6 in Fig. 7) also overlaps ACE2 contact residues and partially overlaps the binding site of the neutralizing antibody CB6, which suggests that antibodies recognizing this epitope also have neutralizing potential (23–25) (Fig. 7G). Several other epitopes also span or are adjacent to critical residues contacted by ACE2 (Fig. 7, F and H). Thus, our data reveal some of the likely binding sites for neutralizing antibodies.

Identification of SARS-CoV-2 epitopes recognized by COVID-19 patients

VirScan detected robust antibody responses to SARS-CoV-2 in COVID-19 patients. These were primarily directed against the S and N proteins, with substantial cross-reactivity to SARS-CoV and milder cross-reactivity with the distantly related MERS-CoV and seasonal HCoVs. Cross-reactive responses to SARS-CoV-2 ORF1 were frequently detected in pre–COVID-19 era controls, suggesting that these result from antibodies induced by other pathogens.

At the population level, most SARS-CoV-2 epitopes were recognized by both IgA and IgG antibodies. We found that individuals often exhibited a “checkerboard” pattern, using either IgG or IgA antibodies against a given epitope. This suggests that a given IgM clone often evolves into either an IgG or IgA antibody, potentially influenced by local signals, and that, within an individual, there may often be a largely monoclonal response to a given epitope.

Examination of the humoral response to SARS-CoV-2 at the epitope level using the triple-alanine scanning mutagenesis library revealed 145 epitopes in S, 116 in N, and 562 across the remainder of the SARS-CoV-2 proteome (table S10). Most S epitopes were located on the surface of the protein or within unstructured regions that often abut, but seldom overlap, glycosylation sites (fig. S11). These epitopes ranged from private to highly public, with one region of S (S 811-830) being recognized by 79.9% of COVID-19 patients. Triple-alanine scanning mutagenesis showed highly conserved antibody footprints for some epitope clusters and diverse antibody footprints for others, indicating varying levels of conservation at the antibody-epitope interface among individuals (fig. S8). Peptides containing public epitopes could be used to isolate and clone antibodies from B cells bearing antigen-specific receptors. If these antibodies are found to lack protective effects or have deleterious effects, these regions could be mutated in future vaccines to divert the immunological response to other regions of S that might have more protective effects. Epitopes also varied in cross-reactivity, which can be explained by the presence or absence of sequence conservation between seasonal HCoVs and SARS-CoV-2 at these regions. Antibodies against several conserved epitopes in HCoVs seemed to be anamnestically boosted in COVID-19 patients. Antibodies recognizing one of these epitopes in the fusion peptide of S2 have been implicated in neutralization, and their presence prior to SARS-CoV-2 infection could mitigate the severity of COVID-19. Collectively, these data help explain why many serological assays for SARS-CoV-2 produce false positives due to preexisting cross-reactive antibodies, some of which may potentially affect the consequences of future SARS-CoV-2 infections.

Development of SARS-CoV-2 signature peptides for detecting seroconversion by Luminex

Using machine learning models trained on VirScan data, we developed a classifier that predicts SARS-CoV-2 exposure history with 99% sensitivity and 98% specificity. We identified peptides frequently and specifically recognized by COVID-19 patients and used these to create a Luminex assay that predicts SARS-CoV-2 exposure with 90% sensitivity and 95% specificity. Notably, the Luminex assay requires only three peptides to perform comparably to full-antigen ELISAs and could be further optimized in the future. This highlights the utility of VirScan-based serological profiling in the development of rapid and efficient diagnostic assays based on public epitopes.

Correlates of severity in COVID-19 patients

An important goal is to uncover serological correlates of COVID-19 severity. To this end, we compared cohorts of COVID-19 patients who did (H) or did not (NH) require hospitalization. Using both VirScan and the COVID-19 Luminex assay, we noticed a pronounced and somewhat counterintuitive increase in recognition of peptides derived from the SARS-CoV-2 S and N proteins among the H group, with more extensive epitope spreading. Whether this is a cause or a consequence of severe disease is not clear. Individuals whose innate and adaptive immune responses are not able to quell the infection early may experience a higher viral antigen load, a prolonged period of antibody evolution, and epitope spreading. Consequently, these patients might develop stronger and broader antibody responses to SARS-CoV-2 and could be more likely to have hyperinflammatory reactions such as cytokine storms that increase the probability of hospitalization. We noticed that hospitalized males had more robust antibody responses to SARS-CoV-2 than hospitalized females. This finding may indicate that males in this group are less able to control the virus soon after infection, and it is consistent with reported differences in disease outcomes for males and females (18, 19).

VirScan allowed us to examine viral exposure history, which revealed two notable correlations. First, the seroprevalence of CMV and HSV-1 was much greater in the H group than the NH group. The demographic differences in our relatively small cohort of H versus NH COVID-19 patients make it impossible for us to conclusively determine whether CMV or HSV-1 infection affects disease outcome or is simply associated with other covariates such as age, race, and socioeconomic status. Although CMV prevalence slightly increases with age after 40, it also differs greatly among ethnic and socioeconomic groups (26, 27). CMV is a chronic herpesvirus that is known to have a profound impact on the immune system: It can skew the naïve T-cell repertoire (28) and decrease T and B cell function (29) and is associated with higher systemic levels of inflammatory mediators (30) and increased mortality of people >65 years of age (31). The effects of CMV on the immune system could potentially influence COVID-19 outcomes.

The second notable correlation we observed was a substantial decrease in the levels of antibodies that target ubiquitous viruses such as rhinoviruses, enteroviruses, and influenza viruses in COVID-19 H patients compared with NH patients. When we examined only the CMV+ or HSV-1+ individuals in the two groups, we found that the strength of the antibody response to CMV and HSV-1 peptides was also reduced in the H group. We examined the effects of age on viral antibody levels in a pre–COVID-19 era cohort and found a diminution with age in the antibody response against viral peptides differentially recognized between the H and NH groups, consistent with previous studies on the effects of aging on the immune system (20). This inferred reduced immunity during aging could affect the severity of COVID-19 outcomes.

In correlative analyses such as these, it is difficult to draw strong conclusions about causality, given the demographic differences in the NH versus H groups. The NH group is younger and has a higher percentage of white and female individuals (average age 42, 66% female) than the H group (average age: 58; 42% female) (fig. S2), consistent with well-documented demographic skews in severely affected COVID-19 patients (18, 19). However, even if age and other demographic factors are covariates, CMV seropositivity and age-related reduction in antibody titers against viral antigens, as described here, could still influence the severity of infection. To test these hypotheses, a much larger cohort of COVID-19 patients with severe and mild disease that could be matched for age, race, and sex is required. Such future studies have the potential to enhance our understanding of the biological mechanisms underlying variable outcomes of COVID-19.

Deep serological profiling can provide a window into the breadth of viral responses, how they differ in patients with diverse outcomes, and how past infections may influence present responses to viral infections. Understanding the epitope landscape of SARS-CoV-2, particularly within S, provides a stepping stone to the isolation and functional dissection of both neutralizing antibodies and antibodies that might exacerbate patient outcomes through ADE and could inform the production of improved diagnostics and vaccines for SARS-CoV-2.

Sources of serum used in this study

Blood sample collection methods

For cohorts 1 to 3: Blood samples were collected into EDTA (ethylenediamine tetraacetic acid) tubes and spun for 15 min at 2600 rpm according to standard protocol. Plasma was aliquoted into 1.5-ml cryovials and stored at −80°C until analyzed. Only de-identified plasma aliquots including metadata (e.g., days since symptom onset, severity of illness, hospitalization, ICU status, survival) were shared for this study. When appropriate for nonconvalescent samples, plasma or serum was also heat-inactivated at 56°C for 60 min and stored at ≤20°C until analyzed.

For cohort 4: Blood samples were collected in EDTA tubes and processed no more than 3 hours post–blood draw in a biosafety level 2+ laboratory on site. Whole blood was diluted with room temperature RPMI medium in a 1:2 ratio to facilitate cell separation for other analyses using the SepMate PBMC isolation tubes (STEMCELL) containing 16 ml of Ficoll (GE Healthcare). Diluted whole blood was centrifuged at 1200 rcf for 20 min at 20°C. After centrifugation, plasma (5 ml) was pipetted into 15-ml conical tubes and placed on ice during PBMC separation procedures. Plasma was then centrifuged at 1000 rcf for 5 min at 4°C, pipetted in 1.5-ml aliquots into three cryovials (4.5 ml total), and stored at −80°C. For the current study, samples (200 μl) were first randomly allocated onto a 96-well plate on the basis of disease outcome grouping.

Design and cloning of the SARS-CoV-2 tiling and triple-alanine scanning library

Multiple VirScan libraries were constructed as described below. We created ~200-nt oligos encoding peptide sequences 56 amino acids in length, tiled with 28–amino acid overlap through the proteomes of all coronaviruses known to infect humans, including HCoV-NL63, HCoV-229E, HCoV-OC43, HCoV- HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2, as well as three closely related bat viruses (BatCoV-Rp3, BatCoV-HKU3, and BatCoV-279). For SARS-CoV-2, we included a number of coding variants available in early sequencing of the viruses. For SARS-CoV-2, we additionally made a 20–amino acid peptide library tiling every five amino acids. Additionally, for SARS-CoV-2 we made triple-alanine mutant sequences scanning through all 56-mer peptides. Non-alanine amino acids were mutated to alanine, and alanines were mutated to glycine. Each peptide in all three libraries was encoded in two distinct ways such that there were duplicate peptides that could be distinguished by DNA sequencing. We reverse-translated the peptide sequences into DNA sequences that were codon-optimized for expression in Escherichia coli, that lacked restriction sites used in downstream cloning steps (EcoRI and XhoI), and that were distinct in the 50 nt at the 5′ end to allow for unambiguous mapping of the sequencing results. Then we added adapter sequences to the 5′ and 3′ ends to form the final oligonucleotide sequences (table S1): These adapter sequences facilitated downstream PCR and cloning steps. Different adapters were added to each sublibrary so that they could be amplified separately. The resulting sequences were synthesized on a releasable DNA microarray (Agilent). We PCR-amplified the DNA oligo library with the primers shown below, digested the product with EcoRI and XhoI, and cloned it into the EcoRI/SalI site of the T7FNS2 vector (5). We packaged the resultant library into T7 bacteriophage using the T7 Select Packaging Kit (EMD Millipore) and amplified the library according to the manufacturer’s protocol.

Primers used for analysis of the different libraries employed:

CoV 56-mer library:

5′ adapter: 5′-GAATTCGGAGCGGT-3′

3′ adapter: 5′-CACTGCACTCGAGA-3′

Forward primer: 5′-AATGATACGGCGTGAATTCGGAGCGGT-3′

Reverse primer: 5′-CAAGCAGAAGACGTCTCGAGTGCAGTG-3′

SARS CoV-2 triple-alanine scanning library:

5′ adapter: 5′-GAATTCCGCTGCGT-3′

3′ adapter: 5′-CAGGGAAGAGCTCG-3′

Forward primer: 5′-AATGATACGGCGGGAATTCCGCTGCGT-3′

Reverse primer: 5′-CAAGCAGAAGACTCGAGCTCTTCCCTG-3′

SARS-CoV-2 20-mer library:

5′ adapter: 5′-GAATTCCGCTGCGT-3′

3′ adapter: 5′-GTACTATACCTACGGAAGGCTCG-3′

Forward primer: 5′-AATGATACGGCGGGAATTCCGCTGCGT-3′

Reverse primer: 5′-TATCTCGCATAGCGCATATACTCGAGCCTTCCGTAGGTATAGTAC-3′

Phage immunoprecipitation and sequencing

We performed phage IP and sequencing as described previously or with slight modifications (5–8). For the IgA and IgG chain isotype-specific IPs, we substituted magnetic protein A and protein G Dynabeads (Invitrogen) with 6 μg of Mouse Anti-Human IgG Fc-BIOT (Southern Biotech) or 4 μg of Goat Anti-Human IgA-BIOT (Southern Biotech) antibodies. We added these antibodies to the phage and serum mixture and incubated the reactions overnight a 4°C. Next, we added 25 or 20 μl of Pierce Streptavidin Magnetic Beads (Thermo-Fisher) to the IgG or IgA reactions, respectively, and incubated the reactions for 4 hours at room temperature, then continued with the washing steps and the remainder of the protocol, as previously described (5–8).

Machine learning classifiers

Gradient-boosting classifier models for the VirScan data were generated using the XGBoost algorithm (version 1.0.2). Classifier models were trained to discriminate either COVID-19+ and COVID-19− patients (n = 232 and 190, respectively) or severe disease and mild disease (n = 101 hospitalized patients and n = 131 nonhospitalized patients). Two models were generated in each case, one using the z-scores for each VirScan peptide from the IgG IP as input features, and the other using the z-scores for each VirScan peptide from the IgA IP as input features. Additionally, a third logistic regression classifier was trained on the output probabilities from the IgG and IgA models to generate a combined prediction. The performance of each of the three model was assessed using a 20-fold cross-validation procedure, whereby predictions for each 5% of the data points were generated from a model trained on the remaining 95%. The SHAP package was used to identify the top discriminatory peptide features from each of the XGBoost models. The logistic regression models for the Luminex data were generated using the scikit-learn python package. The raw median fluorescence intensity (MFI) values were preprocessed using the RobustScalar function, then a logistic regression model was trained using the three most discriminate SARS-CoV-2 peptides. The model performance was quantified by 10-fold cross-validation.

High-resolution epitope identification and clustering

For each position in the 56-mer, the relative enrichment for each amino acid was calculated as the mean fold change of the three mutant peptides containing an alanine mutation at that location relative to the median fold change of all alanine mutants for the 56-mer. Overlapping 56-mers were combined by taking the minimum value at each shared position to account for the possibility that an epitope is interrupted in one of the tiles by the peptide junction. To map the boundaries of antibody footprints from the triple-alanine scanning data for each sample we used the hmmlearn python package to develop a three-state HMM assuming a Gaussian distribution of relative enrichment emissions for each state. Mapped antibody footprints smaller than five amino acids in length were removed from the subsequent analysis. Next, we performed a two-step hierarchical clustering procedure to identify the number of distinct epitopes. First, for each protein all antibody footprints identified across the 169 COVID-19+ patient samples were clustered based on the start and stop locations predicted by the HMM classifier to generate epitope clusters. Next, to identify distinct epitopes, we performed an additional step of hierarchical clustering on the samples with epitopes within each epitope cluster based on the relative enrichment values of the triple-alanine mutants spanning the epitope (fig. S8).

Similarity-score calculation

Pairwise alignments were generated for the S proteins of SARS-CoV-2 and each of the four common HCoVs. Similarity scores were calculated separately for a 21–amino acid window centered at each position of the SARS-CoV-2 S protein. The mean similarity score between SARS-CoV-2 and the corresponding sequence of the other HCoV was calculated for each window using the BLOSUM62 substitution matrix with a gap opening and extending penalty of −10 and −1, respectively. The maximum similarity was score was calculated as the maximum value among the pairwise similarity scores between SARS-CoV-2 and each of the four common HCoVs for the sliding window centered at each position.

Luminex multiplex peptide epitope serology assays

Multiplexed SARS-CoV-2 peptide epitope assays were built using the peptides listed in table S9. Peptides were synthesized by the Ragon/MGH Peptide Core Facility with a Proparglyglycine (Pra, X) moiety in the N terminus to facilitate cross-linking to Luminex beads using a “click” chemistry strategy as described previously (13). In brief, Luminex beads were first functionalized with amine-PEG4-azide and then reacted with the peptides to generate 20 different Luminex beads with attached peptides. Luminex bead–based serology assays were performed in 96-well U-bottom polypropylene plates using PBS + 0.1% bovine serum albumin as the assay buffer. Bead washes were done using PBS + 0.05% Triton X-100 by incubation for 1 min on a strong magnetic plate (Millipore-Sigma, Burlington, MA). All assay incubation times were 20 min. In the first step, beads were incubated with 20 μl of plasma samples. Samples used for the classifier were diluted 1:100, samples used to compare disease severity were diluted 1:300. After a wash step, bound IgA or IgG was detected by adding 40 μl of biotin-labeled anti-IgA or IgG antibodies at 0.1 μg/ml (Southern Biotechnology, Birmingham, AL). Next, 40 μl of phycoerythrin (PE)–labeled streptavidin (0.2 μg/ml) (Biolegend, San Diego, CA) and assay plates were analyzed on a Luminex FLEXMAP 3D instrument (Luminex Corporation, Austin, Texas) to generate MFI values to quantify peptide-specific IgA or IgG levels.

ELISA serology assays

ELISAs were performed separately using the SARS-CoV-2 N protein, S protein, or the S receptor-binding domain (RBD). 96-well plates were coated with antigen overnight. The plates were then blocked in PBS + 3% BSA. After washing with PBS + 0.05% Tween-20, the plasma sample were diluted 1:100, added to the plates and incubated overnight at 4°C. After incubation, the plates were washed three times with PBS + 0.05% Tween-20. The bound IgG was detected by adding anti-human IgG-alkaline phosphatase (Southern Biotech, Birmingham, AL) and incubating for 90 min at room temperature. The plates were washed an additional three times, after which p-nitrophenyl phosphate solution (1.6 mg/ml in 0.1 M glycine, 1 mM ZnCl₂, 1 mM MgCl₂, pH 10.4) was added to each well and allowed to develop for 2 hours. Bound IgG was quantified by measuring the OD405, and the reported values were calculated as the fold change over the pre–COVID-19 controls.