Pre-existing Humoral Immunity To Human Common Cold Coronaviruses Negatively Impacts The Protective SARS CoV-2 Antibody Response

Graphical abstract

SUMMARY 

SARS-CoV-2 infection causes diverse outcomes ranging from asymptomatic infection to respiratory distress and death. A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoVs) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Therefore, we analyzed samples from the same individuals before and after SARS-CoV-2 infection or vaccination. We found how antibody levels increase after SARS-CoV-2 exposure, demonstrating cross-reactivity. However, a case-control study indicates that baseline hCCCoV antibody levels are not associated with protection against SARS-CoV-2 infection. Rather, higher magnitudes of pre-existing betacoronavirus antibodies correlate with more SARS-CoV-2 antibodies following infection, an indicator of greater disease severity. Additionally, immunization with hCCCoV spike proteins before SARS-CoV-2 immunization impedes the generation of SARS-CoV-2-neutralizing antibodies in mice. Together, these data suggest that pre-existing hCCCoV antibodies hinder SARS-CoV-2 antibody-based immunity following infection and provide insight into how pre-existing coronavirus immunity impacts SARS-CoV-2 infection, which is critical considering emerging variants.

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INTRODUCTION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces highly variable disease ranging from very mild or no symptoms to severe respiratory distress and death. Certain comorbidities contribute to the diverse outcomes; however, these factors do not account for all the heterogeneity observed between infected individuals. A major unresolved question is whether susceptibility to SARS-CoV-2 infection and disease severity after infection are impacted by immunity to human common cold coronaviruses (hCCCoVs) that were circulating before the SARS-CoV-2 pandemic. Four hCCCoVs that are prevalent worldwide have been endemic in humans for decades and typically induce mild upper respiratory disease and account for  30% of ‘‘common colds’’ (Forni et al., 2017). HKU1 and OC43 are betacoronaviruses, as is SARS-CoV-2, which are evolutionarily distinct from the alphacoronaviruses, 229E and NL63. Despite dramatic differences in disease severity induced by the viruses, SARS-CoV-2 and the endemic hCCCoVs share  30% homology within the spike proteins (Hicks et al., 2021). Studies identified cross-reactive antibodies that bind both SARS-CoV-2 and hCCCoVs (Ladner et al., 2020; Ng et al., 2020; Wec et al., 2020). However, it is unclear how pre-existing hCCCoV antibodies impact the immune response against SARS-CoV-2 infection (Sealy and Hurwitz, 2021). Prior hCCCoV infections could augment SARS-CoV-2 immunity if hCCCoV antibodies are sufficiently cross-reactive with SARS-CoV-2 to be induced through immunological recall or ‘‘back-boosting’’ and support viral clearance (Fonville et al., 2014). Conversely, pre-existing hCCCoV humoral immunity could hinder the generation of effective SARS-CoV-2-specific antibodies by expanding cross-reactive antibodies that do not neutralize SARS-CoV-2. Further, existing hCCCoV immunity may exacerbate disease by facilitating viral entry into Fc receptor (FcR)-expressing cells to cause antibody-dependent enhancement of disease (Arvin et al., 2020; Iwasaki and Yang, 2020). Since hCCCoV immunity could influence the outcome of SARS-CoV-2 infection in several ways, it is critical to ascertain the impact of pre-existing hCCCoV antibodies on immunity to SARS-CoV-2 infection. Reports investigating whether antibodies specific for hCCCoVs are boosted following SARS-CoV-2 infection yielded conflicting results. Some data suggested antibodies specific for hCCCoVs were not boosted following SARS-CoV-2 infection (Dugas et al., 2021a, 2021b; Loos et al., 2020), while others reported a boost only in OC43-specific antibodies (Anderson et al., 2021; Guo et al., 2021; Nguyen-Contant et al., 2020; Pre´- vost et al., 2020). Additional studies found a boost in both HKU1 and OC43 antibodies (Aydillo et al., 2021; Cohen et al., 2021; Gouma et al., 2021; Westerhuis et al., 2021) or in antibodies specific for all four hCCCoVs following SARS-CoV-2 infection (Ng et al., 2020; Shrock et al., 2020). Yet, other reports surprisingly found a boost predominantly in antibodies specific for the alpha coronaviruses (Becker et al., 2021; Ortega et al., 2021). A major factor contributing to these inconsistencies is that prior studies did not examine the level of HCV antibodies in the same individual before and after SARS-CoV-2 infection.

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Assessing whether prior hCCCoV immunity impacts SARS-CoV-2 disease susceptibility has also yielded inconsistent results (Sealy and Hurwitz, 2021). While some studies reported that the levels of HCV antibodies did not correlate with disease severity or likelihood of becoming infected (Anderson et al., 2021; Gombar et al., 2021; Loos et al., 2020), others concluded that higher levels of hCCCoV antibodies were associated with milder disease (Becker et al., 2021; Dugas et al., 2021a, 2021b; Henss et al., 2021; Ortega et al., 2021; Sagar et al., 2021; Shrock et al., 2020) or with a shorter duration of symptoms (Gouma et al., 2021). Conversely, others found higher levels of hCCCoV antibodies correlated with increased SARS-CoV-2 disease severity (Aydillo et al., 2021; Guo et al., 2021; Pre´ vost et al., 2020; Westerhuis et al., 2021). The health status varied greatly in the cohorts tested in the previous studies, and most of these studies did not test samples from the same individual before and after SARS-CoV-2 infection, which likely contributes to the discrepancy in conclusions. Thus, the impact of pre-existing hCCCoV immunity on susceptibility to SARS-CoV-2 infection remains unresolved. Here, we measured immunoglobin (Ig) G, IgM, and IgA antibodies in samples obtained from the same individual before and after PCR-confirmed SARS-CoV-2 infection. We observed significant increases in betacoronaviruses IgG antibodies; however, high levels of HCV antibodies were not associated with protection against SARS-CoV-2 infection. Conversely, a greater increase in hCCCoV antibodies correlated with higher antibody levels of SARS-CoV-2 following infection, which were associated with increased disease severity. Moreover, mice immunized with hCCCoV spike proteins before the SARS-CoV-2 spike exhibited a profound decrease in SARS-CoV-2-neutralizing antibodies relative to mice only immunized with the SARS-CoV-2 spike. Overall, these data suggest that pre-existing hCCCoV IgG antibodies may hinder the immune response to SARS-CoV-2.

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RESULTS

Common hCCCoV antibody isotypes are associate with age and direct patient contact

We established a prospective, longitudinal cohort (St. Jude Tracking of Viral and Host Factors Associated with COVID-19 study, SJTRC) of St. Jude employees who provided a baseline blood sample at enrollment and underwent weekly nasal swab screening for SARS-CoV-2 infection by PCR (Table S1). Individuals who tested positive during the study provided samples at two-time points following infection. Additionally, participants who did not become infected gave samples after vaccination. This design allowed analysis of samples from the same individuals taken before and after SARS-CoV-2 infection or vaccination. Importantly, weekly nasal swab screening identified asymptomatic infections throughout the study period. To assess immunity before SARS-CoV-2 infection, we analyzed 1,202 baseline samples for antibodies specific for the spike proteins of OC43, HKU1, 229E, and NL63 by ELISA. To control for plate-to-plate variability, the same positive control samples were tested on each plate, and the normalized optical density (OD) for each sample was presented. Although antibody levels varied among individuals, IgG antibodies specific for all four of the hCCCoV spike proteins were identified in nearly all participants (Figures 1A and 1D). HCV IgM antibodies were less prevalent than IgG and IgA, with IgA antibodies exhibiting the greatest variability (Figures 1A–1D). Interestingly, there were stronger correlations between antibody isotypes rather than specificity to a particular virus (Figure 1E). For example, individuals with high levels of HKU1 IgM were more likely to have IgM antibodies specific for the other three hCCCoVs rather than HKU1 IgG and IgA. Further, individuals with high levels of HKU1 IgG did not necessarily have high levels of HKU1 IgA and IgM. Together, these data indicate that nearly every individual had antibodies specific for all four hCCCoVs before SARS CoV-2 infection or vaccination. Moreover, the stronger correlations with antibody isotype compared to virus type suggest there is cross-reactivity among hCCCoV-specific antibodies, with a higher degree of promiscuity in the IgM response followed by IgA then IgG, consistent with previous studies (Becker et al., 2021; Poston et al., 2021).

We next examined whether the level of hCCCoV antibodies at baseline correlated with age, sex, race, or direct patient contact. We compared antibody levels in individuals above and below the median age at the time of enrollment, which was 43 years of age (Table S1). We found that older individuals had significantly higher levels of IgA against HKU1, 229E, and NL63 (Figure 2A). Conversely, younger individuals had significantly higher IgM levels reactive with all four of the honors compared to older individuals. We also found that females had higher levels of all four hCCCoV IgM antibodies and higher OC43 IgA antibodies compared to males (Figure S1A). Additionally, IgG and IgA antibody levels differed across race/ethnicity groups in approximately 10% of the analyses (Figure S1B). Since the study participants are employees at a pediatric hospital and interactions with children may increase exposure to hCCCoVs, we assessed the tact. Individuals with direct patient contact had higher levels of IgM antibodies specific for all four honors, as well as OC43 IgA (Figure 2B). Together, these data indicate that in the SJTRC cohort, younger, female participants with direct patient contact were more likely to have elevated levels of hCCCoV IgM. Since the SJTRC cohort did not include individuals younger than 20 years of age, we also analyzed antibody levels in samples collected from a previous study, the FLU09 cohort, that included a wider age range of participants. Similar to previous reports (Selva et al., 2021), we found higher levels of most of the hCCCoV IgG antibodies and all of the IgA antibodies in older individuals compared to younger individuals (Figures S2A–S2I). Unexpectedly, the levels of IgM antibodies for most of the honors were low in young individuals, peaked around 20 years of age, and then declined with age (Figures S2B and S2E). Therefore, we examined whether there was a correlation between antibody levels and age in individuals 0–14 (Figures S2J– S2L) or 17–54 years of age (Figures S2M–S2O). We found that most IgG and IgA antibody levels increased with age during the younger years (Figures S2G and S2I) and then remained stable (Figures S2M and S2O). Conversely, there was not a significant association between IgM and age in the younger group (Figure S2K), but a significant decline in IgM was found with age for participants 17–54 years of age (Figure S2N). The decline in IgM in the 17–54 age group is consistent with the SJTRC cohort where we found higher levels of hCCCoV IgM in younger individuals (20–43 years of age) compared to older participants (Figure 2A). These data indicate that IgG and IgA hCCCoV antibodies begin to accumulate very early in life. Intriguingly, IgM levels tend to peak between 10–30 years of age rather than declining linearly with age. As younger individuals are more likely to be recently exposed to have and would have a higher proportion of naive IgM+ B cells relative to older individuals, we expected to see higher IgM levels in younger individuals. Overall, these data show the wide degree of heterogeneity in hCCCoV immunity between individuals and demonstrate that most individuals have antibodies specific for all four hCCCoVs from a very early age.

Figure 1. Wide variation in baseline hCCCoV antibody levels

HCV antibodies are increased after infection with SARS-CoV-2

While studies identified cross-reactive antibodies that bind both SARS-CoV-2 and hCCCoVs (Ladner et al., 2020; Ng et al., 2020; Wec et al., 2020), there is significant controversy regarding whether hCCCoV antibodies are boosted after SARS-CoV-2 infection (Anderson et al., 2021; Aydillo et al., 2021; Becker et al., 2021; Dugas et al., 2021a, 2021b; Gouma et al., 2021; Guo et al., 2021; Loos et al., 2020; Ng et al., 2020; Nguyen-Contant et al., 2020; Ortega et al., 2021; Pre´ vost et al., 2020; Shrock et al., 2020; Westerhuis et al., 2021). If pre-existing hCCCoV-specific antibodies cross-react to SARS-CoV-2, the levels of hCCCoV-specific antibodies would increase following SARS-CoV-2 infection. Alternatively, if antibodies specific for hCCCoVs do not cross-react to SARS-CoV-2, the levels of hCCCoV antibodies would not change after infection. We analyzed samples taken before and at two-time points after confirmed SARS CoV-2 infection. The first sample after infection was collected during the acute phase (1–20 days) (Figure 3A), and a subsequent sample was taken during the convalescent phase (>20 days) (Figure 3B). Interestingly, several individuals exhibited reduced hCCCoV antibody levels shortly after SARS-CoV-2 infection relative to baseline, indicated by a negative percent change of baseline (Figures 3C–3E and S3A–S3C; Table S2). This decrease was most evident in samples taken within the first 20 days after infection. The decrease in hCCCoV antibodies shortly after SARS-CoV-2 infection highlights the caveat of not analyzing hCCCoV antibodies in paired samples collected prior to SARS-CoV-2 infection. Similar to associations prior to infection, individuals exhibiting an increase in IgM antibodies to one subtype of hCCCoVs typically showed increases in IgM reactive to all hCCCoVs (Figures 3A and 3B). In contrast, IgA antibodies specific for both betacoronaviruses typically increased concurrently. Interestingly, HKU1 IgG levels increased the most after SARS-CoV-2 infection compared to the other hCCCoV IgG antibodies, while OC43 IgA showed the greatest increase of the IgA antibodies (Figures 3A and 3B; Table S2). Overall, HKU1 and OC43 IgG and IgA antibodies showed the highest and most consistent increase over baseline levels compared to antibodies specific for the alphacoronaviruses (Figures 3A–3H; Table S2), which is consistent with greater homology among the betacoronaviruses. Importantly, hCCCoV antibody levels did not change in individuals infected with the influenza virus (Figures S3D–S3F), demonstrating that the increase in hCCCoV antibodies reflected cross-reactivity with SARS-CoV-2 infection rather than a nonspecific consequence of infection.

Figure 2. hCCCoV IgM levels inversely correlate with age and are higher in individuals with direct patient contact

Figure 3. Antibodies specific for OC43 and HKU1 increase following SARS-CoV-2 infection

The increase in hCCCoV antibodies following SARS-CoV-2 infection could be due to activation of pre-existing memory B cells that were generated after prior hCCCoV infection. Alternatively, the elevated levels of hCCCoV antibodies after SARS-CoV2 infection could be due to the generation of new antibodies that cross-react to hCCCoVs in response to SARS-CoV-2. To distinguish these possibilities, we measured antibody levels in samples collected at various times after SARS-CoV-2 diagnosis. We reasoned that an increase in hCCCoV antibodies due to a boost of pre-existing memory B cells would be detected rapidly following diagnosis, while an increase in hCCCoV antibodies resulting from newly generated antibodies would be evident later. Remarkably, the levels of HKU1 IgG rapidly increased in several individuals within the first 5 days after SARS-CoV-2 diagnosis (Figures 3C and 3F), and OC43 and HKU1 IgA levels increased within 10 days in over 50% of individuals (Figures 3E and 3H). The early rise in betacoronavirus hCCCoV IgG and IgA antibodies suggests that infection with SARS-CoV-2 activates preexisting memory B cells to boost antibodies generated during prior hCCCoV infections. Further, if the increase in hCCCoV antibodies was due to newly generated antibodies in response to SARS-CoV-2 infection, then we would expect these antibodies to also be SARS-CoV-2-specific. Therefore, we examined whether individuals with high levels of HKU1 IgG antibodies within 5 days of diagnosis also had antibodies that recognized the SARS-CoV-2 spike or the receptor binding domain (RBD) of the spike. While a few individuals had positive levels of SARS CoV-2 spike and RBD IgG within 5 days of diagnosis (Figures 3I–3L and S4), there was no correlation between the level of SARS-CoV-2 spike or RBD IgG and HKU1 IgG (Figures 3I and 3J) or a correlation between SARS-CoV-2 spike or RBD IgG and the increase of HKU1 IgG (Figures 3K and 3L). Interestingly, IgM antibodies specific for the SARS-CoV-2 proteins were not typically observed prior to IgG or IgA (Figure S4), which would be expected after exposure to a novel virus or vaccine (Li et al., 2014; Wolf et al., 2011). Thus, the antibody response to SARS-CoV-2 displays a pattern similar to what would be expected after boosting of a memory response. Together, these data are consistent with the notion that SARS-CoV-2 activates pre-existing memory B cells to boost antibodies that were generated after prior HCV infection. The hCCCoV antibodies detected at later time points are likely a combination of boosted, pre-existing antibodies and newly generated antibodies that cross-react to hCCCoVs. If the pre-existing antibodies recognize epitopes on SARS-CoV-2, they could reduce infection severity by promoting viral clearance. Alternatively, if the antibodies do not bind SARS-CoV-2 with sufficient avidity, these antibodies could delay the generation of effective antibodies specific for SARS-CoV-2 by competing with naive B cells for antigen and cytokines. The fact that individuals with an early increase or high levels of hCCCoV antibodies within 5 days of SARS-CoV-2 diagnosis did not have SARS-CoV-2-specific antibodies at this time suggests that the hCCCoV antibodies do not bind SARS-CoV-2 with sufficient avidity to be detected by ELISA.

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HCV antibodies do not impact the probability of becoming infected with SARS-CoV-2

Since hCCCoV-specific antibodies cross-react with SARS-CoV- 2 as demonstrated by the early increase after infection and prior studies (Ladner et al., 2020; Ng et al., 2020), we performed a large, case-control study to test whether pre-existing hCCCoV IgG, IgM, and IgA antibodies were different between individuals who became infected during the study compared to individuals that remained negative. It is important to note that all individuals underwent weekly nasal swab screening, which allowed us to identify asymptomatic infections and confirm all SARS-CoV-2 infections by PCR. We assessed baseline hCCCoV antibodies in 121 individuals who subsequently became positive during the study and compared them to baseline samples of 1,081 individuals who remained uninfected. Even though hCCCoV antibodies exhibit sufficient cross-reactivity with SARS-CoV-2 to increase after infection, baseline levels of hCCCoV antibodies were not different between individuals that became infected compared to those that remained SARS-CoV-2 negative during the study period (Figure 4; Table S3). These data imply that prior infection with hCCCoVs does not protect against infection with SARS CoV-2, which is consistent with the inability of hCCCoV-specific antibodies to neutralize SARS-CoV-2 (Aguilar-Bretones et al., 2021; Legros et al., 2021; Poston et al., 2021).

Figure 4. Baseline HCV antibody levels do not correlate with protection from SARS-CoV-2 infection

Baseline HCV antibodies do not provide protective immunity against SARS-CoV-2 infection

While baseline levels of hCCCoV antibodies were not different between participants who became infected compared to those who remained SARS-CoV-2 negative, hCCCoV antibodies could influence the severity or duration of symptoms. Therefore, we assessed whether there was a correlation between disease severity and baseline levels of HCV antibodies. Infected individuals were given a score of 1–5 based on an a priori ordinal scale as follows: (1) asymptomatic, (2) mild-moderate, (3) moderate-severe illness, (4) severe illness, and (5) critical illness. This scale allowed us to distinguish truly asymptomatic, minimally symptomatic, and more severely symptomatic individuals. Most participants in this cohort had mild-moderate and moderate-severe severity scores (severity scores 2 to 3). Since only a few individuals were asymptomatic, severe, or critical, we compared baseline hCCCoV antibodies between individuals that were either asymptomatic or had mild disease (severity score of 1 to 2) to individuals that experienced moderate, severe, or critical disease (severity score 3–5). We found no significant difference between baseline hCCCoV antibody levels and disease severity when comparing these two groups (Figures 5A and 5B). Moreover, symptom duration did not correlate with baseline hCCCoV antibody levels (Figures 5A and S5). These data suggest that the baseline levels of HCV antibodies do not provide significant protection against SARS-CoV-2 infection. However, as there were few cases of severe COVID-19 requiring hospitalization or critical illness in the included participants, our ability to identify predictors of these states is limited.

Figure 5. Baseline HCV antibody levels do not correlate with disease severity following SARS-CoV-2 infection

Existing HCV antibodies influence SARS-CoV-2 antibody response

Since the SJTRC cohort consists primarily of individuals with mild-moderate disease severity and only four individuals had severe or critical disease, the impact of HCV antibodies on very severe cases may not be evident in this cohort. Many studies reported that the level of SARS-CoV-2 spike or RBD IgG or IgA following infection correlated with disease severity (Aguilar-Bretones et al., 2021; Becker et al., 2021; Doban˜ o et al., 2021; Garcia-Beltran et al., 2021; Guthmiller et al., 2021; Legros et al., 2021; Ortega et al., 2021; Shrock et al., 2020). This may be due to the fact that individuals with more severe disease likely have more viral replication and, therefore, greater antigen exposure. Thus, the antibody response after infection may provide a means to further stratify disease severity within the groups, independent of self-reported symptoms. Therefore, we examined whether the antibody response 16–40 days following SARS CoV-2 infection correlated with disease severity in the SJTRC cohort, in which most participants had mild-moderate disease severity. Importantly, none of the infected individuals had received a vaccine prior to the collection of samples used for this comparison or other comparisons reported here. Similar to other studies, the level of IgG specific for SARS-CoV-2 spike, RBD, and N protein significantly correlated with increased disease severity scores (Figures 6A and S6). Higher spikes and RBD IgM and spike IgA levels also correlated with more severe disease. These data indicate that, although most participants had mild moderate disease, the levels of SARS-CoV-2-specific IgG and IgM correlated with severity. Consequently, we compared baseline hCCCoV antibody levels to SARS-CoV-2 antibody levels following infection to further assess the association of baseline hCCCoVs and a distinct correlate of disease severity.

Figure 6. Existing HCV antibody levels are associated with the magnitude of the SARS-CoV-2 antibody response after infection, but not vaccination

Interestingly, higher levels of OC43 IgG prior to infection correlated with increased SARS-CoV-2 IgG after infection (Figure 6B), raising the possibility that high baseline OC43 IgG may be associated with more severe disease. To further examine the impact of hCCCoV immunity on the immune response to SARS-CoV-2, we tested whether the magnitude of the hCCCoV antibody increase or decrease following SARS-CoV-2 infection impacted SARS-CoV-2 antibody levels. The amount that the hCCCoV antibody levels increase in the initial days after SARS-CoV-2 infection is indicative of the extent to memory B cells are activated to produce antibodies. Thus, we calculated the percent change of HCV antibody in the baseline sample to the sample taken within the first 15 days after diagnosis. Increases in HCV antibody levels in this time frame would reflect the extent of memory B cell activation. We compared this change to the SARS-CoV-2 antibody levels 16–40 days after infection, as these levels correlated with disease severity in our cohort as well as several other studies. Interestingly, a greater increase in betacoronavirus IgG and IgA was associated with higher levels of SARS-CoV-2 IgG and IgM antibodies after infection (Figure 6C). Since increased levels of SARS-CoV-2 IgG and IgM are associated with greater disease severity, these data raise the possibility that the early increase (1–15 days after infection) in antibodies could be associated with higher disease severity. Alternatively, the association between the increase in hCCCoV antibody levels with higher SARS-CoV-2-induced antibodies could be due to newly generated antibodies in response to SARS-CoV-2 infection that cross-react with hCCCoVs. However, analysis of samples taken within the first 5 days of SARS-CoV-2 diagnosis demonstrated that the increase in hCCCoV antibodies preceded the detection of SARS-CoV-2 antibodies (Figures 3I–3L), indicating that the early hCCCoV-reactive antibodies do not bind SARS-CoV-2 spike.

If the correlation between the early increase of betacoronavirus antibodies and higher SARS-CoV-2 antibodies after infection was due to newly generated antibodies in response to SARS-CoV-2 infection that cross-react with hCCCoVs rather than an association with disease severity, then we would predict that the baseline hCCCoV levels or boosts would have a similar correlation in response to vaccination in individuals that were not infected with SARS-CoV-2. Therefore, we compared baseline hCCCoV antibody levels in individuals before vaccination to the level of SARS-CoV-2 antibodies after vaccination. For this analysis, none of the vaccinated participants were previously infected with SARS-CoV-2. The fact that all participants were screened weekly by nasal swab and PCR reduced the probability of individuals with asymptomatic infections being included in this group. We first assessed whether hCCCoV antibodies increased following vaccination similar to infection. We observed an increase in HKU1 IgG after Pfizer/BioNTech BNT162b2 vaccination compared to samples taken at baseline (Figure S7). However, there was not a significant increase in OC43 IgG as seen after SARS-CoV-2 infection. Moreover, the increase in HKU1 IgG antibodies after vaccination was not as great as the increase observed in SARS-CoV-2-infected participants. We also noted a significant decrease in all hCCCoV IgA and IgM antibodies following vaccination. Importantly, neither baseline levels of hCCCoV antibodies nor an increase in hCCCoV antibodies after vaccination correlated with increased SARS-CoV-2 antibodies after vaccination (Figures 6D and 6E). In fact, correlations of baseline or boost of hCCCoV antibodies and SARS-CoV-2 antibodies showed strikingly distinct patterns in infected versus vaccinated individuals (Figures 6B–6E). Interestingly, there were significant correlations with baseline hCCCoV IgM and SARS-CoV-2 IgM after vaccination. As IgM antibodies exhibit greater cross-reactivity among the hCCCoVs compared to IgG and IgA, this could reflect existing hCCCoV IgM antibodies that cross-react with SARS-CoV-2. Alternatively, individuals with higher hCCCoV IgM may have a higher proportion of naive B cells capable of responding to a novel antigen. As the vaccine does not induce a robust IgM response in most individuals, it is currently not known whether IgM antibody levels after vaccination impact vaccine efficacy. Together, these data indicate that pre-existing betacoronavirus IgA and IgG correlate with a higher antibody response to SARS-CoV-2 following infection, but not vaccination. As increased SARS-CoV-2 antibodies after infection correlated with greater disease, these findings raise the possibility that pre-existing betacoronavirus IgG and IgA negatively impact the immune response to SARS-CoV-2, which results in a greater duration of antigen and therefore more SARS-CoV-2 antibodies.

Prior immunization with hCCCoV spike proteins limits the antibody response to SARS-CoV-2 RBD in mice

Since most individuals have positive levels of antibodies specific for all four hCCCoVs (Figure 1D), it is not possible to directly examine whether prior exposure to a particular hCCCoV impacts the antibody response to SARS-CoV-2. Therefore, we performed a series of immunizations in mice that had no prior exposure to coronaviruses. C57BL/6 mice were immunized with the spike proteins of SARS-CoV-2, OC43, HKU1, 229E, or NL63. Four weeks later, all mice were immunized with the SARS-CoV-2 spike protein. Thus, mice either received two immunizations of the SARS-CoV-2 spike or one immunization of the hCCCoV spike followed by one of the SARS-CoV-2 spike. Two weeks following immunization with the SARS-CoV-2 spike, we measured RBD and spike IgG antibodies to determine if prior exposure to hCCCoV spike proteins impacted the antibody response to the SARS-CoV-2 spike and RBD. Prior immunization with hCCCoV spike proteins did not significantly impact antibody levels to SARS-CoV-2 full-length spike (Figure 7A). However, RBD IgG was significantly decreased in mice that received a prior immunization with HKU1 and NL63 spike proteins compared to mice only immunized with SARS-CoV-2 spike protein (Figure 7B). Importantly, prior immunization with any of the hCCCoV spike proteins inhibited neutralizing antibodies following SARS-CoV-2 spike immunization as detected by a pseudo-neutralization assay (Figure 7C). These data, which are consistent with a prior study utilizing a different strain of mice and adjuvant (Lapp et al., 2021), directly demonstrate that prior exposure to hCCCoV spike proteins has the potential to inhibit the generation of neutralizing antibodies specific for the RBD of SARS-CoV-2. Together, these findings illustrate that prior immunity to a virus with a certain degree of homology can impede the immune response to a novel virus.

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DISCUSSION

Immune imprinting refers to the preferential activation of memory B cells that were generated during a prior infection with an antigenically related virus rather than naive B cells specific for the novel virus (Guthmiller and Wilson, 2018; Henry et al., 2018; Monto et al., 2017). This concept is well documented for influenza infections whereby humans are repeatedly exposed to antigenically distinct viruses containing regions of homology. Imprinting can hinder immunity to a novel virus if pre-existing antibodies against conserved epitopes dominate the immune response but do not neutralize the novel virus. Since pre-existing memory B cells are present at higher precursor frequencies relative to naive B cells and are primed to be activated, they can outcompete B cells specific for novel epitopes and hinder immunity to the novel virus (Cobey and Hensley, 2017). In addition, antibodies generated by a related virus could block antibodies specific to the novel virus via steric hindrance by binding conserved epitopes near the novel epitopes.

Humans are repeatedly infected with endemic hCCCoVs (Edridge et al., 2020; Kiyuka et al., 2018), and our data indicate that nearly every individual possesses antibodies specific to all four of the endemic hCCCoVs. A recent study demonstrated that memory B cells specific for hCCCoVs dominated the early immune response following SARS-CoV-2 infection; however, these antibodies did not neutralize SARS-CoV-2 (Dugan et al., 2021). This study illustrates how hCCCoV immunity can hinder protective immunity to SARS-CoV-2 by usurping resources to amplify non-neutralizing antibodies. Our data are consistent with these findings as we show correlations with the baseline level or boost of hCCCoV antibodies and levels of SARS-CoV-2- 2 antibodies after infection, which correlated with greater severity following SARS-CoV-2 infection. Mouse experiments further verified that existing hCCCoV immunity reduced neutralizing antibodies specific for the RBD. It is intriguing that hCCCoV antibodies are boosted following SARS-CoV-2 infection and show clear correlations with the SARS-CoV-2 antibody response yet do not significantly affect the incidence of becoming infected or symptom duration. As several factors contribute to susceptibility to SARS-CoV-2 infection, including age, gender, and underlying disease conditions (Fang et al., 2020), it may be difficult to detect the impact of hCCCoV immunity on self-reported symptoms among other confounding factors. It is important to note that the participants in this cohort were primarily Caucasian females with mild to moderate symptoms. Therefore, we were not able to thoroughly assess associations with more severe disease. Regardless, our data suggest that hCCCoV immunity may be an additional factor that can impede effective immunity to SARS-CoV-2 infection. Considering the continued circulation of SARS-CoV-2 variants, it will be important to further investigate mechanisms in which pre-existing immunity impacts the immune response to a novel, but related, virus.

Figure 7. Prior immunization with hCCCoV spike proteins limits the antibody responses to SARS-CoV-2 RBD

Prior studies investigating whether hCCCoV antibodies contributed to disease severity yielded particularly contradictory results. One main reason for these divergent conclusions is that most of the previous studies lacked baseline samples from the same individual before and after infection. Due to wide variation in hCCCoV antibody levels, it is not possible to accurately assess baseline hCCCoV immunity without analyzing samples from each individual prior to SARS-CoV-2 infection. Importantly, our data demonstrate that hCCCoV antibody levels can increase or decrease as early as 5 days after SARS-CoV-2 infection. Thus, samples taken after SARS-CoV-2 infection are not indicative of pre-existing hCCCoV immunity. Another factor contributing to the divergent conclusions is the composition and range of severity in the different cohorts. While most participants in the SJTRC cohort exhibited mild to moderate symptoms, other studies only included hospitalized individuals. Additionally, the antigens, antibody isotypes, and type of assays varied widely among the previous studies, which may also have influenced the inconsistency in results.

There is extensive cross-reactivity among antibodies specific for hCCCoVs (Ladner et al., 2020; Poston et al., 2021; Wec et al., 2020), and our data illustrate how serology may not be a reliable indicator of the hCCCoV to which an individual was most recently exposed. This is evident in the greater correlation between antibody isotypes specific for different hCCCoVs rather than an association with high levels of IgA, IgM, and IgG specific for a particular hCCCoV. Consistent with previous studies, we found that in older individuals, hCCCoV immunity is more biased toward IgA and IgG compared to IgM in younger individuals (Selva et al., 2021). Each time an individual is exposed to a hCCCoV, the memory B cells are further finetuned through affinity maturation and clonal selection to generate higher affinity hCCCoV-specific antibodies. Accordingly, as individuals age, repeated exposure to hCCCoVs creates a more specific and less adaptable repertoire of hCCCoV-specific memory B cells. Since SARS-CoV-2 is a novel virus that individuals had not encountered, it was unexpected that IgM antibodies did not precede IgG antibodies (Figure S3). These data are consistent with a previous report and suggest that the early immune response to SARS-CoV-2 is dominated by the reactivation of memory B cells generated during prior HCV infection (Dugan et al., 2021). We hypothesize that betacoronavirus IgG and IgA antibody levels are more indicative of an individual’s cumulative response rather than the timing of a recent infection. Accordingly, higher levels of betacoronavirus IgG and IgA antibodies imply a more narrow and less adaptable antibody repertoire, which would be advantageous for immunity to the hCCCoV but detrimental to the immune response to a novel coronavirus. Thus, although younger individuals may be exposed to hCCCoVs more often than older individuals, the hCCCoV IgM bias in younger participants is consistent with a more adaptable repertoire, which may explain why younger individuals exhibit less disease severity than older individuals. Although baseline hCCCoV antibody levels correlated with SARS-CoV-2 antibody levels following infection, we did not observe an association between baseline hCCCoV immunity and SARS-CoV-2 antibodies after vaccination. Many factors differ between the immune response to vaccination compared to infection. One possibility is that pre-existing hCCCoV antibodies may impede the generation of SARS-CoV-2 neutralizing antibodies, thereby extending viral exposure and enhancing the antibody response after infection. However, inhibition of neutralizing antibodies would not impact antigen load in the context of a vaccination, and therefore hCCCoV immunity would not have a similar impact on infection and vaccination. Alternatively, it is also possible that there is no correlation between baseline hCCCoV antibody levels and antibody levels following vaccination because the mRNA vaccines induce such a robust immune response to the SARS-CoV-2 spike protein that the ef-efficacy of these vaccines may override the effect of imprinting. Interestingly, a recent report showed that imprinting also led to divergent outcomes following influenza virus infection versus vaccination (Dugan et al., 2020). In summary, our data demonstrate that SARS-CoV-2 infection and vaccination activate existing memory B cells specific for honors. Baseline levels of HCV antibodies and the magnitude that these antibodies increased after infection or vaccination varied dramatically among individuals. Higher baseline levels or an increase of betacoronavirus IgG and IgA after infection were associated with increased SARS-CoV-2 antibody levels, which correlated with greater disease severity. These findings suggest that similar to the influenza virus, prior exposure to coronaviruses with sufficient homology can hinder the immune response to a novel coronavirus.

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