Boosting of Cross-Reactive Antibodies to Endemic Coronaviruses by SARS-CoV-2 Infection but not Vaccination with Stabilized Spike

Abstract

Pre-existing antibodies to endemic coronaviruses (CoV) that cross-react with SARS-CoV-2 have the potential to influence the antibody response to COVID-19 vaccination and infection for better or worse. In this observational study of mucosal and systemic humoral immunity in acutely infected, convalescent, and vaccinated subjects, we tested for cross reactivity against endemic CoV spike (S) protein at subdomain resolution. Elevated responses, particularly to the β-CoV OC43, were observed in all natural infection cohorts tested and were correlated with the response to SARS-CoV-2. The kinetics of this response and isotypes involved suggest that infection boosts preexisting antibody lineages raised against prior endemic CoV exposure that cross react. While further research is needed to discern whether this recalled response is desirable or detrimental, the boosted antibodies principally targeted the better conserved S2 subdomain of the viral spike and were not associated with neutralization activity. In contrast, vaccination with a stabilized spike mRNA vaccine did not robustly boost cross-reactive antibodies, suggesting differing antigenicity and immunogenicity. In sum, this study provides evidence that antibodies targeting endemic CoV are robustly boosted in response to SARS-CoV-2 infection but not to vaccination with stabilized S, and that depending on conformation or other factors, the S2 subdomain of the spike protein triggers a rapidly recalled, IgG-dominated response that lacks neutralization activity.

Figure 1.. Sequence and structural differences between the spike ectodomain of SARS-CoV-2 and endemic strains.

A. Sequence alignment of SARS-CoV-2 spike protein to other human coronaviruses. Consensus identity is shown on a scale from red (least conserved) to green (most conserved). Color bars are used to indicate different regions of the spike protein: N-terminal domain (NTD, blue), receptor binding domain (RBD, green) in the S1 domain and fusion peptide (FP, cyan), heptad repeat 1 (HR1, yellow), central helix (CH, orange), and connector domain (CD, purple) in the S2 domain. B. Structural model of the spike protein monomer colored by percent sequence conservation across deposited coronaviridae sequences shown as a ribbon model for one protomer (left) and spacefill for the spike trimer (right). C. Superimposed structural model of the spike protein S1 (left) and S2 (right) domains for SARS-CoV-2 (black) and OC43 (teal).

Figure 2.. SARS-CoV-2 infection is associated with elevated IgG and IgA responses to endemic CoV.

A. IgG responses in serum (top), nasal wash (middle), and stool (bottom) across antigens from CoV-2, OC43, and other endemic CoV S, and S1 proteins in the DHMC convalescent cohort. Samples from naïve subjects are indicated in gray, SARS-CoV-2 convalescents at one month post infection in blue, and buffer blanks in hollow circles. B. Volcano plot of fold change and significance (unpaired t test) of differences between antibody responses observed in convalescent subjects of the DHMC (left) and JHMI (right) cohorts one month post infection and naïve subjects in serum (top) and nasal wash (bottom). Each symbol represents an antibody response feature, with Fc domain characteristics represented by color and Fv antigen-specificity indicated by shape. Dotted horizontal line illustrates p = 0.05. Statistical significance was defined by Mann-Whitney U test.

Figure 3:. Response kinetics and isotype profiles suggest in acute and pre and post infection samples suggest recall of pre-existing, cross-reactive antibodies.

A. OC43 and CoV-2 specific IgM (orange), IgA (purple), and IgG (blue) responses in ten acutely infected subjects (color) two weeks post infection (WPI) as compared to naïve subjects (black). Statistical significance was assessed by unpaired t-test (*p<0.05, ***p<0.0005, ****p<0.00005). Fold change in means between groups are presented in inset. B. IgM (orange), IgA (purple), and IgG (blue) responses across CoV-2, other CoV, and control antigens in three subjects (indicated by shape) pre- (black) and post- (color) SARS-CoV-2 infection. Serum samples were taken 2 and 10 weeks post-infection (WPI).

Figure 4.. Correlative relationships between CoV-2 and OC43 specific antibody features.

A. Scatterplots of IgG responses specific to OC43 S, OC43 S2, and OC43 S-2P versus CoV-2 S2. Naïve subjects were excluded from calculations of correlative relationships. B. Correlations (RP) between IgG, IgA, and IgM specific to different stabilized SARS-CoV-2 spike and its subdomains with responses to OC43 S (maroon) and OC43 S-2P. Size and fill of symbols indicate statistical significance. Responses and relationships for naïve subjects are shown in black and convalescent donors shown in maroon (OC43 S), salmon (OC43 S2), and gray (OC43 S-2P).

Figure 5:. Poor neutralization activity of cross-reactive and S2-specific antibodies established by affinity purification.

A. Antigen binding profiles of IgG in unfractionated serum (load, black) and affinity-purified CoV-2 S-2P- (red, top) and OC43 S- (teal, bottom) fractions (eluate) from 30 SARS-CoV-2 convalescent subjects. Reactivity to CoV-2 S-2P, CoV-2 RBD, CoV-2 S2, OC43 S, and a control antigen (influenza HA) are reported. B. Antigen binding profiles of IgG (top) and IgM (bottom) in unfractionated serum (load, black) and affinity purified CoV-2 S2-specific (yellow) eluate. Reactivity to CoV-2 S2 (left) and OC43 S (right) are shown. For A-B, responses to the matched antigen (positive control) used in purification are indicated by green asterisks and to HA (negative control) antigen with red asterisks. Smoothed curves and 95% confidence intervals are shown for both eluate and load fractions. C. Neutralization activity of pooled elution fractions of antibodies affinity purified against CoV-2 S-2P (gray square), CoV-2 RBD (red triangle), CoV-2 S2 (yellow triangle), and OC43 S (teal circle). Error bars depict standard error of the mean across assay duplicates.

Figure 6:. Molecular cross-reactivity profiles of OC43 and SARS-CoV-specific antibody fractions across antigen specificities and Ig isotypes.

The degree of enrichment (area under the curve, AUC) of affinity purified SARS-CoV-2 RBD- (A), S-2P- (B), S2 domain- (C), or OC43 S- (D) specific antibodies of IgM (left), IgA (center), and IgG (right) isotypes across diverse CoV and control (ctl) proteins. Dotted line indicates no enrichment.

Figure 7:. Vaccination with stabilized spike does not result in robust boosting of endemic CoV responses.

IgG responses to SARS-CoV-2 (top) and OC43 (bottom) spike proteins. For each CoV strain, responses to S (left), the S2 domain (center), and stabilized S (S-2P, right) are shown. Responses in SARS-CoV-2 naïve subjects are indicated in black, SARS-CoV-2 infected subjects in light blue, and SARS-CoV-2 vaccinated (mRNA) subjects in dark blue. Pregnant subjects are indicated with triangles. Statistical significance by ANOVA with Dunnett’s correction (*p<0.05, **p<0.005, ****p<0.0001). Fold changes between mean response levels in seropositive and naïve cohorts are shown below each graph.