key: cord-0789576-zwxtbena authors: Butler, S. E.; Crowley, A. R.; Natarajan, H.; Xu, S.; Weiner, J. A.; Lee, J.; Wieland-Alter, W. F.; Connor, R. I.; Wright, P. F.; Ackerman, M. E. title: Features and Functions of Systemic and Mucosal Humoral Immunity Among SARS-CoV-2 Convalescent Individuals date: 2020-08-06 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2020.08.05.20168971 sha: 3bff7d11d724e290589425ff426757cf88388328 doc_id: 789576 cord_uid: zwxtbena Understanding humoral immune responses to SARS-CoV-2 infection will play a critical role in the development of vaccines and antibody-based interventions. We report systemic and mucosal antibody responses in convalescent individuals who experienced varying disease severity. Robust antibody responses to diverse SARS-CoV-2 antigens and evidence of elevated responses to endemic CoV were observed among convalescent donors. SARS-CoV-2-specific IgA and IgG responses were often negatively correlated, particularly in mucosal samples, suggesting subject-intrinsic biases in isotype switching. Assessment of antibody-mediated effector functions revealed an inverse correlation between systemic and mucosal neutralization activity and site-dependent differences in the isotype of neutralizing antibodies. Serum neutralization correlated with systemic anti-SARS-CoV-2 IgG and IgM response magnitude, while mucosal neutralization was associated with nasal SARS-CoV-2-specific IgA. These findings begin to map how diverse Ab characteristics relate to Ab functions and outcomes of infection, informing public health assessment strategies and vaccine development efforts. functions of Abs and T cells may be needed to contribute to protection from infection or disease. As a 98 result, there is concern that neither vaccines nor prior infection will be highly effective, that any protective 99 effects may have limited durability, and that the most vulnerable individuals, such as the 100 immunocompromised, elderly, and those with co-morbidities, may have an inadequate level of CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168971 doi: medRxiv preprint (unpaired two-tailed t-test with Welch's correction, p < 0.0001, p = 0.011 respectively). This apparent 130 boosting of responses to endemic CoV was observed more broadly among IgG1 and IgG3 subclass 131 responses (Supplemental Fig. 1) . Ab responses in serum and nasal wash samples were further examined 132 by measuring levels of other Ab isotypes, subclasses, and by defining binding to diverse FcRs (Fig. 1B, Supplemental Fig. 2-6) . SARS CoV-2-specific IgG1, IgG2, IgG3, IgA, IgM, and Abs able to ligate FcRs (FcaR, To understand how aspects of the humoral response relate to each other, hierarchical clustering 142 was performed on Ab features to define similarities among subjects and features (Supplemental Figures 143 9-10) . When focusing on those features that were significantly increased among convalescent donors 144 (unpaired two-tailed t-test with Welch's correction, p < 0.05), elevated IgG responses were observed in 145 both serum and nasal samples in convalescent donors who had experienced severe disease (Fig. 2) . In 146 contrast, elevated nasal IgA responses were apparent among donors with mild or moderate disease. When correlations between Ab types and specificities elicited among convalescent donors were plotted 148 for serum and nasal samples, clear patterns emerged suggesting biases between IgA and IgG responses. For example, in serum, IgG1, IgG3 and FcgR-binding Ab responses were well correlated with each other 150 across diverse specificities, as were IgA, IgA1, and IgA2 and FcaR-binding Abs (Fig. 3A) . Correlations 151 between these isotypes were more modest, though overall in serum, many response features were 152 positively correlated with each other. In contrast, nasal responses showed clear evidence of a bias to favor 153 either IgG or IgA, as striking inverse correlations were observed between these isotypes across diverse 154 antigen specificities (Fig. 3B) . CoV-2 pseudovirus assay (Letko et al., 2020) . Consistent with other studies (Klein et al., 2020) , elevated 166 serum neutralization activity was observed for subjects who experienced severe, as compared to non-167 severe, disease (unpaired two-tailed t-test with Welch's correction, p = 0.034) (Fig. 5a) . In contrast to 168 observations in serum, nasal samples from subjects with severe disease showed little to no viral 169 neutralization, whereas subjects with elevated mucosal neutralization activity tended to have 170 experienced mild or moderate symptoms (Fig. 5b) . Indeed, nasal and serum neutralization activities 171 exhibited an inverse relationship (Fig. 5c) . This observation further suggests the potential importance of (ADCP) and neutrophil (ADNP) effector cells (Fig 5a) . While nasal wash samples were far less capable of 181 driving functional activity, a number of subjects exhibited nasal Ab responses able to elicit phagocytosis 182 in monocytes (Fig. 5b) . Serum from these subjects also tended to generate a strong phagocytic response 183 (Fig 5c) . Across phagocytosis, NK cell FcgR3a receptor ligation (ADCC), and complement cascade protein 184 C3b deposition (ADCD), a pattern of elevated Ab effector function emerged among subjects who 185 experienced moderate or severe disease; those who experienced mild disease generated little activity. In contrast to serum, but consistent with the lower relative levels of IgG Abs present in nasal wash 187 samples, limited nasal ADNP, ADCC, and ADCD activity was observed (Fig. 5b) . Comparison of serum and 188 nasal effector functions showed positive correlation for ADCP activity, consistent with the observation 189 that subjects with severe disease and high serum IgG also tended to exhibit high nasal IgG, and the known 190 dependence of monocyte phagocytosis on IgG-binding FcgR (Fig. 5c) . Ab features correlated with Ab functions 193 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168971 doi: medRxiv preprint Next we established the characteristics of the Abs mediating each function by measuring the 194 degree and direction of correlation between RBD-specific Ab biophysical features and Ab functions in 195 serum (Fig. 6a) . Consistent with their reliance on FcgR, ADCP, ADNP, and ADCC activities were most 196 strongly correlated with FcgR binding, but also with levels of IgG1 and IgG3, which ligate FcgR best among 197 human IgG subclasses. Interestingly, IgM positively correlated with both ADNP and neutralization activity, 198 though the mechanistic relevance of each observed association is unclear. While IgA responses were 199 robustly induced in serum, this isotype was generally more weakly associated with neutralization and 200 effector activities. In nasal samples, however, neutralization activity was strongly correlated with the IgA response 202 ( Fig. 6a . ADCP showed significant correlation with total RBD-specific IgG and FcgR-binding RBD-specific Fig. 12 ). This elevation was also evident in nasal samples (Fig. 7A) . Critically, RBD-specific Ab binding to 220 FcaR was found to be significantly elevated in the nasal wash samples from subjects who had experienced 221 mild or moderate as opposed to severe disease (Fig. 7A) , and a number of other IgA-related responses 222 exhibited differences near the arbitrary significance threshold (Supplemental Fig. 11 ). When examining 223 the relationships between these features among donors who recovered from mild, moderate, and severe 224 disease, IgG-related features typically showed a uniformly increasing magnitude with increasing disease 225 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168971 doi: medRxiv preprint severity (Fig. 7B) . In contrast, the IgA-associated feature defined by nasal RBD-specific Abs binding to FcaR 226 ( Fig. 7C ) and its correlated function neutralization (Fig. 7D) were lowest in subjects with either mild or 227 severe disease, and elevated among those who recovered from moderate illness. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. Separate from the dichotomy of whether Abs serve a protective or pathogenic role, we observed 278 that the characteristics of the responses observed in serum and nasal samples tended to be highly distinct. Not only did the Ab profile of the nasal wash samples from individual subjects tend to favor either IgG or in light of the observation that those subjects whose nasal specimens had the greatest neutralization 288 potency also tended to report experiencing only mild or moderate symptoms. It is important to note 289 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168971 doi: medRxiv preprint however that the available cohort was not large enough to adequately power a robust examination of this Polio is a particularly informative model in this respect as the mucosally administered form of the vaccine 319 is capable of providing sterilizing immunity -at the expense of a risk of reversion to virulence -while the 320 systemically administered form fails to induce mucosal immunity and thus serves primarily to protect the 321 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168971 doi: medRxiv preprint chromatography according to the manufacturers' protocols ( Supplemental Tables 1 and 2) . Human FcgR 353 were expressed and purified as described previously (Boesch et al., 2014) . Fc array assay: CoV and control antigens, including S trimers, S subdomains (i.e., S1 and S2), and other viral 357 proteins from SARS-CoV-2 as well as S and S subdomains from SARS CoV-1, MERS, HKU1, OC43, NL63, 358 229E, and WIV1 (Supplemental Table 2 ) and influenza HA and herpes simplex virus (HSV) gE proteins were 359 covalently coupled to Luminex Magplex magnetic microspheres using a two-step carbodiimide chemistry is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168971 doi: medRxiv preprint Antibody-dependent Complement Deposition (ADCD) was quantified essentially as previously 415 described (Fischinger et al., 2019) . In brief, serum and nasal samples were heat-inactivated at 56°C for 30 416 min prior to a 2 hr incubation at a dilution of 1:20 at RT with multiplex assay microspheres. After washing, CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168971 doi: medRxiv preprint 481 482 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168971 doi: medRxiv preprint 488 489 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168971 doi: medRxiv preprint 495 496 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168971 doi: medRxiv preprint 504 505 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted August 6, 2020. . https://doi.org/10.1101/2020.08.05.20168971 doi: medRxiv preprint 513 514 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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