ABSTRACT
Antibody responses serve as the primary protection against SARS-CoV-2 infection through neutralization of viral entry into cells. We have developed a two-dimensional multiplex bead binding assay (2D-MBBA) that quantifies multiple antibody isotypes against multiple antigens from a single measurement. Here, we applied our assay to profile IgG, IgM and IgA levels against the spike antigen, its receptor-binding domain and natural and designed mutants. Machine learning algorithms trained on the 2D-MBBA data substantially improve the prediction of neutralization capacity against the authentic SARS-CoV-2 virus of serum samples of convalescent patients. The algorithms also helped identify a set of antibody isotype-antigen datasets that contributed to the prediction, which included those targeting regions outside the receptor-binding interface of the spike protein. We applied the assay to profile samples from vaccinated, immune-compromised patients, which revealed differences in the antibody profiles between convalescent and vaccinated samples. Our approach can rapidly provide deep antibody profiles and neutralization prediction from essentially a drop of blood without the need of BSL-3 access and provides insights into the nature of neutralizing antibodies. It may be further developed for evaluating neutralizing capacity for new variants and future pathogens.
ABSTRACT
The COVID-19 pandemic remains a global threat, and host immunity remains the main mechanism of protection against the disease. The spike protein on the surface of SARS-CoV-2 is a major antigen and its engagement with human ACE2 receptor plays an essential role in viral entry into host cells. Consequently, antibodies targeting the ACE2-interacting surface (ACE2IS) located in the receptor-binding domain (RBD) of the spike protein can neutralize the virus. However, the understanding of immune responses to SARS-CoV-2 is still limited, and it is unclear how the virus protects this surface from recognition by antibodies. Here, we designed an RBD mutant that disrupts the ACE2IS and used it to characterize the prevalence of antibodies directed to the ACE2IS from convalescent sera of 94 COVID19-positive patients. We found that only a small fraction of RBD-binding antibodies targeted the ACE2IS. To assess the immunogenicity of different parts of the spike protein, we performed in vitro antibody selection for the spike and the RBD proteins using both unbiased and biased selection strategies. Intriguingly, unbiased selection yielded antibodies that predominantly targeted regions outside the ACE2IS, whereas ACE2IS-binding antibodies were readily identified from biased selection designed to enrich such antibodies. Furthermore, antibodies from an unbiased selection using the RBD preferentially bound to the surfaces that are inaccessible in the context of whole spike protein. These results suggest that the ACE2IS has evolved less immunogenic than the other regions of the spike protein, which has important implications in the development of vaccines against SARS-CoV-2.
ABSTRACT
Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Here, we determine the ability of sera from 101 recovered healthcare workers to neutralize both authentic SARS-CoV-2 and SARS-CoV-2 pseudotyped virus and address their antibody titers against SARS-CoV-2 nucleoprotein and spike receptor-binding domain. Interestingly, the majority of individuals have low neutralization capacity and only 6% of the healthcare workers showed high neutralizing titers against both authentic SARS-CoV-2 virus and the pseudotyped virus. We found the antibody response to SARS-CoV-2 infection generates antigen-specific isotypes as well as a diverse combination of antibody isotypes, with high titers of IgG, IgM and IgA against both antigens correlating with neutralization capacity. Importantly, we found that neutralization correlated with antibody titers as quantified by ELISA. This suggests that an ELISA assay can be used to determine seroneutralization potential. Altogether, our work provides a snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides evidence that possessing multiple antibody isotypes may play an important role in SARS-CoV-2 neutralization.
