Most research on immunity to the SARS-CoV-2 virus and Covid-19 vaccine development has focused on antibody responses to the spike protein and other viral surface proteins. But antibodies that recognize the virus’s internal proteins could also be important for immunity and disease outcomes, according to a new study led by University of Pittsburgh, Georgia Institute of Technology, and Emory University researchers.

In the study, online now in Cell Reports, the team performed the most comprehensive analysis to date of Covid-19 antibodies in a small set of patients with severe disease. They found that antibody profiles of internal viral proteins, including those conserved across coronaviruses, predicted which patients survived or died just as well as corresponding profiles for surface proteins, suggesting that targeting other parts of the virus beyond the spike protein could be important for enhancing Covid-19 vaccines and therapies.

“The novel aspect of this study is that we conducted very deep profiling of SARS-CoV-2 antibodies and looked at many different aspects of these antibodies,” said co-senior author Jishnu Das, assistant professor of immunology and of computational and systems biology in Pitt’s School of Medicine. “The whole world has been focused on the spike protein and the receptor binding domain, but this study is the first concrete evidence that specific antibodies against internal proteins are also positively associated with survival in severe Covid-19.”

When the immune system encounters a virus, it produces antibodies that help neutralize and clear the infection. Each antibody specifically recognizes just one antigen, often a viral protein. Most Covid-19 immunity research has focused on the spike and other surface proteins, which form the virus’s outer coat, but beyond these so-called “canonical antigens,” SARS-CoV-2 has about 25 other internal proteins.

To see whether immune responses to these non-canonical antigens could predict survival outcomes in patients with severe Covid-19, Das teamed up with co-senior authors Aniruddh Sarkar, assistant professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory, and Harinder Singh, professor of immunology and the director of the Center for Systems Immunology at Pitt.

The researchers analyzed blood samples that had been collected from 21 patients who were hospitalized with severe Covid-19 in 2020 — prior to the approval of vaccines. Seven of these patients died from the disease, and the other 14 survived. Using a microscale antibody profiling platform developed by Sarkar, the team comprehensively analyzed antibodies to three canonical and four non-canonical antigens.

According to Sarkar, the platform analyzes three key features of antibodies. One is antigen specificity, or what the antibody is binding to. The second is effector function, which relates to the antibody’s role in immune response. The third feature is glycosylation, or the addition of carbohydrate molecules to the antibody, which dramatically impacts antibody function.

“By simultaneously profiling these three features, we can get a far deeper understanding of a given antibody than just looking at antibody titers,” Sarkar explained.