Chaves, F. | Search bioRxiv

bioRxiv2025

A funnel approach to enable analyses of epitope-specific human CD4 T cells specific for influenza and SARS-CoV-2.

Protection from pathogenic organisms relies heavily on the adaptive immune response, for which key regulators are CD4 T cells. CD4 T cells, notable in the complexity of their repertoire and functional potential, can most easily be dissected with the ability to identify, quantify, characterize and isolate epitope-specific cells. In the study reported here, we present a systematic and unbiassed strategy that has enabled identification of highly immunogenic peptide epitopes derived from influenza virus and SARS-CoV-2, presented by human HLA-DR proteins. Coupling the use of HLA-DR transgenic mice with infection and vaccination with highly sensitive epitope specific cytokine ELISpot assays, we have narrowed the potential epitopes from 450-600 peptides to 5-15 peptides, by an iterative process of elimination and selection which we have termed a funnel approach. These epitopes have been validated in HLA-DR typed human CD4 T cells directly ex vivo and enabled derivation and implementation of HLA-DR peptide tetramers. Tetramer staining of human PBMCs enriched CD4 T memory populations from healthy adult subjects highlighting this approach as a sensitive and specific method of identifying novel epitopes and subsequent CD4 T cell responses to human viral infections. ImportanceTracking single epitope-specific CD4 T cells enables sophisticated analyses of the human response to infectious pathogens, vaccines and probing the human CD4 T cell immune memory compartment. The studies presented here provide a unbiased strategy for accomplishing this goal and provide a verified compilation of candidate HLA-DR restricted CD4 T cell peptide epitopes for future studies by researchers in the field of human immunology.

immunology↗

bioRxiv2020

S protein-reactive IgG and memory B cell production after human SARS-CoV-2 infection includes broad reactivity to the S2 subunit

The high susceptibility of humans to SARS-CoV-2 infection, the cause of COVID-19, reflects the novelty of the virus and limited preexisting B cell immunity. IgG against the SARS-CoV-2 spike (S) protein, which carries the novel receptor binding domain (RBD), is absent or at low levels in unexposed individuals. To better understand the B cell response to SARS-CoV-2 infection, we asked whether virus-reactive memory B cells (MBCs) were present in unexposed subjects and whether MBC generation accompanied virus-specific IgG production in infected subjects. We analyzed sera and PBMCs from non-SARS-CoV-2-exposed healthy donors and COVID-19 convalescent subjects. Serum IgG levels specific for SARS-CoV-2 proteins (S, including the RBD and S2 subunit, and nucleocapsid [N]) and non-SARS-CoV-2 proteins were related to measurements of circulating IgG MBCs. Anti-RBD IgG was absent in unexposed subjects. Most unexposed subjects had anti-S2 IgG and a minority had anti-N IgG, but IgG MBCs with these specificities were not detected, perhaps reflecting low frequencies. Convalescent subjects had high levels of IgG against the RBD, S2, and N, together with large populations of RBD- and S2-reactive IgG MBCs. Notably, IgG titers against the S protein of the human coronavirus OC43 in convalescent subjects were higher than in unexposed subjects and correlated strongly with anti-S2 titers. Our findings indicate cross-reactive B cell responses against the S2 subunit that might enhance broad coronavirus protection. Importantly, our demonstration of MBC induction by SARS-CoV-2 infection suggests that a durable form of B cell immunity is maintained even if circulating antibody levels wane. IMPORTANCERecent rapid worldwide spread of SARS-CoV-2 has established a pandemic of potentially serious disease in the highly susceptible human population. Key questions are whether humans have preexisting immune memory that provides some protection against SARS-CoV-2 and whether SARS-CoV-2 infection generates lasting immune protection against reinfection. Our analysis focused on pre- and post-infection IgG and IgG memory B cells (MBCs) reactive to SARS-CoV-2 proteins. Most importantly, we demonstrate that infection generates both IgG and IgG MBCs against the novel receptor binding domain and the conserved S2 subunit of the SARS-CoV-2 spike protein. Thus, even if antibody levels wane, long-lived MBCs remain to mediate rapid antibody production. Our study also suggests that SARS-CoV-2 infection strengthens preexisting broad coronavirus protection through S2-reactive antibody and MBC formation.

immunology↗