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Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to affect the quality of the antibody response focusing it to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and three variants of concern (VOC), B.1.351, B.1.617.2, and BA.1. We demonstrate that immunogens built on structure selection can be used to influence the quality of the antibody response by focusing it to a conserved site of vulnerability shared between wildtype virus and VOCs, resulting in neutralizing antibodies across variants.

Understanding adaptive immunity against SARS-CoV-2 is a major requisite for the development of effective vaccines and treatments for COVID-19. CD4+ T cells play an integral role in this process primarily by generating antiviral cytokines and providing help to antibody-producing B cells. To empower detailed studies of SARS-CoV-2-specific CD4+ T cell responses in mouse models, we comprehensively mapped I-Ab-restricted epitopes for the spike and nucleocapsid proteins of the BA.1 variant of concern via IFNγ ELISpot assay. This was followed by the generation of corresponding peptide:MHCII tetramer reagents to directly stain epitope-specific T cells. Using this rigorous validation strategy, we identified 6 immunogenic epitopes in spike and 3 in nucleocapsid, all of which are conserved in the ancestral Wuhan strain. We also validated a previously identified epitope from Wuhan that is absent in BA.1. These epitopes and tetramers will be invaluable tools for SARS-CoV-2 antigen-specific CD4+ T cell studies in mice. [ABSTRACT FROM AUTHOR]

SARS-CoV-2-reactive T cells are detected in some healthy unexposed individuals. Human studies indicate these T cells could be elicited by the common cold coronavirus OC43. To directly test this assumption and define the role of OC43-elicited T cells that are cross-reactive with SARS-CoV-2, we develop a model of sequential infections with OC43 followed by SARS-CoV-2 in HLA-B*0702 and HLA-DRB1*0101 Ifnar1−/− transgenic mice. We find that OC43 infection can elicit polyfunctional CD8+ and CD4+ effector T cells that cross-react with SARS-CoV-2 peptides. Furthermore, pre-exposure to OC43 reduces subsequent SARS-CoV-2 infection and disease in the lung for a short-term in HLA-DRB1*0101 Ifnar1−/− transgenic mice, and a longer-term in HLA-B*0702 Ifnar1−/− transgenic mice. Depletion of CD4+ T cells in HLA-DRB1*0101 Ifnar1−/− transgenic mice with prior OC43 exposure results in increased viral burden in the lung but no change in virus-induced lung damage following infection with SARS-CoV-2 (versus CD4+ T cell-sufficient mice), demonstrating that the OC43-elicited SARS-CoV-2 cross-reactive T cell-mediated cross-protection against SARS-CoV-2 is partially dependent on CD4+ T cells. These findings contribute to our understanding of the origin of pre-existing SARS-CoV-2-reactive T cells and their effects on SARS-CoV-2 clinical outcomes, and also carry implications for development of broadly protective betacoronavirus vaccines. The origin of SARS-CoV-2 cross-reactive T cells in unexposed humans is unclear. Here, the authors use HLA transgenic mouse models of sequential infections with human coronavirus OC43 and SARSCoV-2 and show that OC43 elicits cross-protective immunity against SARS-CoV-2, which partially depends on CD4 + T cells. [ABSTRACT FROM AUTHOR]

Abstract: Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV), two members of the Pneumoviridae family, account for the majority of severe lower respiratory tract infections worldwide in very young children. They are also a frequent cause of morbidity and mortality in the elderly and immunocompromised adults. High levels of neutralizing antibodies, mostly directed against the viral fusion (F) glycoprotein, correlate with protection against either hRSV or hMPV. However, no cross‐neutralization is observed in polyclonal antibody responses raised after virus infection or immunization with purified F proteins. Based on crystal structures of hRSV F and hMPV F, we designed chimeric F proteins in which certain residues of well‐characterized antigenic sites were swapped between the two antigens. The antigenic changes were monitored by ELISA with virus‐specific monoclonal antibodies. Inoculation of mice with these chimeras induced polyclonal cross‐neutralizing antibody responses, and mice were protected against challenge with the virus used for grafting of the heterologous antigenic site. These results provide a proof of principle for chimeric fusion proteins as single immunogens that can induce cross‐neutralizing antibody and protective responses against more than one human pneumovirus. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has accumulated more than 700 million infection cases and 6.9 million deaths. New variants have affecte d antibody interaction with the surface spike protein. We defined the domain specificities and measured hexa-proline stabilized spike protein (HexaPro) binding kinetics of a large panel of antibodies sourced by the Coronavirus Immunotherapeutics Consortium. Epitope binning analysis of antibodies competing for HexaPro binding separated the fine specificities of the majority of antibodies to four regions: top, outer, mesa/valley, or cryptic site of receptor binding domain (RBD). Most of the topRBDspecific antibodies showed >3-fold loss of binding and authentic-virus neutralization activity for the B.1.351 variant. Remarkably, among RBD mesa/valleyspecific or crypticsitespecific antibodies, 55% showed >3-fold stronger affinities, and at least 60% maintained neutralization activity for the B.1.351 variant. These data also highlighted the diversity of SARSCoV2specific antibodies that retain high spike affinities and antiviral functions across variants. [ABSTRACT FROM AUTHOR]

We illustrate the utility of native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), for the characterization of protein complex topology and glycoprotein heterogeneity. ECCR efficiently reduces the charge states of tetradecameric GroEL, illustrating Orbitrap m/z measurements to greater than 100,000 m/z . For pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) reduces the charge states and yields more topologically informative fragmentation. This is the first demonstration that ECCR yields more native-like SID fragmentation. ECCR also significantly improved mass and glycan heterogeneity measurements of heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer. Protein glycosylation is important for structural and functional properties and plays essential roles in many biological processes. The immense heterogeneity in glycosylation sites and glycan structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Without ECCR, average mass determination of glycoprotein complexes is available only through charge detection mass spectrometry or mass photometry. With narrow m/z selection windows followed by ECCR, multiple glycoform m/z values are apparent, providing quick global glycoform profiling and providing a future path for glycan localization on individual intact glycoforms.

Understanding adaptive immunity against SARS-CoV-2 is a major requisite for the development of effective vaccines and treatments for COVID-19. CD4 + T cells play an integral role in this process primarily by generating antiviral cytokines and providing help to antibody-producing B cells. To empower detailed studies of SARS-CoV-2-specific CD4 + T cell responses in mouse models, we comprehensively mapped I-A b -restricted epitopes for the spike and nucleocapsid proteins of the BA.1 variant of concern via IFNγ ELISpot assay. This was followed by the generation of corresponding peptide:MHCII tetramer reagents to directly stain epitope-specific T cells. Using this rigorous validation strategy, we identified 6 immunogenic epitopes in spike and 3 in nucleocapsid, all of which are conserved in the ancestral Wuhan strain. We also validated a previously identified epitope from Wuhan that is absent in BA.1. These epitopes and tetramers will be invaluable tools for SARS-CoV-2 antigen-specific CD4 + T cell studies in mice., Competing Interests: GG receives research funding from Merck and Moderna. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bricio-Moreno, Barreto de Albuquerque, Neary, Nguyen, Kuhn, Yeung, Hastie, Landeras-Bueno, Olmedillas, Hariharan, Nathan, Getz, Gayton, Khatri, Gaiha, Ollmann Saphire, Luster and Moon.)