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Browsing by Subject "SARS-CoV-2"

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  • Structural analysis of SARS-CoV-2 neutralization by a spike-targeting antigen-binding fragment from a patient-derived monoclonal antibody 
    Jayachandran, Rupesh Balaji (2022)
    COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has thus far claimed over six million lives. Vaccines against SARS-CoV-2 have successfully mitigated severe disease and eased the burden on healthcare systems. Neutralizing antibodies play crucial roles both in vaccine derived immunity, and as drugs widely utilized in monoclonal antibody therapy. Neutralizing antibodies primarily target the spike protein, where most of the neutralizing epitopes are located in the receptor binding domain (RBD). Elucidating the sites of vulnerability to neutralization on SARS-CoV-2 can facilitate the development of therapeutics. 7A12 is a newly-discovered IgG antigen-binding fragment (Fab) isolated from a COVID-19 patient in Finland that can neutralize SARS-CoV-2 by targeting the spike protein. In this thesis, a complex of the Fab 7A12 with SARS-CoV-2 spike ectodomain trimer was studied using cryogenic electron microscopy (cryo-EM) single-particle analysis to elucidate the epitope of 7A12 and to gain insight into the neutralization mechanism of 7A12. Cryo-EM data of the complex revealed that Fab 7A12 can bind to both “open” and “closed” conformations of RBD. Rigid-body fitting of the spike trimer and Fab 7A12 models into the cryo-EM density indicates that 7A12 recognizes an epitope in the RBD which is mainly located outside the ACE2 binding site. Together, these results suggest that the 7A12 epitope belongs to class III of SARS-CoV-2 neutralizing epitopes located in the RBD, indicating that 7A12 could neutralize by sterically hindering ACE2 and by preventing the adjacent RBD from changing to ”up” conformation. Furthermore, our results revealed an overlap of 7A12 epitope with the putative binding site of heparan sulfate, a host factor of SARS-CoV-2 infection, which might contribute to neutralization. 7A12-RBD interface mapping delineated the residues comprising the epitope, which do not include mutants found in Beta, Gamma, and Delta variants, while four mutants were found in Omicron within the epitope indicating that 7A12 is likely to neutralize Beta, Gamma, and Delta variants of concern.
  • Vaccine- and infection-induced cellular immunity against SARS-CoV-2 in human 
    Vara, Saimi (2023)
    In this thesis, I studied T cell responses to SARS-CoV-2 structural proteins in subjects who had been both vaccinated and infected (n=30), who had only been infected (n=22), and as controls, in subjects who had been neither vaccinated nor infected (n=6). In addition, I compared cellular responses between groups of subjects who had been infected with either wild-type (WT) SARS-CoV-2, Alpha (B.1.1.7), or Beta (B.1.351) variants. Before analyzing the samples to be studied, I optimized the conditions for the cell stimulations. Peripheral blood mononuclear cells (PBMCs) were collected from infected subjects six months after infection. PBMCs were stimulated with SARS-CoV-2 wild-type nucleoprotein, spike-, envelope-, and membrane protein peptide pools. I quantified cytokines and effector molecules characteristic of CD4+ and CD8+ cell responses; perforin, tumor necrosis factor alpha (TNF-a), granzyme B, interferon gamma (IFN-γ), interleukin 2 (IL-2) and interleukin 4 (IL-4) secreted by PBMCs were quantified. In this study, I found that subjects with infection, or combination of infection and vaccination had higher cellular immune responses compared to uninfected controls. Infection induced higher granzyme B, IFN-y, and IL-2 secretion, and the combination of infection and vaccination induced higher granzyme B, perforin, IFN-y, IL-2 and IL-4 secretion. I found that subjects with hybrid immunity, defined as immunity acquired from combined vaccination and infection, had on average higher IL-4 responses compared to those who had been infected only. In this study, I found that nucleoprotein, spike-, and membrane proteins stimulated T cell responses whereas envelope protein did not stimulate T cell responses. I found that WT, Alpha or Beta infection produced equally good T cell responses to WT spike peptide. In conclusion, I found that COVID-19 patients have long-lasting T cell responses. I found that T cells recognize different SARS-CoV-2 variants. Mutations present in the spike proteins of the different variants do not affect T-cell ability to recognize these antigens. Immunity based on T cells is not as susceptible to antigenic changes as the humoral immunity. T cells have a vital role in protection against variants, when new SARS-CoV-2 variants evaded antibody-based immunity.

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