Assessment of Immunogenic and Antigenic Properties of Recombinant Nucleocapsid Proteins of Five SARS-CoV-2 Variants in a Mouse Model.

COVID-19 cases caused by new variants of highly mutable SARS-CoV-2 continue to be identified worldwide. Effective control of the spread of new variants can be achieved through targeting of conserved viral epitopes. In this regard, the SARS-CoV-2 nucleocapsid (N) protein, which is much more conserved than the evolutionarily influenced spike protein (S), is a suitable antigen. The recombinant N protein can be considered not only as a screening antigen but also as a basis for the development of next-generation COVID-19 vaccines, but little is known about induction of antibodies against the N protein via different SARS-CoV-2 variants. In addition, it is important to understand how antibodies produced against the antigen of one variant can react with the N proteins of other variants. Here, we used recombinant N proteins from five SARS-CoV-2 strains to investigate their immunogenicity and antigenicity in a mouse model and to obtain and characterize a panel of hybridoma-derived monoclonal anti-N antibodies. We also analyzed the variable epitopes of the N protein that are potentially involved in differential recognition of antiviral antibodies. These results will further deepen our knowledge of the cross-reactivity of the humoral immune response in COVID-19.

Development of a T Cell-Based COVID-19 Vaccine Using a Live Attenuated Influenza Vaccine Viral Vector.

The COVID-19 pandemic emerged in 2020 and has caused an unprecedented burden to all countries in the world. SARS-CoV-2 continues to circulate and antigenically evolve, enabling multiple reinfections. To address the issue of the virus antigenic variability, T cell-based vaccines are being developed, which are directed to more conserved viral epitopes. We used live attenuated influenza vaccine (LAIV) virus vector to generate recombinant influenza viruses expressing various T-cell epitopes of SARS-CoV-2 from either neuraminidase (NA) or non-structural (NS1) genes, via the P2A self-cleavage site. Intranasal immunization of human leukocyte antigen-A*0201 (HLA-A2.1) transgenic mice with these recombinant viruses did not result in significant SARS-CoV-2-specific T-cell responses, due to the immunodominance of NP366 influenza T-cell epitope. However, side-by-side stimulation of peripheral blood mononuclear cells (PBMCs) of COVID-19 convalescents with recombinant viruses and LAIV vector demonstrated activation of memory T cells in samples stimulated with LAIV/SARS-CoV-2, but not LAIV alone. Hamsters immunized with a selected LAIV/SARS-CoV-2 prototype were protected against challenge with influenza virus and a high dose of SARS-CoV-2 of Wuhan and Delta lineages, which was confirmed by reduced weight loss, milder clinical symptoms and less pronounced histopathological signs of SARS-CoV-2 infection in the lungs, compared to LAIV- and mock-immunized animals. Overall, LAIV is a promising platform for the development of a bivalent vaccine against influenza and SARS-CoV-2.

Detection of IFNγ-Secreting CD4+ and CD8+ Memory T Cells in COVID-19 Convalescents after Stimulation of Peripheral Blood Mononuclear Cells with Live SARS-CoV-2.

BACKGROUND: New coronavirus SARS-CoV-2, a causative agent of the COVID-19 pandemic, has been circulating among humans since November 2019. Multiple studies have assessed the qualitative and quantitative characteristics of virus-specific immunity in COVID-19 convalescents, however, some aspects of the development of memory T-cell responses after natural SARS-CoV-2 infection remain uncovered. METHODS: In most of published studies T-cell immunity to the new coronavirus is assessed using peptides corresponding to SARS-CoV-1 or SARS-CoV-2 T-cell epitopes, or with peptide pools covering various parts of the viral proteins. Here, we determined the level of CD4+ and CD8+ memory T-cell responses in COVID-19 convalescents by stimulating PBMCs collected 1 to 6 months after recovery with sucrose gradient-purified live SARS-CoV-2. IFNγ production by the central and effector memory helper and cytotoxic T cells was assessed by intracellular cytokine staining assay and flow cytometry. RESULTS: Stimulation of PBMCs with live SARS-CoV-2 revealed IFNγ-producing T-helper effector memory cells with CD4+CD45RA-CCR7- phenotype, which persisted in circulation for up to 6 month after COVID-19. In contrast, SARS-CoV-2-specific IFNγ-secreting cytotoxic effector memory T cells were found at significant levels only shortly after the disease, but rapidly decreased over time. CONCLUSION: The stimulation of immune cells with live SARS-CoV-2 revealed a rapid decline in the pool of effector memory CD8+, but not CD4+, T cells after recovery from COVID-19. These data provide additional information on the development and persistence of cellular immune responses after natural infection, and can inform further development of T cell-based SARS-CoV-2 vaccines.