Study of the antigenic specificity of T-cell immune reactions in response to immunization of laboratory mice with a recombinant adenoviral vector encoding the Spike-protein of SARS-CoV-2

Introduction.Recombinant adenoviral vectors become the leading technological platform in the development and production of modern vaccines.In Russia and other countries of the world, vaccines based on recombinant adenoviruses have been designed and registered against the Ebola virus and SARS-CoV-2 coronavirus infections.Clinical trials of vaccines against infl uenza, Marburg virus, human papillomaviruses are ongoing.The target antigen encoded in the DNA of the adenoviral vector is expressed in the body of the vaccine recipient, and adaptive immune responses develop against this target antigen protein.The vector particle is viral and contains dozens of viral antigens.Therefore, along with immune responses to the encoded target antigen, immune responses to the antigens of the vector itself can develop in the body of the vaccinated.The aim of this work is to investigate the intensity and quality of two immune responses that are different in their antigenic specifi city, i.e. directed against the target coronavirus antigen and adenovirus vector antigens. Material and methods.In C57BL/6 mice, the intensity of immune responses of CD4-and CD8-T-cells was studied in response to immunization with a recombinant adenoviral vector encoding the SARS-CoV-2 S-protein (Ad5-S). 2 and 3 months after immunization, the number and antigenic specifi city of CD4-and CD8-T memory cells were determined in the spleen of mice.The T-cell response was induced in vitro in co-culture with antigen-presenting dendritic cells.Purifi ed CD4-and CD8-T-cell populations were obtained by sorting on a BD FACS Aria II laser fl ow sorter machine.Antigen-presenting cells were transduced with the Ad5-S adenoviral vector encoding the SARS-CoV-2 S-protein or a control recombinant adenoviral vector without a target insert (Ad5-0).The T cells response was determined by ELISPOT according to the number of cells secreting IFN-γ.In some experiments, to reactivate CD4 T cells, antigen-presenting dendritic cells were loaded with a recombinant RBD-fragment of the SARS-CoV-2 S-protein.To enhance the T-cell response, antigen presenting dendritic cells were stimulated with a TLR4 agonist.Results.A single intranasal immunization with the Ad5-S vector at a dose of 10 8 PFU induced strong systemic T-cell immune responses in C57BL/6 mice.Two months after immunization, about 100 000-200 000 antigen-reactive memory T-cells were found in the spleen of mice, secreting IFN-γ when reactivated in vitro by dendritic cells presenting the target SARS-CoV-2 S-antigen.Most antigen-reactive CD8-T memory cells were specifi c for the SARS-CoV-2 S-antigen.The concentration of such cells after immunization with the Ad5-S vector exceeds 1 % of all CD8-T cells.The number of antigen-reactive CD8-T memory cells specifi c to the adenoviral antigens of the vector was approximately 3-fold lower than the number of antigen-reactive CD8-T memory cells specifi c to the target S-antigen.The intensity