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
Abstract
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 influenza, 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 specificity, 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 specificity 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. Purified CD4 and CD8 T cell populations were obtained by sorting on a BD FACS Aria II laser flow 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 108 PFU induced strong systemic T-cell immune responses in C57BL/6 mice. Two months after immunization, about 100–200 thousand 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 specific for the SARS-CoV-2 S-antigen. The contents 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 specific to the adenoviral antigens of the vector was approximately 3-fold lower than the number of antigen-reactive CD8-T memory cells specific to the target S-antigen. The intensity of the immune response of CD4-T cells to immunization with the Ad5-S vector was comparable to the intensity of the immune response of CD8-T cells. The vast majority of antigen-reactive CD4-T memory cells were specific for adenovirus vector antigens. These CD4-T cells secreted IFN-γ in response to in vitro restimulation by dendritic cells transduced with the recombinant adenoviral Ad5-0 vector without a targeted insert. The number of CD4-T cells responding to restimulation by dendritic cells loaded with recombinant RBD was vanishingly small.
An intensity of the response of CD8-T cells specific to the target S-antigen can be increased by elevating of the Ad5-S vector dose during transduction of antigen-presenting dendritic cells, as well as by stimulation of antigen-presenting dendritic cells with a TLR4 agonist.
Possible mechanisms of cooperative interaction between CD8-T cells specific for the target coronavirus S-antigen and CD4-T cells specific for adenovirus vector antigens have been proposed. Possible ways to enhance the response of CD4-T cells to the target S-antigen are considered.
Conclusion. Immunization with a recombinant adenoviral vector encoding the coronavirus S-antigen induces strong CD8- and CD4-T cell immune responses with the formation of a massive pool of antigen-reactive memory T cells. CD8- and CD4-T cells responding to immunization with the Ad5-S vector differ in their antigen specificity. Memory CD8-T cells are generally specific for the target coronavirus S-antigen. Memory CD4-T cells are specific to adenovirus vector antigens.
Keywords:recombinant adenoviral vector; coronavirus S-antigen; memory T-cells; antigen specificity
For citation: Ataullakhanov R.I., Ushakova E.I., Pichugin A.V., Lebedeva E.S., Ivanov S.V., Ozharovskaia T.A., Popova O., Shcherbinin D.N., Bandelyuk A.S., Zubkova O.V., Shmarov M.M., Logunov D.Yu., Naroditsky B.S., Gintsburg A.L. 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. Immunologiya. 2023; 44 (5): 557–74. DOI: https://doi.org/10.33029/1816-2134-2023-44-5-557-574 (in Russian)
Funding. The study was supported by State assignment (Agreement No. 388-03-2021-010 of 01/20/2021). Open publication of the research results is allowed.
Conflict of interests. The authors declare no conflict of interests.
Authors’ contribution. Idea of the study – Ataullakhanov R.I., Naroditsky B.S., Gintsburg A.L.; design of experiments – Ataullakhanov R.I., Ushakova E.I., Pichugin A.V., Shmarov M.M., Logunov D.Yu., Naroditsky B.S., Gintsburg A.L.; DNA construct design, production and purification of the recombinant RBD-fragment of the coronavirus S-protein – Ivanov S.V.; obtaining genetic constructs carrying coronavirus genes as part of the adenoviral vector genome – Ozharovskaia T.A.; purification and titration of recombinant adenoviral vectors – Shcherbinin D.N., cultivation of human cells line 293 and the growth of recombinant adenoviral vectors – Popova O.; design of recombinant adenoviral vectors carrying coronavirus genes as part of the adenoviral vector genome – Zubkova O.V.; immunization of animals, sampling of organs and tissues – Bandelyuk A.S.; cytometry and cell sorting – Pichugin A.V.; ELISPOT – Ushakova E.I.; analysis of results – Ataullakhanov R.I., Ushakova E.I., Pichugin A.V., Lebedeva E.S., Shmarov M.M.; statistical processing and technical design of the article – Ushakova E.I.; conception of the article – Ataullakhanov R.I.; writing of the article – Ataullakhanov R.I., Ushakova E.I., Lebedeva E.S.
11. Lebedeva E., Bagaev A., Pichugin A., Chulkina M., Lysenko A., Tutykhina I., Shmarov M., Logunov D., Naroditsky B., Ataullakhanov R. The differences in immunoadjuvant mechanisms of TLR3 and TLR4 agonists on the level of antigen-presenting cells during immunization with recombinant adenovirus vector. BMC Immunol. 2018; 19 (1): 26. DOI: https://doi.org/10.1186/s12865-018-0264-x
12. Janssen E.M., Lemmens E.E., Wolfe T., Christen U., von Herrath M.G., Schoenberger S.P. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature. 2003; 421 (6925): 852–56. DOI: https://doi.org/10.1038/nature01441
13. Sun J.C., Bevan M.J. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science. 2003; 300 (5617): 339–42. DOI: https://doi.org/10.1126/science.1083317
14. Shedlock D.J., Shen H. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science. 2003; 300 (5617): 337–39. DOI: https://doi.org/10.1126/science.1082305
15. Novy P., Quigley M., Huang X., Yang Y. CD4 T cells are required for CD8 T cell survival during both primary and memory recall responses. J Immunol. 2007; 179 (12): 8243–51. DOI: https://doi.org/10.4049/jimmunol.179.12.8243
16. Phares T.W., Stohlman S.A., Hwang M., Min B., Hinton D.R., Bergmann C.C. CD4 T cells promote CD8 T cell immunity at the priming and effector site during viral encephalitis. J Virol. 2012; 86 (5): 2416–27. DOI: https://doi.org/10.1128/JVI.06797-11
17. Gressier E., Schulte-Schrepping J., Petrov L., Brumhard S., Stubbemann P., Hiller A., Obermayer B., Spitzer J., Kostevc T., Whitney P.G., Bachem A., Odainic A., van de Sandt C., Nguyen T.H.O., Ashhurst T., Wilson K., Oates C.V.L., Gearing L.J., Meischel T., Hochheiser K., Greyer M., Clarke M., Kreutzenbeck M., Gabriel S.S., Kastenmüller W., Kurts C., Londrigan S.L., Kallies A., Kedzierska K., Hertzog P.J., Latz E., Chen Y.E., Radford K.J., Chopin M., Schroeder J., Kurth F., Gebhardt T., Sander L.E., Sawitzki B., Schultze J.L., Schmidt S.V., Bedoui S. CD4+ T cell calibration of antigen-presenting cells optimizes antiviral CD8+ T cell immunity. Nat Immunol. 2023; 24 (6): 979–90. DOI: https://doi.org/10.1038/s41590-023-01517-x