Epidemic influenza virus nucleoprotein gene incorporated into vaccine influenza virus strain genome to optimize systemic and local T-cell immune response against live attenuated influenza vaccine

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Introduction. Optimization of the vaccine-induced T-cell repertoire is one of the strategies to expand the spectrum of protective potential for live attenuated influenza vaccine (LAIV). LAIV cross-protective properties can be improved by introducing the nucleoprotein (NP) gene derived from epidemic parental virus into vaccine strain genome, i.e. by replacing the classical 6:2 genome formula with 5:3. The main objective of the present study was to detail evaluation for virus-specific systemic and tissue-resident memory T-cells subsets in mice immunized with seasonal H1N1 LAIV of the genome formula 6:2 and 5:3. Materials and methods. Two H1N1 LAIV strains with varying NP genes (LAIV 6:2 and LAIV 5:3) were generated using reverse genetics techniques. C57BL/6J mice were immunized intranasally with the vaccine candidates, twice, 3 weeks apart. Cells from the spleen and lung tissues were isolated 7 days after booster immunization to be stimulated with whole H1N1 influenza virus for assessing cytokine-producing memory CD44+CD62L– T-cells as well as expression of CD69 and CD103 surface markers using flow cytometry. Humoral murine serum immunity against H1N1 virus was assessed by ELISA. Results. The LAIV 5:3 vs classical 6:2 vaccine strain carrying the epidemic parental NP gene induced significantly more pronounced humoral immune response against recent influenza virus. The group of mice immunized with LAIV 5:3 demonstrated higher levels of virus-specific CD4+ and CD8+ effector memory T cells (TEM) in the spleen, including a subset of polyfunctional (IFNγ+TNFα+IL-2+) CD4+ TEM, compared to LAIV 6:2 group. Virus-specific memory T cell levels in lung tissues after immunization with LAIV 5:3 vs LAIV 6:2 also tended to increase, but no significant difference in stimulated tissue-resident CD69+CD103– and CD69+CD103+ T cells between the groups were found. Conclusion. Modification of the seasonal LAIV strain genome for updating its epitope composition allowed to enhance the virus-specific T-cell immune response both at systemic level and in lung tissues, thereby shoeing that the effectiveness of the vaccine against circulating influenza viruses can be potentially increased.

About the authors

P. I. Prokopenko

Institute of Experimental Medicine

Author for correspondence.
Email: pi.prokopenko@gmail.com

Junior Researcher, A.A. Smorodintsev Department of Virology

Russian Federation, St. Petersburg

E. A. Stepanova

Institute of Experimental Medicine

Email: pi.prokopenko@gmail.com

PhD (Biology), Leading Researcher, A.A. Smorodintsev Department of Virology

Russian Federation, St. Petersburg

V. A. Matyushenko

Institute of Experimental Medicine

Email: pi.prokopenko@gmail.com

Researcher, A.A. Smorodintsev Department of Virology

Russian Federation, St. Petersburg

A. K. Chistyakova

Institute of Experimental Medicine

Email: pi.prokopenko@gmail.com

Research Laboratory Assistant, A.A. Smorodintsev Department of Virology

Russian Federation, St. Petersburg

A. D. Kostromitina

Institute of Experimental Medicine

Email: pi.prokopenko@gmail.com

Research Laboratory Assistant, A.A. Smorodintsev Department of Virology

Russian Federation, St. Petersburg

T. S. Kotomina

Institute of Experimental Medicine

Email: pi.prokopenko@gmail.com

Researcher, A.A. Smorodintsev Department of Virology

Russian Federation, St. Petersburg

A. Ya. Rak

Institute of Experimental Medicine

Email: pi.prokopenko@gmail.com

PhD (Biology), Senior Researcher, A.A. Smorodintsev Department of Virology

Russian Federation, St. Petersburg

A. A. Rubinstein

Institute of Experimental Medicine

Email: pi.prokopenko@gmail.com

Junior Researcher, Cell Immunology Laboratory, Department of Immunology

Russian Federation, St. Petersburg

I. V. Kudryavtsev

Institute of Experimental Medicine

Email: pi.prokopenko@gmail.com

PhD (Biology), Head of the Cell Immunology Laboratory, Department of Immunology

Russian Federation, St. Petersburg

V. V. Novitskaya

Institute of Experimental Medicine

Email: pi.prokopenko@gmail.com

Research Laboratory Assistant, A.A. Smorodintsev Department of Virology

Russian Federation, St. Petersburg

L. G. Rudenko

Institute of Experimental Medicine

Email: pi.prokopenko@gmail.com

DSc (Medicine), Professor, Head of A.A. Smorodintsev Department of Virology

Russian Federation, St. Petersburg

I. N. Isakova-Sivak

Institute of Experimental Medicine

Email: pi.prokopenko@gmail.com

RAS Corresponding Member, DSc (Biology), Head of the Laboratory of Immunology and Prevention of Viral Infections, A.A. Smorodintsev Department of Virology

Russian Federation, St. Petersburg

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2. Figure 1. Detection of blood serum virus-specific IgG antibodies in mice immunized twice with LAIV 6:2 and LAIV 5:3 strains. Notes. Antibody levels were detected by ELISA using whole virus A/Guangdong-Maonan/SWL1536/2019 (H1N1). A. Average values of optical density in wells at each serum dilution. B. Values of antibody titers in each study group. The data were compared using the ANOVA variance analysis with Tukey’s correction (*р < 0.05; ****р < 0.0001).

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3. Figure 2. Induction of systemic T-cell response by immunization with LAIV 6:2 and LAIV 5:3 strains. Notes. A. Representative dot-plot graphs of mouse splenocyte flow cytometry data after stimulation with whole H1N1/wt virus. B. Levels of cytokine-producing CD4+ T cells among the effector memory population (CD44+CD62L–). C. Cytokine-producing CD8+ T cell levels among effector memory population (CD44+CD62L–). The subpopulations of ТЕМ producing IFNγ (left), IFNγ and TNFα (middle), and IFNγ, TNFα and IL-2 (right) in response to double vaccination are presented. * р < 0.05, ** р < 0.01.

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4. Figure 3. Levels of tissue-resident CD4+ (upper panel) and CD8+ (lower panel) memory T cells in mice immunized with LAIV 6:2 and LAIV 5:3 as well as placebo treated (PBS). Notes. The number of cells expressing IFNγ among CD4+(A) and CD8+ (D) effector memory T cells (CD44+CD62L–) in lung samples from immunized mice. Column 2: the proportion of CD69+ cells among the corresponding populations (B, E). Column 3: the proportion of CD69+CD103+ cells (C, F). Significant differences between the groups (Mann–Whitney criteria) are shown, *р < 0.05, **р < 0.01, ***р < 0.001.

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Copyright (c) 2024 Prokopenko P.I., Stepanova E.A., Matyushenko V.A., Chistyakova A.K., Kostromitina A.D., Kotomina T.S., Rak A.Y., Rubinstein A.A., Kudryavtsev I.V., Novitskaya V.V., Rudenko L.G., Isakova-Sivak I.N.

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