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Safety and Immunogenicity Study of a Bivalent Vaccine for Combined Prophylaxis of COVID-19 and Influenza in Non-Human Primates - PubMed Skip to main page content
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. 2024 Sep 26;12(10):1099.
doi: 10.3390/vaccines12101099.

Safety and Immunogenicity Study of a Bivalent Vaccine for Combined Prophylaxis of COVID-19 and Influenza in Non-Human Primates

Ekaterina Stepanova  1 , Irina Isakova-Sivak  1 , Victoria Matyushenko  1 , Daria Mezhenskaya  1 , Igor Kudryavtsev  1 , Arina Kostromitina  1 , Anna Chistiakova  1 , Alexandra Rak  1 , Ekaterina Bazhenova  1 , Polina Prokopenko  1 , Tatiana Kotomina  1 , Svetlana Donina  1 , Vlada Novitskaya  1 , Konstantin Sivak  2 , Dzhina Karal-Ogly  3 , Larisa Rudenko  1
Affiliations

Safety and Immunogenicity Study of a Bivalent Vaccine for Combined Prophylaxis of COVID-19 and Influenza in Non-Human Primates

Ekaterina Stepanova et al. Vaccines (Basel). .

Abstract

Background: Influenza and SARS-CoV-2 viruses are two highly variable pathogens. We have developed a candidate bivalent live vaccine based on the strain of licensed A/Leningrad/17-based cold-adapted live attenuated influenza vaccine (LAIV) of H3N2 subtype, which expressed SARS-CoV-2 immunogenic T-cell epitopes. A cassette encoding fragments of S and N proteins of SARS-CoV-2 was inserted into the influenza NA gene using the P2A autocleavage site. In this study, we present the results of preclinical evaluation of the developed bivalent vaccine in a non-human primate model.

Methods: Rhesus macaques (Macaca mulatta) (n = 3 per group) were immunized intranasally with 7.5 lg EID50 of the LAIV/CoV-2 bivalent vaccine, a control non-modified H3N2 LAIV or a placebo (chorioallantoic fluid) using a sprayer device, twice, with a 28-day interval. The blood samples were collected at days 0, 3, 28 and 35 for hematological and biochemical assessment. Safety was also assessed by monitoring body weight, body temperature and clinical signs of the disease. Immune responses to influenza virus were assessed both by determining serum antibody titers in hemagglutination inhibition assay, microneutralization assay and IgG ELISA. T-cell responses were measured both to influenza and SARS-CoV-2 antigens using ELISPOT and flow cytometry. Three weeks after the second immunization, animals were challenged with 105 PFU of Delta SARS-CoV-2. The body temperature, weight and challenge virus shedding were monitored for 5 days post-challenge. In addition, virus titers in various organs and histopathology were evaluated on day 6 after SARS-CoV-2 infection.

Results: There was no toxic effect of the immunizations on the hematological and coagulation hemostasis of animals. No difference in the dynamics of the average weight and thermometry results were found between the groups of animals. Both LAIV and LAIV/CoV-2 variants poorly replicated in the upper respiratory tract of rhesus macaques. Nevertheless, despite this low level of virus shedding, influenza-specific serum IgG responses were detected in the group of monkeys immunized with the LAIV/CoV-2 bivalent but not in the LAIV group. Furthermore, T-cell responses to both influenza and SARS-CoV-2 viruses were detected in the LAIV/CoV-2 vaccine group only. The animals were generally resistant to SARS-CoV-2 challenge, with minimal virus shedding in the placebo and LAIV groups. Histopathological changes in vaccinated animals were decreased compared to the PBS group, suggesting a protective effect of the chimeric vaccine candidate.

Conclusions: The candidate bivalent vaccine was safe and immunogenic for non-human primates and warrants its further evaluation in clinical trials.

Keywords: SARS-CoV-2; bivalent vaccine; influenza; non-human primates; preclinical study; rhesus monkeys; virus-vectored vaccine.

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Conflict of interest statement

Authors I.-I.S., E.S., D.M., V.M. and L.R. have patent #RU 2782531 issued to FSBSI “Institute of experimental medicine”. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The scheme of the experiment on assessment of safety and immunogenicity of the FluCoVac-96 in rhesus monkeys. D—days of the experiment; PBMCs—peripheral blood mononuclear cells; MNT—virus microneutralization test; ICS—intracellular cytokine staining.
Figure 2
Figure 2
The scheme of modified NA segment of the FluCoVac-96 and the SARS-CoV-2 cassette insertion. UTR—untranslated regions of influenza NA segment; P2A—sequence encoding self-cleaving site of porcine teschovirus-1; N, S—SARS-CoV-2 proteins, the amino acid residues are listed in subscript; Stop—3 stop-codons at the end of the cassette. The additional fragment of NA sequence is shown in blue.
Figure 3
Figure 3
Replication of FluCoVac-96 and control H3N2 LAIV strain in chicken embryos and MDCK cells. (A) Reproduction in eggs at different temperatures; (B) reproduction in MDCK cells at different temperatures.
Figure 4
Figure 4
Body weight and temperature dynamics of the rhesus monkeys at D0-D49. Group mean values are presented. (A) Body weight dynamics (mean ± SD). (B) Body temperature dynamics. There were no significant differences in body weight and temperature dynamics between groups.
Figure 5
Figure 5
Serum antibody immune responses in rhesus macaques immunized with study vaccines or placebo. (A) Hemagglutination inhibiting antibodies to whole H3N2 LAIV virus. (B) Neutralizing antibodies against wild-type H3N2 virus. Antibody levels were measured at baseline (D0), after first immunization (D28) and after two doses (D49).
Figure 6
Figure 6
Serum IgG antibody levels in rhesus macaques immunized with study vaccines or placebo. (A) OD450 values for FluCoVac-96 vaccine group. (B) OD450 values for H3N2 LAIV group. (C) OD450 values for placebo group. (D) Endpoint serum IgG titers for each group at three time points. ELISA was performed with whole sucrose gradient-purified H3N2 LAIV virus. Antibody levels were measured at baseline (D0), after first immunization (D28) and after two doses (D49).
Figure 7
Figure 7
Fold rises of antibodies to influenza virus at D49 of the experiment in rhesus macaques immunized with study vaccines or placebo. HAI—hemagglutination inhibition; MN50—microneutralization test; ELISA—serum IgG antibodies.
Figure 8
Figure 8
The levels of IFNγ-producing cells in PBMCs of studied monkeys after stimulation with influenza virus of recombinant SARS-CoV-2 N protein at D0 and D35 (7 days after the boost immunization) of the experiment. (A) Representative wells, FluoroSpot (IFNγ-FITC). (B) Levels of IFNγ-producing cells after stimulation with influenza virus; (C) stimulation with SARS-CoV-2 N. (*) p < 0.05 (Kruskal-Wallis test).
Figure 8
Figure 8
The levels of IFNγ-producing cells in PBMCs of studied monkeys after stimulation with influenza virus of recombinant SARS-CoV-2 N protein at D0 and D35 (7 days after the boost immunization) of the experiment. (A) Representative wells, FluoroSpot (IFNγ-FITC). (B) Levels of IFNγ-producing cells after stimulation with influenza virus; (C) stimulation with SARS-CoV-2 N. (*) p < 0.05 (Kruskal-Wallis test).
Figure 9
Figure 9
The levels of IFNγ/TNFα-producing T cells in PBMCs of rhesus macaques before the immunization (day 0) and 7 days after the boost dose (day 35). (A) The levels of CD4+ memory T cells specific to influenza virus. (B) The levels of CD8+ memory T cells specific to influenza virus. (C) The levels of CD4+ memory T cells specific to SARS-CoV-2 N protein. (D) The levels of CD8+ memory T cells specific to SARS-CoV-2 N protein. The statistically significant differences between levels at day 0 and day 35 are shown (two-way ANOVA, uncorrected Fisher’s LSD test for multiple comparisons * p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 10
Figure 10
The levels of IFNγ-producing T cells in PBMCs of rhesus macaques after H3N2 influenza virus stimulation. (A) CD4+ CD45RA- T-cells subset. (B) CD8+ CD45RA- T-cells subset.
Figure 11
Figure 11
Representative gates of CD4+ CR45RA- (top panel) and CD8+ CR45RA- (bottom panel) IFNγ/TNFα-producing T cells after stimulation of the PBMCs with live purified influenza H3N2 virus.
Figure 12
Figure 12
The polyfunctional memory T cells (CD8+ CD45RA- IFNγ+ TNFα+ IL-2+) in PBMCs of macaque #45884 immunized with FluCoVac-96 after influenza stimulation (day 35 of the experiment). The percentage of IFNγ+ TNFα+ IL-2+ cells of CD8+ CD45RA- population is 0.0686%.
Figure 13
Figure 13
The levels of IFNγ-producing T cells in PBMC of rhesus macaques after recombinant SARS-CoV-2 N protein stimulation. (A) CD4+ CD45RA- T-cells subset. (B) CD8+ CD45RA- T-cells subset.
Figure 14
Figure 14
The body weight and body temperature monitoring in groups of rhesus macaques after SARS-CoV-2 intranasal challenge. (A) Average body weight dynamics in groups. (B) Body temperature dynamics.
Figure 15
Figure 15
Titers of live SARS-CoV-2 virus in the respiratory organs of the rhesus macaques after challenge infection.
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