Safety, Immunogenicity and Protective Activity of a Modified Trivalent Live Attenuated Influenza Vaccine for Combined Protection Against Seasonal Influenza and COVID-19 in Golden Syrian Hamsters

Abstract

Background/objectives: Influenza viruses and SARS-CoV-2 are currently cocirculating with similar seasonality, and both pathogens are characterized by a high mutational rate which results in reduced vaccine effectiveness and thus requires regular updating of vaccine compositions. Vaccine formulations combining seasonal influenza and SARS-CoV-2 strains can be considered promising and cost-effective tools for protection against both infections.

Methods: We used a licensed seasonal trivalent live attenuated influenza vaccine (3×LAIV) as a basis for the development of a modified 3×LAIV/CoV-2 vaccine, where H1N1 and H3N2 LAIV strains encoded an immunogenic cassette enriched with conserved T-cell epitopes of SARS-CoV-2, whereas a B/Victoria lineage LAIV strain was unmodified. The trivalent LAIV/CoV-2 composition was compared to the classical 3×LAIV in the golden Syrian hamster model. Animals were intranasally immunized with the mixtures of the vaccine viruses, twice, with a 3-week interval. Immunogenicity was assessed on day 42 of the study, and the protective effect was established by infecting vaccinated hamsters with either influenza H1N1, H3N2 or B viruses or with SARS-CoV-2 strains of the Wuhan, Delta and Omicron lineages.

Results: Both the classical 3×LAIV and 3×LAIV/CoV-2 vaccine compositions induced similar levels of serum antibodies specific to all three influenza strains, which resulted in comparable levels of protection against challenge from either influenza strain. Protection against SARS-CoV-2 challenge was more pronounced in the 3×LAIV/CoV-2-immunized hamsters compared to the classical 3×LAIV group. These data were accompanied by the higher magnitude of virus-specific cellular responses detected by ELISPOT in the modified trivalent LAIV group.

Conclusions: The modified trivalent live attenuated influenza vaccine encoding the T-cell epitopes of SARS-CoV-2 can be considered a promising tool for combined protection against seasonal influenza and COVID-19.

Keywords: LAIV; SARS-CoV-2; Syrian hamster; bivalent vaccine; influenza virus; preclinical research.

Figure 1

A list of LAIV viruses used in this study. (A) Schematic representation of LAIV reassortant viruses either used as monovalent preparations (upper panel) or in trivalent compositions (lower panel). The dose of each vaccine strain is shown above the virus figure. (B) Scheme of the modified influenza A NA gene, where the SARS-CoV-2 T-cell cassette is inserted into the NA open reading frame via the P2A self-cleavage site, which facilitates independent intracellular processing of the influenza NA protein and the inserted cassette [9].

Figure 2

The scheme of the experiment for assessment of the safety, immunogenicity and protective potential of the studied monovalent and trivalent LAIV candidates in Syrian hamsters. Doses of vaccine viruses administered alone or in trivalent compositions are shown on Figure 1A. D—days of the experiment. Red circles denote the extracted nasal turbinate tissues.

Figure 3

Safety assessment of the modified trivalent 3×LAIV/CoV2 vaccine in Syrian hamsters. Animals were immunized twice with the indicated vaccine variant on days 0 and 21, and body weight was evaluated during the immunization phase, until day 42 of the experiment.

Figure 4

Serum IgG antibody responses to influenza viruses in immunized hamsters (ELISA with whole sucrose gradient-purified virus antigens). OD₄₅₀ values (left panel) and endpoint serum IgG titers (right panel) in reaction with H1N1 antigens (A), H3N2 antigens (B) and B/Victoria antigens (C). Antibody levels were measured at day 42 of the study, after two doses of the vaccines. (****) p < 0.0001, (***) p < 0.001, (**) p < 0.01, (*) p < 0.05 (ANOVA with post hoc Tukey test).

Figure 5

Protective efficacy of a modified trivalent LAIV and control monovalent and trivalent LAIVs against three seasonal influenza viruses in the Syrian hamster model. Titers of influenza A/H1N1 (top panel), A/H3N2 (middle panel) and type B viruses (bottom panel) in the lungs and nasal turbinates are shown. Animals in the placebo group received PBS. (****) p < 0.0001, (**) p < 0.01, (*) p < 0.05 (ANOVA with post hoc Tukey test).

Figure 6

Protective efficacy of a modified trivalent LAIV and control monovalent and trivalent LAIVs against SARS-CoV-2 of three different antigenic lineages. Viral titers in nasal turbinates (NTs) and lung tissues on day 6 post-challenge are shown: (A) Wuhan strain; (B) Delta variant; (C) Omicron variant. (D) Sum of clinical symptom scores within 6 days of infection. (****) p < 0.0001, (***) p < 0.001, (**) p < 0.01, (*) p < 0.05 (ANOVA with post hoc Tukey test).

Figure 7

Histopathological assessment of protective effect of 3×LAIV/CoV2 against Wuhan challenge in a hamster model. Representative micrographs of hematoxylin–eosin-stained lung sections of animals on day 6 after challenge are shown using 50× (upper panel, scale bar: 500 µm), 200× (middle panel, scale bar: 100 µm) and 400× magnifications (lower panel, scale bar: 100 µm).

Figure 8

Semi-quantitative analyses of the airway, lung/alveolar and vascular damage to the lungs of immunized and control hamsters on day 6 after challenge with Wuhan (upper panel), Delta (middle panel) and Omicron (lower panel) SARS-CoV-2 variants. Data were analyzed by one-way ANOVA with Tukey’s post hoc multiple-analyses test. (*) p < 0.05.

Figure 9

Levels of IFNγ-secreting cells in splenocytes of immunized Syrian hamsters on day 6 after infection with hCoV-19/Russia/StPetersburg-3524/2020 (line B.1, Wuhan). Isolated splenocytes were stimulated in vitro with influenza viruses (upper panel) and live SARS-CoV-2 or PepTivator (lower panel), followed by quantification of IFNγ-secreting cells using the Hamster IFN-γ ELISpot Plus kit.