An inactivated multivalent influenza A virus vaccine is broadly protective in mice and ferrets

Abstract

Influenza A viruses (IAVs) present major public health threats from annual seasonal epidemics and pandemics and from viruses adapted to a variety of animals including poultry, pigs, and horses. Vaccines that broadly protect against all such IAVs, so-called "universal" influenza vaccines, do not currently exist but are urgently needed. Here, we demonstrated that an inactivated, multivalent whole-virus vaccine, delivered intramuscularly or intranasally, was broadly protective against challenges with multiple IAV hemagglutinin and neuraminidase subtypes in both mice and ferrets. The vaccine is composed of four β-propiolactone-inactivated low-pathogenicity avian IAV subtypes of H1N9, H3N8, H5N1, and H7N3. Vaccinated mice and ferrets demonstrated substantial protection against a variety of IAVs, including the 1918 H1N1 strain, the highly pathogenic avian H5N8 strain, and H7N9. We also observed protection against challenge with antigenically variable and heterosubtypic avian, swine, and human viruses. Compared to control animals, vaccinated mice and ferrets demonstrated marked reductions in viral titers, lung pathology, and host inflammatory responses. This vaccine approach indicates the feasibility of eliciting broad, heterosubtypic IAV protection and identifies a promising candidate for influenza vaccine clinical development.

Fig. 1.. Multivalent IAV vaccination elicits antibody responses and confers protection against viral challenge in mice.

Serum IgG concentrations against (A) the four vaccine hemagglutinin (HA) antigens and (B) the four vaccine neuraminidase (NA) antigens were measured by ELISA and shown as area under the curve (AUC) 3 weeks after the boost immunization in PBS-, IM-, or IN-immunized mice. Welch ANOVA test and post hoc Dunnett’s T3 multiple comparison test were used to compare antibody concentrations between groups; *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. (C to F) Percent survival and percent weight loss in PBS-, IM-, or IN-immunized mice after lethal challenge (10× LD₅₀) with six different IAV challenge strains (n = 5 per group except H5N8 challenge): (C) 1918 pandemic H1N1, (D) H7N9, (E) HPAI H5N8 (n = 10 per group), (F) chimeric avian H7N1, (G) chimeric avian H6N1, and (H) chimeric H10N7 virus. Error bars represent SD.

Fig. 2.. Immunized mice have reduced IAV RNA, inflammatory responses, and evidence of lung pathology.

(A) Top: bar graph showing relative expression of IAV M gene mRNA in immunized mouse lung compared to PBS-immunized animals as measured by qRT-PCR. Bottom, differences in lung gene expression in lungs of PBS-, IM-, and IN-immunized mice identified by ANOVA (greater than twofold difference in median expression, P < 0.01) on day 6 after challenge; heatmaps show the relative expression of type I IFN response genes, lymphocyte activation genes, ROS response and DNA damage genes, and programmed cell death genes. Genes with increased expression are shown in red, genes with no change as black, and genes showing decreased expression in blue. (B) Lung histopathology of PBS-, IM-, and IN-immunized mice lethally challenged with chimeric avian H6N1, H7N1, or H7N10 viruses (10× LD₅₀ dose), and analyzed at day 5 after infection. In each case, PBS-immunized animals showed widespread, severe viral pneumonia with necrotizing bronchitis and bronchiolitis as well as alveolitis. In contrast, IM- or IN-immunized animals showed an absence of pneumonia, with no bronchitis, bronchiolitis, or alveolitis. Aggregates of lymphoid tissue were observed in peribronchiolar and peribronchiolar spaces in immunized animals. Original magnifications are ×20; scale bar, 500 μM. See fig. S4 to S6 for additional pathological analyses.

Fig. 3.. Cytotoxic T cell and NK cell responses are elicited during heterosubtypic challenge in mice.

Flow cytometry was performed on immune cells isolated from lungs 5 days after a lethal heterosubtypic H10N7 challenge. Among lung leukocytes (intravenous CD45 staining negative), CD8 T cells, CD4 T cells, and NK cells were analyzed for two representative cytotoxic molecules: granzyme B and perforin. Fluorescence minus one (FMO) controls were used to gate granzyme B or perforin positive populations. The frequencies of (A) granzyme B– or (B) perforin-positive CD8 T cells relative to the total CD8 T cells are shown. BUV, brilliant ultraviolet; AF, Alexa Fluor; PE, phycoerythrin. The frequencies of (C) granzyme B– or (D) perforin-positive CD4 T cells relative to the total CD4 T cells are shown. Similarly, the frequencies of (E) granzyme B– or (F) perforin-positive NK cells relative to the total NK cells are shown. APC, allophycocyanin. The differences in the abundance of (G) granzyme B or (H) perforin in NK cells between groups were measured by comparing sum of fluorescent intensities (sum FI). The frequencies of (I) CD8 resident memory T (T_RM) cells and (J) CD4 T_RM relative to the total CD8 and CD4 T cells, respectively, are shown. Animal experiments were done twice independently; red and blue symbols indicate each independent experiment (four to five mice per group). Each symbol represents an individual animal. Contour plots are representative of the frequency of granzyme B– or perforin-positive CD8 T cells, CD4 T cells, and NK cells. Gating strategies to identify lung CD8, CD4, and NK cells are shown in fig. S20. Welch ANOVA test and post hoc Dunnett’s T3 multiple comparison test were used to compare between groups; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4.. Quadrivalent vaccination is immunogenic and confers protection against viral challenge in immunized ferrets.

Serum IgG concentrations against (A) the four vaccine HA antigens and (B) the four vaccine NA antigens were measured 3 weeks after the boost immunization in PBS-, IM-, or IN-immunized mice using ELISA. Welch ANOVA test and post hoc Dunnett’s T3 multiple comparison test were used to compare antibody concentrations between groups. *P < 0.05, **P < 0.01, ***P < 0.001. (C to F) Reductions in nasal wash and lung viral titers were measured in IM- or IN-immunized ferrets as compared to PBS-immunized ferrets (n = 6 per group). Nasal wash titers were measured at days 1, 3, 5, and 7 after challenge, and lung titers were determined on day 5 after challenge. (C) Reductions in titers after A/swine/1931 (H1N1) challenge. (D) Reductions in titers after A/Port Chalmers/1/1973 (H3N2) challenge. (E) Reductions in titers after chimeric 1957 pandemic H2N7 challenge. (F) Reductions in titers after chimeric avian H10N7 challenge. For (C) and (D), two of six ferrets per group were used for lung viral titration. For (E) and (F), four of six ferrets per group were used for lung viral titration. Unpaired t test (C and D; n = 2) and ordinary ANOVA test and post hoc Dunnett’s multiple comparison test (E and F; n = 4) were used to compare viral titers in IM- or IN-immunized ferrets to PBS-immunized ferrets at the indicated time points. Error bars represent geometric SD. Horizontal dashed lines indicate the limit of detection.

Fig. 5.. Reduced influenza viral RNA, lung inflammatory responses, and lung pathology were observed in vaccinated ferrets.

(A) Top, bar graph showing relative expression of IAV M gene mRNA in immunized ferret lung compared to PBS-immunized animals as measured by qRT-PCR. Bottom, differences in lung gene expression responses in lungs of PBS-, IM-, and IN-immunized ferrets identified by ANOVA (greater than twofold difference in median expression, P < 0.01) on day 5 after challenge; the heatmaps show the relative expression of type I IFN response genes, cytokine signaling genes, lymphocyte activation genes, and DNA repair genes. (B) Lung histopathology is shown for PBS-, IM-, and IN-immunized mice lethally challenged with A/swine/1931 (H1N1), A/Port Chalmers/1/1973 (H3N2), or completely heterosubtypic challenge with chimeric H2N7 or H10N7 challenge viruses. In each case, PBS-immunized animals showed widespread, severe viral pneumonia with necrotizing bronchitis and bronchiolitis as well as alveolitis. In contrast, IM- or IN-immunized animals showed an absence of pneumonia, with no bronchitis, bronchiolitis, or alveolitis. Aggregates of lymphoid tissue were observed in peribronchiolar and peribronchiolar spaces in immunized animals. Original magnifications are ×20; scale bar, 500 μM. See fig. S14 to S17 for additional pathological analyses.