Vaccination With Viral Vectors Expressing Chimeric Hemagglutinin, NP and M1 Antigens Protects Ferrets Against Influenza Virus Challenge

Abstract

Seasonal influenza viruses cause significant morbidity and mortality in the global population every year. Although seasonal vaccination limits disease, mismatches between the circulating strain and the vaccine strain can severely impair vaccine effectiveness. Because of this, there is an urgent need for a universal vaccine that induces broad protection against drifted seasonal and emerging pandemic influenza viruses. Targeting the conserved stalk region of the influenza virus hemagglutinin (HA), the major glycoprotein on the surface of the virus, results in the production of broadly protective antibody responses. Furthermore, replication deficient viral vectors based on Chimpanzee Adenovirus Oxford 1 (ChAdOx1) and modified vaccinia Ankara (MVA) virus expressing the influenza virus internal antigens, the nucleoprotein (NP) and matrix 1 (M1) protein, can induce strong heterosubtypic influenza virus-specific T cell responses in vaccinated individuals. Here, we combine these two platforms to evaluate the efficacy of a viral vectored vaccination regimen in protecting ferrets from H3N2 influenza virus infection. We observed that viral vectored vaccines expressing both stalk-targeting, chimeric HA constructs, and the NP+M1 fusion protein, in a prime-boost regimen resulted in the production of antibodies toward group 2 HAs, the HA stalk, NP and M1, as well as in induction of influenza virus-specific-IFNγ responses. The immune response induced by this vaccination regime ultimately reduced viral titers in the respiratory tract of influenza virus infected ferrets. Overall, these results improve our understanding of vaccination platforms capable of harnessing both cellular and humoral immunity with the goal of developing a universal influenza virus vaccine.

Keywords: CD8 T-cells; influenza; stalk antibodies; universal influenza virus vaccine; vectored vaccine.

Figure 1

Ferret vaccination regime. In the first experiment ferrets were either prime-boosted with the virus-matched TIV, primed with ChAdOx1-cH14/3-NP + M1 and boosted 4 weeks later with MVA-cH15/3-NP + M1 or were naïve (A). In the second experiment ferrets were either prime-boosted with the virus-matched TIV, primed with ChAdOx1-cH14/3 and boosted 4 weeks later with MVA-cH15/3 or primed with ChAdOx1-NP + M1 and boosted with MVA-NP + M1 (B). The study was split into two experiments due to space limitations. The TIV groups served as bridging groups.

Figure 2

Nasal wash and oropharyngeal titres at days 1 and 3 post-challenge. Ferrets were prime-boosted with TIV or viral vectors (ChAdOx1, then MVA) expressing influenza virus antigens (cHA-NP + M1, cHA, or NP + M1). Six weeks after the boost, ferrets were challenged IN with 10⁶ PFU of A/Wyoming/03/2003 (H3N2) virus and nasal washes were collected at days 1 (A) and 3 (B) post-challenge to detect viral titres via plaque assay. Oropharyngeal swabs were also collected at days 1 (C) and 3 (D) post-challenge to determine viral titres. The data are presented as individual replicates and the mean for each group is presented as line. n = 4 ferrets/group. ^**p ≤ 0.01, ^***p ≤ 0.001 when naïve controls were compared to individual vaccination groups as determined by one-way analysis of variance, followed by a Bonferroni multiple-comparison test.

Figure 3

Viral titres in the respiratory tract at day 4 post-challenge. Ferrets were prime-boosted with TIV or viral vectors (ChAdOx1 and MVA) expressing influenza antigens (cHA-NP + M1, cHA, or NP + M1). Six weeks after the boost, ferrets were challenged IN with 10⁶ PFU of A/Wyoming/03/2003 (H3N2) virus and at 4 days post-challenge ferrets were euthanized and respiratory tissues were taken to detect virus titres in the nasal turbinates (A), olfactory bulb (B), trachea (C), and lung (D). The data are presented as individual replicates and the mean for each group is presented as line. n = 4 ferrets/group. ^*p ≤ 0.05, ^***p ≤ 0.001 when naïve controls were compared to individual vaccination groups as determined by one-way analysis of variance, followed by a Bonferroni multiple-comparison test.

Figure 4

Antibody responses toward influenza virus antigens in the pre-challenge serum of vaccinated ferrets. Six weeks after the booster vaccination, pre-challenge serum was collected from TIV vaccinated, viral vector vaccinated, and naïve ferrets and IgG antibody responses toward influenza virus antigens were assessed via ELISA. Shown are antibody responses toward the H3 (A), H7 (B), H10 (C), cH4/3 (D), NP (E), and M1 (F) recombinant proteins. HI (G) and microneutralization titres (H) were also assessed. The data are presented as individual replicates and the mean for each group is presented as line. n = 4 ferrets/group. ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p < 0.005 when naïve controls were compared to individual vaccination groups as determined by one-way analysis of variance, followed by a Bonferroni multiple-comparison test.

Figure 5

Influenza virus specific T cells in the nasal washes. TIV vaccinated, viral vector vaccinated (cHA-NP + M1, cHA, or NP + M1), and naïve ferrets were challenged IN with 10⁶ PFU of A/Wyoming/03/2003 (H3N2) virus and nasal washes were collected at days 1 and 3 post-challenge. Cells were isolated from these nasal washes and stimulated with peptide arrays spanning the NP + M1 fusion protein. Following stimulation, cells were stained with anti-CD8 and -IFN-γ antibodies and influenza virus-specific IFN-γ⁺CD8⁺ T cells were measured at days 1 (A) and 3 (B) post-challenge. The data are presented as individual replicates and the mean for each group is presented as line. n = 4 ferrets/group. ^**p < 0.01 when naïve controls were compared to individual vaccination groups as determined by one-way analysis of variance, followed by a Bonferroni multiple-comparison test.

Figure 6

IFNγ cellular responses in the MLNs and spleen. TIV vaccinated, viral vector vaccinated (cHA-NP + M1, cHA, or NP + M1), and naïve ferrets were challenged IN with 10⁶ PFU of A/Wyoming/03/2003 (H3N2) virus and at 4 days post-challenge, ferrets were euthanized, and spleens and MLNs were removed to enumerate influenza virus-specific cellular responses. Splenocytes and MLN cells were stimulated with peptide arrays spanning the NP + M1 fusion protein and influenza virus-specific IFN-γ expressing cells were detected in the MLNs (A) and spleens (B) by ELISpot. The ICS assay was also used to detect influenza virus-specific IFNγ⁺CD8⁺ T cells in the MLN (C) and spleen (D). The data are presented as individual replicates and the mean for each group is presented as line. n = 4 ferrets/group. ^**p ≤ 0.01, ^***p ≤ 0.001 when naïve controls were compared to individual vaccination groups as determined by one-way analysis of variance, followed by a Bonferroni multiple-comparison test.