Intranasal administration of octavalent next-generation influenza vaccine elicits protective immune responses against seasonal and pre-pandemic viruses

Abstract

Development of next-generation influenza virus vaccines is crucial to improve protection against circulating and emerging viruses. Current vaccine formulations have to be updated annually due to mutations in seasonal strains and do not offer protection against strains with pandemic potential. Computationally optimized broadly reactive antigen (COBRA) methodology has been utilized by our group to generate broadly reactive immunogens for individual influenza subtypes, which elicit protective immune responses against a broad range of strains over numerous seasons. Octavalent mixtures of COBRA hemagglutinin (HA) (H1, H2, H3, H5, H7, and influenza B virus) plus neuraminidase (NA) (N1 and N2) recombinant proteins mixed with c-di-AMP adjuvant were administered intranasally to naive or pre-immune ferrets in prime-boost fashion. Four weeks after final vaccination, collected sera were analyzed for breadth of antibody response, and the animals were challenged with seasonal or pre-pandemic strains. The octavalent COBRA vaccine elicited antibodies that recognized a broad panel of strains representing different subtypes, and these vaccinated animals were protected against influenza virus challenges. Overall, this study demonstrated that the mixture of eight COBRA HA/NA proteins mixed with an intranasal adjuvant is a promising candidate for a universal influenza vaccine.

Importance: Influenza is a respiratory virus which infects around a billion people globally every year, with millions experiencing severe illness. Commercial vaccine efficacy varies year to year and can be low due to mismatch of circulating virus strains. Thus, the formulation of current vaccines has to be adapted accordingly every year. The development of a broadly reactive influenza vaccine would lessen the global economic and public health burden caused by the different types of influenza viruses. The significance of our research is producing a promising universal vaccine candidate which provides protection against a wider range of virus strains over a wider range of time.

Keywords: COBRA; Sting agonist; ferret; influenza; intranasal; mice; multivalent; vaccine.

Fig 1

Diagram of ferret vaccine study. For the pre-immune vaccine groups, ferrets that had been exposed to A/California/2009 3 months previously were intranasally infected with A/Panama/1999 and B/Hong Kong/2001 60 days prior to vaccination. On days 0 and 28, pre-immune and naïve groups were vaccinated intranasally with c-di-AMP as adjuvant. Sera were collected on days 0, 28, and 56. Ferrets were challenged with A/Brisbane/02/2018 (H1N1), B/Washington/02/2019 (IBV), or A/Vietnam/1203/2004 (H5N1) on day 56. Ferrets were weighed and monitored for clinical signs daily. Nasal washes were taken on days 57, 59, 61, and 63.

Fig 2

Octavalent COBRA vaccination elicits IgG antibody response in ferrets. Ferrets were vaccinated as described in Fig. 1. Total IgG antibody titers were determined against each of the eight COBRA components, as indicated on the x-axis, from sera collected before vaccination (d0, open bars) and after final vaccination (d56, closed bars) from (a) pre-immune ferrets given COBRA vaccination, (b) naïve ferrets given COBRA vaccination, and (c) pre-immune ferrets given mock vaccination. Total IgG antibody titers from d56 sera across all groups were analyzed (d). Two-way analysis of variance with multiple comparisons was used to analyze the statistical differences between d0 and d56 ELISA results for each group and d56 ELISA results across all groups by GraphPad Prism version 9 software (GraphPad, San Diego, CA, USA). A P value of <0.05 was defined as statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ELISA, enzyme-linked immunosorbent assay; ns, not significant.

Fig 3

Octavalent COBRA vaccination elicits IgG antibody response against group 1 and group 2 stem proteins. Ferrets were vaccinated as described in Fig. 1. Total IgG antibody titers were determined against group 1 and group 2 stem proteins, as indicated on the x-axis, from sera collected before vaccination (d0, open bar) and after final vaccination (d56, closed bar) from (a) pre-immune ferrets given COBRA vaccination, (b) naïve ferrets given COBRA vaccination, and (c) pre-immune ferrets given mock vaccination. Total IgG antibody titers from d56 sera across all groups were analyzed (d). Two-way analysis of variance with multiple comparisons was used to analyze the statistical differences between d0 and d56 ELISA results for each group and d56 ELISA results across all groups by GraphPad Prism version 9 software (GraphPad). A P value of <0.05 was defined as statistically significant. **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.

Fig 4

HAI antibody titers for seasonal viruses are increased in octavalent COBRA vaccinated ferrets. Ferrets were vaccinated as described in Fig. 1. Sera were collected before vaccination (open bars) and 4 weeks after the second vaccination (closed bars) for HAI assay against a panel of (a) H1N1, (b) H3N2, and (c) influenza B viruses. The virus strains are listed along the x-axis. The y-axis indicates the log₂ HAI titers for each vaccinated group and presents them as absolute mean values ± SEM. The dotted lines indicate HAI titers ranging from 1:40 (lower line) and 1:80 (upper line). Statistical differences between day 0 and day 56 HAI titers for each vaccine group were analyzed using two-way analysis of variance with multiple comparisons by GraphPad Prism version 9 software (GraphPad). A P value of <0.05 was defined as statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. HAI, hemagglutination inhibition.

Fig 5

Octavalent COBRA vaccination elicits HAI antibody titers for pre-pandemic viruses in ferrets. Ferrets were vaccinated as described in Fig. 1. Sera were collected before vaccination (a) and 4 weeks after the second vaccination (b) for HAI assay against a panel of pre-pandemic reassortant viruses. The virus strains are listed along the x-axis. The y-axis indicates the log₂ HAI titers for each vaccinated group and presents them as absolute mean values ± SEM. The dotted lines indicate HAI titers ranging from 1:40 (lower line) and 1:80 (upper line). Statistical differences between day 56 HAI titers for each vaccine group were analyzed using unpaired t-tests by GraphPad Prism version 9 software (GraphPad). A P value of <0.05 was defined as statistically significant. *P < 0.05. ns, not significant.

Fig 6

Octavalent COBRA vaccination increases serum NAI antibody titers after vaccination in ferrets. Ferrets were vaccinated as described in Fig. 1. Sera were collected before vaccination (d0) and 4 weeks after the second vaccination (d56) for NAI assay against (a–c) N1 and (d–f) N2 recombinant proteins. (c and f) Statistical differences between NAI₅₀ titers were analyzed using one-way analysis of variance with multiple comparisons by GraphPad Prism version 9 software (GraphPad). A P value of <0.05 was defined as statistically significant. **P < 0.01, ****P < 0.0001. ns, not significant.

Fig 7

Octavalent COBRA vaccination or pre-existing immunity protects against seasonal or pandemic virus challenges. Ferrets were vaccinated as described in Fig. 1. Vaccine groups are pre-immune ferrets given octavalent COBRA recombinant HA and NA (blue line), naïve ferrets given octavalent COBRA recombinant HA and NA (red line), pre-immune ferrets given mock vaccination (gray line), or naïve ferrets given mock vaccination (black dashed line). Pre-immune ferrets were previously infected with A/California/2009, A/Panama/1999, and B/Hong Kong/2001. Four weeks after final vaccination, ferrets were intranasally infected with (a) A/Brisbane/02/2018 (10⁸ PFU), (b) B/Washington/02/2019 (10⁷ PFU), or a lethal dose of (c) A/Vietnam/1203/2004 (10⁵ PFU) in a volume of 1 mL. The animals were observed for clinical signs, and their body weight was recorded daily post-infection.

Fig 8

Viral titers in the upper respiratory tract of ferrets after seasonal infection are reduced after octavalent COBRA vaccination. Ferrets were vaccinated as described in Fig. 1. Vaccine groups are indicated on the x-axis. Four weeks after the second vaccination, the groups were challenged with H1N1 A/Brisbane/02/2018 (top row, a–c) or IBV B/Washington/02/2019 (bottom row, d–f). Each figure represents viral titers from nasal washes taken on the indicated day post-infection—day 1, day 3, or day—as quantified by viral plaque assay. Viral titers in nasal washes are presented as PFU per milliliter as shown on the y-axis. Each dot represents an individual ferret. Statistical differences were analyzed by one-way analysis of variance with multiple comparisons by GraphPad Prism version 9 software (GraphPad). A P value of less than 0.05 was defined as statistically significant. *P < 0.05, **P < 0.01.