Adenoviral-Vectored Centralized Consensus Hemagglutinin Vaccine Provides Broad Protection against H2 Influenza a Virus

Abstract

Several influenza pandemics have occurred in the past century, one of which emerged in 1957 from a zoonotic transmission of H2N2 from an avian reservoir into humans. This pandemic caused 2-4 million deaths and circulated until 1968. Since the disappearance of H2N2 from human populations, there has been waning immunity against H2, and this subtype is not currently incorporated into seasonal vaccines. However, H2 influenza remains a pandemic threat due to consistent circulation in avian reservoirs. Here, we describe a method of pandemic preparedness by creating an adenoviral-vectored centralized consensus vaccine design against human H2 influenza. We also assessed the utility of serotype-switching to enhance the protective immune responses seen with homologous prime-boosting strategies. Immunization with an H2 centralized consensus showed a wide breadth of antibody responses after vaccination, protection against challenge with a divergent human H2 strain, and significantly reduced viral load in the lungs after challenge. Further, serotype switching between two species C adenoviruses enhanced protective antibody titers after heterologous boosting. These data support the notion that an adenoviral-vectored H2 centralized consensus vaccine has the ability to provide broadly cross-reactive immune responses to protect against divergent strains of H2 influenza and prepare for a possible pandemic.

Keywords: H2N2; adenovirus; consensus; influenza; species C; vaccine.

Figure 1

Characterization of centralized consensus human H2 immunogen and study strains. (A) The genetic relationships between the centralized consensus and wildtype H2 influenza A virus strains are shown in an unrooted neighbor-joining phylogenetic tree. H2-Con (blue arrow) localizes to the center of all human H2 strains isolated between 1957 and 1968. Reference strains isolated from 1957 to 1964 (red arrows) and used in this study are labeled on the tree. (B) Western blot analysis of H2-Con (~72 kDa) in Ad6 and Ad5 viral vectors from two independent experiments. GAPDH (~33 kDa) was probed as a cellular loading control. (C) Percent sequence similarity of the wildtype strains used in the study compared to H2-Con.

Figure 2

Hemagglutinin inhibition (HI) titers after prime vaccination. BALB/c mice (n = 5) were immunized with 10¹⁰ viral particles (vp) of Ad5-H2-Con, Ad6-H2-Con, or DPBS. Sera from immunized animals were assayed for HI antibody titers against a range of divergent human H2 influenza A viruses. Data are represented as the mean HI titer with standard error (SEM). (n = 5; statistical analysis based on one-way ANOVA with Tukey multiple comparison; * p < 0.05, ** p < 0.01).

Figure 3

Hemagglutinin inhibition (HI) titers after boost vaccination with homologous or heterologous vaccine regimen. BALB/c mice (n = 5) were immunized with 10¹⁰ vp of Ad5-H2-Con or Ad6-H2-Con and boosted with their homologous or heterologous adenovirus serotype. HI titers against Taiwan/64 (A), Japan/62 (B), Korea/68 (C), Singapore/57 (D), Japan/57 (E), and RI/57 (F) after homologous or heterologous boosting are shown. Data are represented as the mean HI titer with standard error (SEM). The dotted line indicates a protective titer of ≥ 1:40 (n = 5; one-way ANOVA with Tukey multiple comparison; * p < 0.05, ** p < 0.01, *** p < 0.005).

Figure 4

Protection against challenge with a lethal human H2 isolate. BALB/c mice (n = 10) were vaccinated with 10¹⁰ vp of Ad5-H2-Con, Ad6-H2-Con, or PBS and boosted with either homologous or heterologous vaccine regimen. Mice were challenged with 20MLD₅₀ of A/Korea/426/1968, then monitored for morbidity (A), clearance of viral RNA from the lungs (B), and mortality (C). Mice that lost 25% of their initial weight were humanely euthanized (n = 5; one-way ANOVA with Tukey multiple comparison; * p < 0.05, ** p < 0.01, *** p < 0.005).