Impact of Adjuvants on the Immunogenicity and Efficacy of Split-Virion H7N9 Vaccine in Ferrets

Abstract

Background: An effective vaccine is urgently needed against the H7N9 avian influenza virus. We evaluated the immunogenicity and protective efficacy of a split-virion H7N9 vaccine with or without the oil-in-water adjuvants in ferrets.

Methods: Ferrets were vaccinated with 2 doses of unadjuvanted, MF59 or AS03-adjuvanted A/Shanghai/2/2013 (H7N9) vaccine, and the induction of antibodies to hemagglutinin (HA) or neuraminidase proteins was evaluated. Ferrets were then challenged with wild-type H7N9 virus to assess the vaccine's protective efficacy. The vaccine composition and integrity was also evaluated in vitro.

Results: Adjuvanted vaccines stimulated robust serum antibody titers against HA and neuraminidase compared with the unadjuvanted vaccines. Although there was a difference in adjuvanticity between AS03 and MF59 at a lower dose (3.75 µg of HA), both adjuvants induced comparable antibody responses after 2 doses of 15 µg. On challenge, ferrets that received adjuvanted vaccines showed lower viral burden than the control or unadjuvanted vaccine group. In vitro examinations revealed that the vaccine contained visible split-virus particles and retained the native conformation of HA recognizable by polyclonal and monoclonal antibodies.

Conclusions: The adjuvanted H7N9 vaccines demonstrated superior immunogenicity and protective efficacy against H7N9 infection in ferrets and hold potential as a vaccination regimen.

Keywords: H7N9; adjuvants; ferrets; influenza; vaccine.

Figure 1.

Viral titers in the respiratory tracts of vaccinated and saline control ferrets after challenge with A/Anhui/1/2013 (H7N9) virus. Virus titers in the upper respiratory tract were determined from nasal wash samples collected on days 3, 5, and 7 after challenge. Viral titers are presented as means and standard deviations. Linear mixed model with Kenward–Roger corrections were applied to investigate log₁₀ -transformed viral titers across various groups, and differences were considered statistically significant at P ≤ .05. Abbreviations: PBS, phosphate-buffered saline; TCID50, tissue culture infectious dose-50.

Figure 2.

Relationship between hemagglutination inhibition (HI) (A), virus neutralization (VN) (B), neuraminidase (NA) (C), and hemagglutinin (HA)–immunoglobulin (Ig) G antibody (D) titers with total viral load. The x-axes represent log-transformed antibody titers; the y-axes, the area under the curve (AUC) of a viral shedding curve as a measure of cumulative viral load throughout the study period. The AUC values here have been normalized to a percentage, with the minimum and maximum AUCs set to 0% and 100%, respectively. The R² value indicates the goodness of fit, and the antibody titer that correlates to 50% reduction in total viral load is expressed as the 50% inhibitory concentration (IC₅₀). Abbreviation: CI, confidence interval.

Figure 3.

Conformation and morphological evaluation of the vaccines. A, Reactivity of immune serum samples to native and denatured H7N9 vaccine, rg-A/Vietnam/1203/2004 (H5N1) vaccine, and seasonal trivalent influenza vaccine (TIV; Fluzone) by enzyme-linked immunosorbent assay. The polyclonal serum samples used against the respective vaccine types were ferret polyclonal serum samples raised against rg-A/Anhui/1/2013 (H7N9), rg-A/Vietnam/1203/04 (H5N1), and A/California/04/2009 (pdmH1N1), and the monoclonal antibodies used were H7 (clone 1H11; Abcam), H5 (clone 8D2; Abcam), and H1 specific. Wells coated with buffer only served as background control. B, Coomasie-stained polyacrylamide gel electrophoresis gel and Western blot analysis of native (N) and denatured (D) preparation of the TIV, H7N9, and H5N1 vaccines. The blot was allowed to be overexposed to visualize the less abundant hemagglutinin (HA) monomer in the reduced samples. C, Electron micrograph of the vaccines. Small spherical structures (arrowheads) and large spherical structures with glycoproteinlike projections emanating from the convex face (asterisks) are seen. Images were captured at ×29 000 magnification for TIV and H7N9 and ×50 000 magnifications for H5N1 vaccines. Abbreviations: NP, nucleoprotein; OD, optical density.