Recombinant virus-like particles elicit protective immunity against avian influenza A(H7N9) virus infection in ferrets

Abstract

In March 2013, diagnosis of the first reported case of human infection with a novel avian-origin influenza A(H7N9) virus occurred in eastern China. Most human cases have resulted in severe respiratory illness and, in some instances, death. Currently there are no licensed vaccines against H7N9 virus, which continues to cause sporadic human infections. Recombinant virus-like particles (VLPs) have been previously shown to be safe and effective vaccines for influenza. In this study, we evaluated the immunogenicity and protective efficacy of a H7N9 VLP vaccine in the ferret challenge model. Purified recombinant H7N9 VLPs morphologically resembled influenza virions and elicited high-titer serum hemagglutination inhibition (HI) and neutralizing antibodies specific for A/Anhui/1/2013 (H7N9) virus. H7N9 VLP-immunized ferrets subsequently challenged with homologous virus displayed reductions in fever, weight loss, and virus shedding compared to these parameters in unimmunized control ferrets. H7N9 VLP was also effective in protecting against lung and tracheal infection. The addition of either ISCOMATRIX or Matrix-M1 adjuvant improved immunogenicity and protection of the VLP vaccine against H7N9 virus. These results provide support for the development of a safe and effective human VLP vaccine with potent adjuvants against avian influenza H7N9 virus with pandemic potential.

Keywords: Avian influenza; Ferret; Influenza; Vaccine; Virus.

Fig. 1

Recombinant baculovirus (rBV) construct and expression of A/Anhui/1/2013 (H7N9) VLPs in Sf9 cells. (A) Influenza HA, NA and M1 were codon-optimized for high-level expression in Sf9 cells and synthesized biochemically. The HA, NA, and M1 genes were combined within rBV in a tandem fashion so that each gene was expressed from its own expression cassette that included polyhedrin promoter and polyadenylation signal. (B) Expression of H7N9 VLPs by SDS-PAGE (left panel) and Western blot. The molecular weight (MW) of uncleaved HA0 was approximately 62–64 kilodalton (kDa). Western blot was done using rabbit anti-H7N7 and mouse anti-influenza A M1 (right panel). (C) Properties and functions of the H7N9 VLPs, and (D) negative staining electron microscopy of H7N9 VLPs. Bar, 100 nm.

Fig. 2

Morbidity of VLP vaccinated ferrets following Anhui/1 virus challenge. Ferrets (6 per group) received two intramuscular inoculations (4 weeks apart) in the presence or absence of ISCOMATRIX adjuvant and control animals received PBS in place of vaccine. Following H7N9 virus challenge ferrets were weighed daily to measure morbidity. Average weights in each vaccine group were measured for the duration of the study, and percent original body weight was calculated based on the average starting weight for each group on day 0. Statistical significance from PBS group was determined by analysis of area under curve (AUC) with ANOVA and Bonferroni post-test; p < 0.05 for VLP versus PBS, p < 0.001 for VLP + ISCO versus PBS group.

Fig. 3

H7N9 VLP vaccine efficacy following Anhui/1/virus challenge. Ferrets (6 per group) received two intramuscular inoculations (4 weeks apart) in the presence or absence of ISCOMATRIX adjuvant and control animals received PBS in place of vaccine. The nasal cavities of challenged ferrets were washed with 1 ml PBS every other day beginning on day 2 p.c. and virus titers were determined. Statistical significances were determined by two-way ANOVA. ***Indicates statistical significance (p < 0.001) in viral titers between PBS and VLP-vaccinated groups for each day post-challenge.

Fig. 4

H7N9 VLP vaccine protection against lung replication. Ferrets (6 per group) received two intramuscular inoculations (5 weeks apart) in the presence or absence of Matrix-M1 adjuvant and control animals received PBS in place of vaccine. Animals were challenged i.n. with 10⁶ pfu of influenza A/Anhui/1/2013 (H7N9) virus. Viral titers of influenza virus in tissues on day 3 (A), and day 5 post-challenge (B) were determined by a standard plaque assay. Infectious virus was detectable in lung tissues of two of three vaccinated ferrets on day 3. The data shown are mean values plus standard deviations (error bars) for 3 ferrets per group for each time period. The differences in viral titers between groups were analyzed by two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5

H7N9 VLP vaccine protection against virus shedding. Ferrets (6 per group) received two intramuscular inoculations (5 weeks apart) in the presence or absence of Matrix-M1 adjuvant and control animals received PBS in place of vaccine. Animals were challenged i.n. with 10⁶ pfu of Anhui/1 virus. Nasal washes were collected prior to euthanasia (3 ferrets/time point) and titrated in a standard plaque assay. The differences in viral titers between groups were analyzed by two-way ANOVA. *p < 0.05, ***p < 0.001.