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. 2020 Aug 21;8(3):465.
doi: 10.3390/vaccines8030465.

Anti-Influenza Protective Efficacy of a H6 Virus-Like Particle in Chickens

Wan-Zhen Zhu  1 , Yi-Chi Wen  1 , Shu-Yi Lin  1 , Ting-Chih Chen  1 , Hui-Wen Chen  1
Affiliations

Anti-Influenza Protective Efficacy of a H6 Virus-Like Particle in Chickens

Wan-Zhen Zhu et al. Vaccines (Basel). .

Abstract

H6 avian influenza viruses (AIVs) have a worldwide distribution, and they pose a potential concern for public health. In Taiwan, H6 AIVs have circulated in domestic chickens for more than 40 years, and certain strains have crossed the species barrier to infect mammals. With the goal of containing the disease, there is a pressing need to develop a safe and effective vaccine for pandemic preparedness. In this study, we prepared a virus-like particle (VLP) that consisted of the hemagglutinin (HA) and matrix protein 1 (M1) derived from a H6 AIV as a vaccine antigen, and we examined the immunogenicity and protective efficacy when combined with an adjuvant in a chicken model. Full-length HA and M1 protein genes were cloned and expressed using a baculovirus expression system, and VLPs were purified from the supernatant of insect cell cultures. We performed nanoparticle-tracking analysis and transmission electron microscopy to validate that the particle structure and properties resembled the native virions. In animal experiments, specific-pathogen-free chickens that received the H6 VLPs in combination with an adjuvant showed superior H6N1 virus-specific serum IgG and hemagglutination-inhibition antibody responses, which lasted more than 112 days. Following the H6N1 viral challenge, the vaccinated chickens showed reduced viral replication in the lungs, kidneys and conjunctival/cloacal shedding. The antibodies induced in the chickens by the vaccine were able to cross-react with the H6N1 human isolate and drifted avian H6N1 isolates. In summary, the H6 VLP vaccine elicited superb immunogenicity in vivo, and the use of an adjuvant further enhanced the antiviral protective efficacy. This vaccine formulation could potentially be used to manage H6 influenza virus infections in chickens.

Keywords: H6 avian influenza virus; adjuvant; hemagglutinin; matrix 1; vaccine; virus-like particles.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Generation and purification. A schematic diagram of H6 virus-like particle (VLP) construction and expression. (A) A schematic diagram of H6 VLP construction and expression. The recombinant plasmid carrying the hemagglutinin (HA) and matrix protein 1 (M1) genes derived from the H6 avian influenza virus (AIV) was transposed onto the bacmid DNA. (B) VLPs that assembled HA and M1 proteins were harvested in the rBac-H6M1-infected Sf21 culture supernatant. (C) The H6 VLPs were purified through sucrose density gradient centrifugation. (D) The hemagglutination activity of purified VLPs was verified with the HA test. (E) The expression of the HA and M1 proteins on the VLPs was analyzed using SDS-PAGE and validated by Western blot using the antiserum directed against H6 AIVs.
Figure 2
Figure 2
Characterization of H6 VLPs. (A) The size and surface zeta potential of H6 VLPs and native virions were measured using NanoSight. (B) The H6 VLPs and native virions were imaged using TEM under negative staining and immunogold staining using the H6/HA monoclonal antibody. Bar = 100 nm.
Figure 3
Figure 3
The immunogenicity of H6 VLPs with ISA 71 VG in a chicken model. (A) Specific-pathogen-free (SPF) chickens were prime-boost vaccinated on Days 0 and 21 with PBS, VLPs alone, or VLPs with commercial adjuvant ISA 71 VG, and blood samples were collected. H6N1 AIV-specific IgG titers were analyzed by ELISA on day 21. (B) H6N1 AIV-specific IgG titers were analyzed by ELISA on day 56. (C) H6N1 AIV-specific HI titers were analyzed by a HI test. Error bars represent the means ± S.D. The horizontal dotted lines mark the detection limit for the antibody titer. Serum IgG titers among groups were compared using one-way ANOVA followed by Dunnett’s multiple-comparison test. The HI titers among vaccination groups and the control group were compared using two-way ANOVA followed by Tukey’s multiple-comparison test, and the differences between the VLP alone and ISA 71 VG-adjuvanted groups are shown (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
Figure 4
Figure 4
HI titers against the H6N1 human isolate and drifted avian H6N1 isolates. Cross-reactive antibody titers against the H6N1 human isolate and drifted avian H6N1 isolates from the VLP plus ISA 71 VG adjuvant vaccinated chickens were analyzed at 35 days post immunization (dpi) and 77 dpi using a HI test. Error bars represent the means ± S.D. The horizontal dotted lines mark the detection limit for the antibody titer. The horizontal dotted line marks the detection limit for viral copies. Titers among groups were compared by one-way ANOVA followed by Tukey’s multiple-comparison test. ns: non-significant.
Figure 5
Figure 5
The protective efficacy of H6 VLPs in a chicken model. (A) The vaccine immunization and virus challenge schedule in chickens. Three weeks after the booster immunization, chickens were intranasally challenged with 106 50% egg-infective dose (EID50) H6 AIV. (B) Viral RNA in tears at 4 dpc was analyzed by RT-qPCR. Error bars represent the means ± S.D. The horizontal dotted line marks the detection limit for viral copies. Viral copies among groups were compared by one-way ANOVA followed by Tukey’s multiple-comparison test.
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