Impact of Individual Viral Gene Segments from Influenza A/H5N8 Virus on the Protective Efficacy of Inactivated Subtype-Specific Influenza Vaccine

Abstract

Since its emergence in 2014, the highly pathogenic avian influenza H5N8 virus has continuously and rapidly spread worldwide in the poultry sector resulting in huge economic losses. A typical inactivated H5N8 vaccine is prepared using the six internal genes from A/PR8/1934 (H1N1) and the two major antigenic proteins (HA and NA) from the circulating H5N8 strain with the HA modified to a low pathogenic form (PR8_HA/NA-H5N8). The contribution of the other internal proteins from H5N8, either individually or in combination, to the overall protective efficacy of PR8-based H5N8 vaccine has not been investigated. Using reverse genetics, a set of PR8-based vaccines expressing the individual proteins from an H5N8 strain were rescued and compared to the parent PR8 and low pathogenic H5N8 strains and the commonly used PR8_HA/NA-H5N8. Except for the PR8-based vaccine strains expressing the HA of H5N8, none of the rescued combinations could efficiently elicit virus-neutralizing antibodies. Compared to PR8, the non-HA viral proteins provided some protection to infected chickens six days post infection. We assume that this late protection was related to cell-based immunity rather than antibody-mediated immunity. This may explain the slight advantage of using full low pathogenic H5N8 instead of PR8_HA/NA-H5N8 to improve protection by both the innate and the humoral arms of the immune system.

Keywords: H5N8; PR8-based influenza vaccine; humoral immunity; innate immunity; vaccine efficacy.

Figure 1

List of successfully generated reassortant viruses using reverse genetics. Plasmids of A/Puerto Rico/8/34 (H1N1, PR8) are shown in grey while A/green-winged teal/Egypt/871/2016 (H5N8) plasmids are shown in red.

Figure 2

Virus Microneutralization (VMN) and hemagglutination inhibition (HI) assays for evaluating antibody responses at different weeks post vaccination (WPV) to AI H5N8 virus in vaccinated chicken groups with full LP-H5N8, PR8HA/NA-H5N8, and seven groups including one segment of H5N8 virus plus seven segments of PR8, and the control group.

Figure 3

Survival rate of vaccinated chicken groups different days post infection (DPI) with the HP H5N8 virus.

Figure 4

Viral shedding in vaccinated chicken groups at day 3 post challenge with the HP H5N8 virus in oral and cloacal swabs (A) and lungs (B).

Figure 5

Cytokine expression levels in the lungs of vaccinated chicken groups post challenge with the HP H5N8 virus normalized to vaccinated non-challenged chickens. The control group is unvaccinated–challenged chickens normalized to unvaccinated–uninfected chickens. Stars represent the statistical significance in expression levels of marked groups (* indicates p value < 0.05; *** indicates p value < 0.001).