Broadening the H5N3 Vaccine Immunogenicity against H5N1 Virus by Modification of Neutralizing Epitopes

Abstract

The highly pathogenic avian influenza (HPAI) H5N1 virus remains to be one of the world's largest pandemic threats due to the emergence of new variants. The rapid evolution of new sub-lineages is currently the greatest challenge in vaccine development. In this study, we developed an epitope modified non-pathogenic H5N3 (A/duck/Singapore/97) vaccine for broad protection against influenza H5 subtype. H5N3 hemagglutinin (HA) mutant reassortant viruses with A/Puerto Rico/8/34 (PR8) backbone were generated by mutating amino acids at the 140th loop and 190th α-helix of hemagglutinin. The cross-neutralizing efficacy of reverse genetics-derived H5N3HA (RG-H5N3HA) mutants was confirmed by testing reactivity with reference chicken anti-H5N1 clade 2 virus sera. Furthermore, RG-H5N3HA mutant immunized mice induced cross-neutralizing antibodies and cross-protection against distinct H5N1 viral infection. Our findings suggest that the use of non-pathogenic H5 viruses antigenically related to HPAI-H5N1 allows for the development of broadly protective vaccines and reduces the need for biosafety level 3 (BSL3) containment facilities.

Keywords: antibody-guided design; epitope modified vaccine; highly pathogenic avian influenza H5N1 (HPAI-H5N1); neutralizing epitopes.

Figure 1

The reactivity of RG-H5N3HA and RGH5N3 HA mutants with H5N1 specific monoclonal antibodies (mAbs). Indirect immunofluorescence assay (IFA) of MDCK cells infected with RG-H5N3 or RG-H5N3HAM1 or RG-H5N3HAM2. Infected cells were fixed and stained with mAb 2D9 (H5 specific positive control) or 3H11 or 3E8, or H.

Figure 2

Measurement of systemic immune responses of vaccinated mice sera: Mice subcutaneously immunized on days 0 and 28 with RG-H5N3 or RG-H5N3HA_M1 or RG-H5N3HA_M2 viral vaccine. (A) Serum hemagglutination inhibition (HAI) assay. The serum HAI on day 49 against H5N1 viruses from clade 2.1 (A/Indonesia/CDC669/2006, A/Indonesia/CDC1031/2007), clade 2.2 (A/Nigeria/6e/2007, A/chicken/Egypt/12186F/2012) and clade 2.3 (A/Whooper Swan/Akita/1/2008, A/Hubei/1/2010, A/Anhui/01/2005). Each point represents the geometric mean titer (n = 6) ± standard error (SE). (** p < 0.01; *** p < 0.001, when compared with RG-H5N3), # represents <8 HAI titer; (B) serum virus microneutralization (VMN) assay on day 49 against H5N1 strains. Each point represents the geometric mean titer (n = 6) ± standard error SE. (** p < 0.01; *** p < 0.001, when compared with RG-H5N3), # represents <10 VMN titer; (C) measurement of Serum hemagglutinin (HA)-specific immunoglobulin G (IgG) antibody titer on day 49 against H5N1HA protein (A/Indonesia/5/2005) by indirect ELISA. Each point represents the geometric mean titer (n = 6) ± standard error SE. (* p < 0.05 when compared with RG-H5N3).

Figure 3

Protection of mice from lethal H5N1 virus challenge. Mice (n = 6/group) were subcutaneously immunized on days 0 and 28 with RG-H5N3 or RG-H5N3HA_M1 or RG-H5N3HA_M2 viral vaccine and PBS. Four weeks after the second vaccination, mice were intranasally infected with 5 50% mouse lethal dose (MLD₅₀) of a clade 2.3.2.1 H5N1 (RG-A/Hubei/1/2010) strain on day 49. Mice were monitored for survival for 14 days and the results were expressed in percent survival (A). Weight loss of the mice groups was also monitored throughout the 14-day observation period and the results were expressed in percent body weight compared to the start of the viral challenge (B).