Updating the National Antigen Bank in Korea: Protective Efficacy of Synthetic Vaccine Candidates against H5Nx Highly Pathogenic Avian Influenza Viruses Belonging to Clades 2.3.2.1 and 2.3.4.4

Abstract

Since 2018, Korea has been building an avian influenza (AI) national antigen bank for emergency preparedness; this antigen bank is updated every 2 years. To update the vaccine strains in the antigen bank, we used reverse genetics technology to develop two vaccine candidates against avian influenza strains belonging to clades 2.3.2.1d and 2.3.4.4h, and then evaluated their immunogenicity and protective efficacy in SPF chickens challenged with H5 viruses. The two vaccine candidates, named rgCA2/2.3.2.1d and rgES3/2.3.4.4h, were highly immunogenic, with hemagglutination inhibition (HI) titers of 8.2−9.3 log2 against the vaccine strain, and 7.1−7.3 log2 against the lethal challenge viruses (in which the HA genes shared 97% and 95.4% homology with that of rgCA2/2.3.2.1d and rgES3/2.3.4.4h, respectively). A full dose of each vaccine candidate provided 100% protection against the challenge viruses, with a reduction in clinical symptoms and virus shedding. A 1/10 dose provided similar levels of protection, whereas a 1/100 dose resulted in mortality and virus shedding by 7 dpi. Moreover, immunity induced by the two vaccines was long lasting, with HI titers of >7 log2 against the vaccine strain remaining after 6 months. Thus, the two vaccine candidates show protective efficacy and can be used to update the AI national antigen bank.

Keywords: SPF chicken; antigen bank; high pathogenic avian influenza; synthesis of HA; vaccine candidates.

Figure 1

Phylogenetic trees of clades 2.3.2.1 and 2.3.4.4 (HA; H5). The vaccine strains developed by reverse genetics are indicated in red. Each tree was built using the distance-based neighbor-joining method in MEGA6 software (bootstrap value = 1000). Red boxes indicate challenge strain and blue boxes indicate vaccine strain. (A) rgCA2/2.3.2.1d. (B) rgES3/2.3.4.4h.

Figure 2

Survival of chickens inoculated with a single dose of one of the two inactivated vaccines (a full dose, a 1/10 dose, a 1/100 dose, or sham) and then challenged with highly pathogenic H5 viruses. (A) rgCA2/2.3.2.1d. (B) rgES3/2.3.4.4h.

Figure 3

Hemagglutination inhibition (HI) assay titers in chickens vaccinated at different times with a full dose, a 1/10 dose, or a 1/100 dose of each candidate vaccine. HI titers were assessed at 14 days post-vaccination (dpv), at 21 dpv, and at 14 days post-infection. (A) HI titer of rgCA2/2.3.2.1d against challenge virus antigen; (B) HI titer of rgES3/2.3.4.4h against challenge virus antigen; (C) HI titer of rgCA2/2.3.2.1d against vaccine strain antigen; and (D) HI titer of rgES3/2.3.4.4h against vaccine strain antigen. Individual data points are shown, along with the mean and standard error.

Figure 4

Shedding of virus in oropharyngeal and cloacal swabs taken from chickens inoculated with inactivated vaccines (full dose, 1/10 dose, 1/100 dose, or sham) was assessed at 1, 3, 5, and 7 days post-infection with highly pathogenic H5 viruses (Figure 4). Viral titers are expressed as log₁₀TCID₅₀ (50% tissue culture infectious dose) per 0.1 mL, with error bars. (A) rgCA2/2.3.2.1d. (B) rgES3/2.3.4.4h. The lower limit of detection was 0.3 log₁₀ TCID₅₀ per 0.1 mL. * p value < 0.05.

Figure 5

Antibody persistence following vaccination with a full dose of each candidate vaccine. Hemagglutination inhibition (HI) assay titers were measured during the 24 weeks post-vaccination. Titers are expressed as log₂ values. The horizontal dotted line indicates a HI titer of 7 log₂, which is the standard threshold for prevention of virus shedding.