Immunogenicity and Protective Efficacy of Five Vaccines Against Highly Pathogenic Avian Influenza Virus H5N1, Clade 2.3.4.4b, in Fattening Geese

Abstract

Background/Objectives: The risk of the introduction of highly pathogenic avian influenza virus (HPAIV) in geese breeding and fattening flocks is heightened due to the necessity of free-range access to grazing grounds. This study aimed to evaluate the safety, immunogenicity, and protective efficacy of five commercial vaccines against HPAIV subtype H5N1 (clade 2.3.4.4b) in subadult fattening geese. Methods: A prime-boost vaccination trial was conducted using five commercial vaccines, including H5 expressing vaccines of novel technology (subunit, vector, RNA) and whole inactivated virus (WIV) vaccines. Based on serological results, one RNA and one WIV vaccine were selected for a homologous challenge experiment. Results: Two vaccines of novel technology (vector, RNA) required a booster dose to raise specific antibodies titers above a threshold of four log₂ using a hemagglutination inhibition (HI) assay, whereas a subunit vaccine and two WIV vaccines induced seroconversion after primary vaccination. In the challenge experiment, all unvaccinated control geese succumbed to infection by day four. In contrast, all vaccinated geese that had seroconverted exhibited full clinical protection. Although sterile immunity was not achieved, viral excretion was significantly reduced in the vaccinated groups compared to controls. Conclusions: Vaccination substantially mitigated the impact of HPAIV H5N1, clade 2.3.4.4b infection in geese, greatly improving animal welfare by preventing severe disease. Additionally, there was a significant reduction in viral burden. Further studies are necessary to verify the potential of these vaccines to reduce susceptibility to infection and virus excretion in order to achieve suppression of the between-flock reproduction number to < 1 in geese flocks at high risk of infection.

Keywords: H5N1; HPAIV; free range; goose; poultry; protection; vaccination.

Figure 1

Vaccination scheme. Vaccination included a prime immunization followed by a second booster immunization 4 weeks later. While four of the groups received the first immunization at 6 weeks of age, due to a delay during international shipment, the #BI group was first vaccinated at 10 weeks of age (*). A sixth group was kept under the same housing conditions but did not receive any vaccination (unvaccinated control group). Plasma samples taken at each vaccination time point and three weeks after the booster vaccination were used to determine the antibody response. Two vaccine groups with the highest HI antibody titers and the unvaccinated control group were exposed to a challenge infection with an observation period of up to 2 weeks, depending on the clinical status.

Figure 2

Comparative analysis of the antibody response after prime and booster vaccination with one of five commercial vaccines in five different serological assays. Plasma samples were taken after prime vaccination and three weeks after booster immunization and tested for the presence of AI virus antibodies through H5-ELISA (A), NP-ELISA (B), HI against H5 antigen (C), HI against Newcastle virus antigen (D) and SNT (E). Dotted lines on graphs (A–E) mark the respective cut-off, separating negative (below dotted line) from positive titers (above dotted line) (“indeterminate” area between dotted lines for ELISAs). Box-plots showing 25–75th percentiles and median titers.

Figure 3

Overview of plasma reactivity in HI assays against different antigens. Plasma samples from four vaccine groups were tested after booster immunization via HI assay against antigens of clade 2.3.4.4b: (A) A/chicken/Germany-NI/AI 4286/2022 and (B) A/chicken/Germany-SH/AI08298/2021. Heterologous antigen originated from clade 1.0 (C) A/chicken/Vietnam/P41-05 (R75/05). In addition, an NDV antigen was used: (D) Genotype 2.II (NDV Clone 30). Dotted lines on graphs mark the respective cut-off (4 log₂) [22], separating negative (below dotted line) from positive titers (above dotted line).

Figure 4

Comparison of Clinical scores (A) total (B) and individual daily (C) oropharyngeal viral shedding of infected groups. Individual clinical status was scored daily between 1 and 14 dpi (A). Total oropharyngeal shedding per goose (VE/mL [log₁₀]) was compared up to four days post infection, measured by calculating the area under the curve (AUC) values of all animals of the three challenged groups (B). The viral load in VE/mL [log₁₀] per oropharyngeal swab of each individual animal per day was plotted in (C). Colored symbols (red) indicate that infectious virus was recovered from these samples in LMH cell cultures, from swabs derived from the control group only as well as from one non-responder of the group. The levels of significance are indicated by *** (p < 0.001), **** (p < 0.0001), and ns (not significant).

Figure 5

Antibody responses in vaccinated geese following HPAIV H5N1 challenge infection. Sero-reactivity was tested using NP-(A) and H5-specific ELISA (B) as well as HI (C). Significance levels are indicated by ** (p < 0.01) and **** (p < 0.0001).