A broad-spectrum vaccine candidate against H5 viruses bearing different sub-clade 2.3.4.4 HA genes

Abstract

The global spread of H5 clade 2.3.4.4 highly pathogenic avian influenza (HPAI) viruses threatens poultry and public health. The continuous circulation of these viruses has led to their considerable genetic and antigenic evolution, resulting in the formation of eight subclades (2.3.4.4a-h). Here, we examined the antigenic sites that determine the antigenic differences between two H5 vaccine strains, H5-Re8 (clade 2.3.4.4g) and H5-Re11 (clade 2.3.4.4h). Epitope mapping data revealed that all eight identified antigenic sites were located within two classical antigenic regions, with five sites in region A (positions 115, 120, 124, 126, and 140) and three in region B (positions 151, 156, and 185). Through antigenic cartography analysis of mutants with varying numbers of substitutions, we confirmed that a combination of mutations in these eight sites reverses the antigenicity of H5-Re11 to that of H5-Re8, and vice versa. More importantly, our analyses identified H5-Re11_Q115L/R120S/A156T (H5-Re11 + 3) as a promising candidate for a broad-spectrum vaccine, positioned centrally in the antigenic map, and offering potential universal protection against all variants within the clade 2.3.4.4. H5-Re11 + 3 serum has better cross-reactivity than sera generated with other 2.3.4.4 vaccines, and H5-Re11 + 3 vaccine provided 100% protection of chickens against antigenically drifted H5 viruses from various 2.3.4.4 antigenic groups. Our findings suggest that antigenic regions A and B are immunodominant in H5 viruses, and that antigenic cartography-guided vaccine design is a promising strategy for selecting a broad-spectrum vaccine.

Fig. 1. Key amino acid alterations in the HA proteins of H5 viruses.

a Alignment of HA1 protein sequences (H5 numbering). Antigenic regions A, B, C, D, and E are delineated and highlighted in orange, green, cyan, blue, and pink, respectively. Sites with amino acid variations are marked and highlighted in red. b The site exhibiting amino acid variation is depicted in the HA monomer of an H5 virus, retrieved from the PDB database (5HUF). Amino acid substitutions at antigenic sites are denoted in red, and the respective antigenic regions are marked with corresponding colors.

Fig. 2. Antigenic heat map of H5-Re11 mutants.

The antigenicity of the H5-Re11 variants was determined by quantifying HI titers utilizing a panel of 19 mAbs generated in our laboratory. A 4-fold difference in antigenicity was deemed significant.

Fig. 3. Antigenic cartography of the indicated mutants and reference viruses.

a Antigen map constructed using HI titer data. b Antigen map generated based on MN titers. Each square represents a 2-fold difference in either HI or MN titers. Viruses and serum samples are represented by circles and rectangles, respectively.

Fig. 4. Protective breadth of serum generated with the Re11 + 3 mutant.

a The scope of protective efficacy depicted in the antigenic map was constructed using HI titer data. b The scope of protective efficacy illustrated in the antigenic map was created with MN titer data. Each square represents a 2-fold difference in either HI or MN titers. Viruses and serum samples are represented by circles and rectangles, respectively. The dashed red circle on the map marks the region where the titers of the H5-Re11 + 3 serum fall below the thresholds of 32 or 64 for the HI and MN titers, respectively.

Fig. 5. Evaluation of the protective efficacy of H5-Re11 + 3 vaccine against clade 2.3.4.4 viruses in chickens.

Groups of birds were administered either the H5-Re11 + 3 vaccine, the Re11 vaccine, or a PBS control. Three weeks post-immunization, sera were collected from all experimental chickens and analyzed for HI antibody titers against both the vaccine strain and the challenge virus (a, d, g, and j). To assess virus shedding, oropharyngeal and cloacal swabs were collected from all surviving chickens on days 3 and 5 post-challenge (b, e, h, and k). Subsequently, all the chickens were challenged with 10⁵ EID₅₀ of the indicated virus in a 100-μl volume of PBS. Following the challenge, chickens were monitored for two weeks to observe signs of disease progression and mortality (c, f, i, and l). Virus titers presented are the mean values derived from the birds that survived, with error bars indicating the standard deviations. The blue pound symbols denote the instances where birds died before the specified day. In cases where fewer than ten birds survived, the exact number of survivors is noted. The dashed lines represent the lower limit of virus detection. Statistical significance (p < 0.05) was determined by using GraphPad software, utilizing an unpaired t-test method for the analysis.