Mapping of antigenic sites

The vaccine strain H5-Re8 contains surface genes from a clade 2.3.4.4g virus, A/chicken/Guizhou/4/13(H5N1) (GZ/4/13), and six internal genes from the high-growth A/Puerto Rico/8/1934 (H1N1) (PR8) virus. It differs antigenically from the H5-Re11 strain that harbors surface genes from a clade 2.3.4.4h virus, A/duck/Guizhou/S4184/2017(H5N6) (GZ/S4184/17), and internal genes from PR834. Sequence analysis indicates that there are 20 amino acid differences in HA1 between these two vaccines (Fig. 1).

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.

To elucidate which amino acid substitution(s) determines this antigenic difference, we used reverse genetics to rescue 20 H5-Re11 mutants. Firstly, we evaluated the growth properties of the mutants and then analyzed their antigenicity by using a HI assay with H5-Re8 and H5-Re11 antisera. As shown in Table 1, none of the mutations had an adverse effect on the growth properties; all the mutants grew to relatively high titers (Table 1). To our surprise, none of the substitutions changed the antigenicity of the mutants in the HI assay, except mutant H5-Re11_−126D (−, an amino acid deletion at position 126), which showed higher titer with H5-Re8 antiserum, with an HI titer that was 2-fold higher than that of the other mutants (Table 1). To distinguish the subtle antigenic difference, a panel of 19 mAbs was generated and used to evaluate the antigenicity of the mutants. The 19 mAbs identified seven mutants that displayed considerable antigenic differences in the HI assay (i.e., at least a 4-fold difference), namely H5-Re11_Q115L, H5-Re11_R120S, H5-Re11_N124D, H5-Re11_V140M, H5-Re11_T151I, H5-Re11_A156T, and H5-Re11_E185A (Fig. 2). Thus, eight antigenic sites in H5 HA, 115, 120, 124, 126, 140, 151, 156, and 185, were identified.

Table 1 Cross-reactive HI antibody titers of mutant viruses with different antiseraFig. 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.

Molecular basis of the antigenic difference between H5-Re8 and H5-Re11

We next investigated which combination of antigenic substitutions changed the antigenicity of H5-Re11 to that of H5-Re8 or vice versa. First, we categorized the eight antigenic positions into three groups: Group 1 containing positions 115, 120, and 156, which were the three positions that most changed the antigenicity of H5-Re11 (Fig. 2); Group 2 containing positions 140, 151, and 185, which changed the antigenicity of H5-Re11 to a lesser extent (Fig. 2); and Group 3 containing positions 124 and 126, which affected the antigenicity of H5-Re11 and resulted in the formation of a glycosylation site (Table 1 and Supplementary Fig. 1). Then, three mutants, H5-Re11_Q115L/R120S/A156T (H5-Re11 + 3), H5-Re11_Q115L/R120S/V140M/T151I/A156T/E185A (H5-Re11 + 6), and H5-Re11_Q115L/R120S/N124D/-126D/V140M/T151I/A156T/E185A (H5-Re11 + 8) were rescued by gradually adding groups of mutations into the H5-Re11 vaccine strain. Their antigenicity was analyzed by using the HI assay with chicken sera generated with one clade 2.3.4.4b virus, one clade 2.3.4.4g virus, and two 2.3.4.4h viruses that were antigenically different. Antigenic cartography showed that the antigenicity of mutant H5-Re11 + 8 was closest to that of GZ/4/13 (donor virus of H5-Re8), whereas mutants H5-Re11 + 6 and H5-Re11 + 3 were still antigenically different from GZ/4/13 (Fig. 3a and Supplementary Table 1). To confirm that the antigenic transition was caused by the mutations, a H5-Re8 mutant with eight corresponding mutations, H5-Re8_L115Q/S120R/D124N/D126-/M140V/I151T/T156A/A185E (H5-Re8 + 8), was generated and its antigenicity was evaluated. As expected, the antigenicity of H5-Re8 + 8 was similar to that of both H5-Re11 and GZ/S4184/17 (donor virus of H5-Re11) (Fig. 3a and Supplementary Table 1). Compared with the HI assay, the MN assay is considered the gold standard for the analysis of the antigenic properties of the viruses. Thus, the antigenicity of the mutants was further confirmed by the MN assay (Supplementary Table 2). Although the antigenic map generated with the HI data differed from that generated with the MN data (Fig. 3b), the MN map confirmed that the antigenic transition between H5-Re11 and H5-Re8 was caused by substitutions at the eight antigenic sites.

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.

Selection of a broad-spectrum vaccine candidate based on antigenic distance

Since the implementation of the first clade 2.3.4.4g vaccine (H5-Re8) in 2015, three more vaccines, H5-Re11 (2.3.4.4h), H5-Re13 (2.3.4.4h), and H5-Re14 (2.3.4.4b), were generated to control clade 2.3.4.4 antigenic variants in China34,35,36. The frequent updating of vaccine strains highlights the need to develop a vaccine strain with broader protective efficacy. Our antigenic cartography analysis revealed that the mutant H5-Re11 + 3 was located in the relative center of the map, indicating its potential to serve as a broad-spectrum vaccine candidate against H5 viruses bearing the clade 2.3.4.4 HA gene. To evaluate the protective breath of H5-Re11 + 3, homologous serum of H5-Re11 + 3 was generated and applied for antigenic analysis with all eight subclades of 2.3.4.4 viruses (2.3.4.4a–h). In the HI assay, viruses in subclades 2.3.4.4a, 2.3.4.4c, and 2.3.4.4f reacted poorly with the H5-Re11 + 3 serum, with an HI titer of 16, whereas the HI titer of H5-Re11 + 3 against viruses in other subclades was 32–512 (Supplementary Table 3). In the antigenic map generated with the HI data, the breadth of the H5-Re11 + 3 serum response was calculated as described by Ron et al. (Fig. 4a)37. The H5-Re11 + 3 serum covered viruses in six subclades of 2.3.4.4, except 2.3.4.4c and 2.3.4.4f, which have not circulated since 2019 and 2017, respectively38,39 (Supplementary Fig. 2). We also evaluated the protective efficacy by using the MN assay, the gold standard for evaluating a vaccine’s protective capabilities. Notably, the data indicated that H5-Re11 + 3 serum reacted well with viruses in all eight subclades, with MN titers ranging from 64 to 1028, above the typical protective value (titers > 40) (Supplementary Table 4). As expected, in the antigenic map generated with the MN data (Fig. 4b), H5-Re11 + 3 serum was found to be located in the relative center of the antigenic map, and its breadth covered all eight subclades of the 2.4.4.4 viruses. Together, these data suggest that H5-Re11 + 3 is a potential broad-spectrum vaccine candidate against viruses belonging to clade 2.3.4.4.

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.

Protective efficacy of H5-Re11 + 3 inactivated vaccine against clade 2.3.4.4 viruses

Lastly, we assessed the protective efficacy of the H5-Re11 + 3 vaccine in chickens that were immunized with a single dose of the inactivated H5-Re11 + 3 vaccine. The chickens were then challenged with clade 2.3.4.4 antigenic variants that previously caused epidemics in poultry: one 2.3.4.4g virus, one 2.3.4.4b virus, and two 2.3.4.4h viruses belonging to two different antigenic groups (Fig. 4). The antigenic map (Fig. 4) indicated that H5-Re11 serum may confer relatively broad protection against other clade viruses. Therefore, vaccine strain H5-Re11 was used for comparison in the challenge study. The H5-Re11 + 3- inactivated vaccine was immunogenic in chickens; three weeks after the single dose, the mean HI titer in chickens against the homologous virus was 7.4 log2, which is 0.3 log2 higher than that of the H5-Re11 vaccine (Fig. 5a, d, g, and j).

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.

The H5-Re8 vaccine was used to control clade 2.3.4.4g viruses between 2015 and 2018 and provided full protection against GZ/S4184/17 (clade 2.3.4.4h), which emerged in 20176. In the GZ/4/13 (clade 2.3.4.4g) virus-challenged group, the H5-Re11 + 3 vaccine induced a significantly higher HI titer against GZ/4/13 than that induced by H5-Re11 (Fig. 5a). All chickens in the control groups died within 4 days post-challenge (p.c.), and the two chickens that survived on day 3 p.c. shed high titers of viruses through both the oropharynx and cloaca (Fig. 5b, c). In the H5-Re11-vaccinated group, 60% of the birds died during the 14-day observation period, and all the birds shed virus in the oropharynx and cloaca on days 3 and 5 p.c., except for one chicken that died on day 7 post-immunization (p.i). However, in the H5-Re11 + 3-vaccinated group, no virus was recovered from the organs, and all the vaccinated birds were healthy during the 14-day observation period (Fig. 5b, c). These data indicate that H5-Re11 provides only partial protection against the 2.3.4.4g virus, whereas H5-Re11 + 3 offers complete protection against the same virus.

The H5-Re11 vaccine was used to control clade 2.3.4.4h viruses between 2018 and 20216. In the GZ/S4184/17 (clade 2.3.4.4h) virus-challenged group, the H5-Re11 + 3 vaccine induced a similar HI titer against GZ/S4184/17 to that induced by H5-Re11 (Fig. 5d). All chickens in the control groups died, and the three chickens that survived on day 3 p.c. shed high titers of viruses in collected organs. In the H5-Re11 + 3 and H5-Re11-vaccinated groups, no virus was recovered from the organs, and all the vaccinated birds were healthy during the 14-day observation period (Fig. 5e, f). These data demonstrate that H5-Re11 + 3, despite harboring three mutations, provides 100% protection against the GZ/S4184/17 virus.

The H5-Re13 vaccine was used instead of H5-Re11 to control antigenic variants in clade 2.3.4.4h, beginning in 20226. In the A/duck/Fujian/S1424/2020(H5N6) (FJ/S1424/20, donor virus of H5-Re13) virus-challenged groups, the H5-Re11 + 3 vaccine induced a significantly higher HI titer against FJ/S1424/20 than that induced by H5-Re11 (Fig. 5g). All chickens in the control groups died, and the three chickens that survived on day 3 p.c. shed high titers of viruses in collected swabs. In the H5-Re11-vaccinated group, all the chickens survived the 14-day observation period, but they all shed viruses in the two tested organs on day 3 p.c., while 8 and 4 chickens shed viruses in the oropharynx and cloaca swabs, respectively, on day 5 p.c. In contrast, all the chickens in the H5-Re11 + 3-vaccinated group were healthy and shed no virus for 14 days (Fig. 5h, i). These results show that H5-Re11 mitigates lethality but fails to prevent virus shedding after FJ/S1424/20 challenge, whereas H5-Re11 + 3 affords comprehensive protection against the virus.

The H5-Re14 vaccine was used to control clade 2.3.4.4b virus, beginning in 20206. In the A/whooper swan/Shanxi/4-1/2020(H5N8) (SX/4-1/20, donor virus of H5-Re14) virus-challenged group, the H5-Re11 + 3 vaccine induced a significantly higher HI titer against SX/4-1/20 than that induced by H5-Re11 (Fig. 5j). Chickens in the control group all died within 4 days p.c., and the surviving birds shed viruses to high titers. In the H5-Re11-immunized group, no chicken died and no virus was shed in the cloaca swabs; however, the virus was detected in oropharyngeal swabs from 4 chickens on days 3 and 5 p.c. For chickens immunized with the H5-Re11 + 3 vaccine, no virus was detected in the tested swabs, and no symptoms were observed for 14 days (Fig. 5k, l). These data indicate that H5-Re11 + 3 offers 100% protection against the 2.3.4.4b virus.

Taken together, these results indicate that the H5-Re11 + 3 vaccine provides solid protection against four antigenically drifted clade 2.3.4.4 H5 viruses.