Dominant substitutions underlying the antigenic evolution of H5 influenza virus

Dominant substitutions underlying the antigenic evolution of H5 influenza virus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Dominant substitutions underlying the antigenic evolution of H5 influenza virus Qianqian Li, Aiping Wu, Youchun Wang, Mengyi Zhang, Luyao Qin, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6040842/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Nov, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Highly pathogenic avian influenza (HPAI) H5 viruses have recently been documented in mammals including humans, posing a major threat to global public health. To prevent a potential H5 pandemic, it is critical to elucidate the antigenic evolutionary pattern and identify key drivers underlying its evolution. In this study, we constructed a comprehensive antigenic map of H5 influenza viruses spanning their evolutionary history for the first time, revealing three distinct antigenic clusters (AC1, AC2, and AC3) with no cross-neutralization. In contrast to its sequential genetic evolution, AC3 lies between AC1 and AC2 in antigenic space. This divergence stems from two distinct mutation patterns at six key amino acid positions: (1) persistent mutations at positions 88 (N > R > S), 199 (D > N > S), and 205 (K > N > D), and (2) reversible mutations at positions 131 (Q > L > Q), 139 (S > P > S), and 289 (N > H > N). Moreover, single mutations at positions 205 and 289 can lead to significant immune escape. The risk clade of current interest, 2.3.4.4b belongs to AC2 and remains sensitive to current AC2-targeted vaccine strains. Additionally, clades 2.3.2.1c of AC1 and 2.3.4.4h of AC3 are also prevalent and capable of human infection, necessitating continuous surveillance of their epidemiological dynamics. These findings not only reveal the antigenic evolution mechanism of H5 influenza unseen in other influenza viruses, but also provide important guidance for vaccine strain selection and broad-spectrum vaccine development. Biological sciences/Evolution Biological sciences/Genetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Main Since the emergence of the highly pathogenic avian influenza (HPAI) H5N1 virus A/Goose/Guangdong/1/1996(GD/96), this subtype has rapidly spread through wild bird populations in Europe, Africa, North America, and Asia, with several lineages evolving [ 1 – 3 ] . Clade 2.3.4.4b, first detected in wild birds in Europe and Asia, caused multiple spillover events among poultry and mink at the end of 2021 [ 4 ] , followed by subsequent spread to wild mammals such as water rails, sea lions, red foxes and striped skunks [ 5 – 8 ] . In late March 2024, the United States Department of Agriculture (USDA) reported an outbreak of H5N1 virus in dairy cattle. More concerningly, a case of H5N1 infection in a dairy farm worker was reported in Texas in the same month [ 9 ] . Since then, clade 2.3.4.4b has expanded to 15 states, resulting in dozens of infections and one death in humans [ 10 , 11 ] . The expanding pattern of zoonotic transmission underscores the risk that continued viral evolution could enable sustained human-to-human transmission and potentially precipitate an influenza pandemic. In response to the potential threat of pandemic H5 viruses, nearly 30 vaccine stockpiles have been established globally, including A/Vietnam/1203/2004 (clade 1), A/Indonesia/05/2005 (clade 2.1), and more recently A/Astrakhan/3212/2020 (clade 2.3.4.4b) [ 12 – 15 ] . However, the protective efficacy of these stockpile vaccines against currently prevalent strains has yet to be evaluated. Given that the protective efficacy of vaccines is largely determined by the degree of antigenic match between vaccine strains and circulating viruses [ 16 – 18 ] , delineating the antigenic relationships of H5 viruses and identifying the key amino acid substitutions driving their antigenic evolution is essential for assessing the effectiveness of existing vaccines and informing future vaccine strain selection. To date, the antigenic evolution pattern of H5 virus and its key drivers remain poorly understood. The antigenic evolution from the first H5 isolate (GD/96) to present, especially the antigenic relationships between early clades and the currently prevalent clade 2.3.4.4b, urgently needs elucidation. Traditionally, viral antigenicity studies relied on hemagglutination inhibition (HI) assays, but experiments are extremely challenging due to the high pathogenicity of H5 viruses. By contrast, pseudotyped virus systems, which can be operated in biosafety level 2 laboratories, allow rapid generation of experimental data by incorporating H5 virus hemagglutination (HA) and neuraminidase (NA) genes [ 19 ] . Based on this advantage, we constructed an H5 pseudotyped virus library containing different clades and obtained sera from guinea pigs immunized with vaccine-recommended strains. Through systematic neutralization assays, we constructed a comprehensive antigenic map spanning the evolutionary history of H5 influenza viruses, revealed their unique antigenic evolutionary pattern and identified the key amino acid substitutions driving transitions between adjacent antigenic clusters. Additionally, we assessed the neutralizing capacity of stockpile vaccine-induced serum against prevalent clade 2.3.4.4b viruses. These findings not only advance our understanding of H5 influenza virus antigenic evolution, but also provide insights for vaccine strain selection and broad-spectrum vaccine development. Results Antigenic evolution of H5 influenza virus Following evolutionary analysis and intensive sampling of three H5 influenza subtypes (H5N1, H5N6, and H5N8), 136 representative strains were selected to establish the H5 pseudotyped virus library (Extended Data Fig. 1 , Extended Data Table 1, Extended Data Table 2). High-throughput neutralization assays were conducted using serum from guinea pigs immunized with 25 vaccine strains to systematically characterize the antigenic properties of H5 viruses. Based on the neutralization heatmap of H5 vaccine-immunized serum against representative strains, we identified three antigenic clusters of H5 viruses that do not cross-neutralize each other: AC1, AC2, and AC3 (Fig. 1 a). Similar patterns were observed across different H5 subtypes, including H5N1, H5N6, and H5N8 (Extended Data Fig. 2 ). In terms of genetic evolution, AC1 comprises five clades (0 ~ 9, 2.1*, 2.2*, 2.3.2*, 2.3.4*), AC2 comprises two clades (2.3.4.4b and 2.3.4.4*), and AC3 corresponds to clade 2.3.4.4h. The genetic map (Fig. 1 b, Extended Data Table 3) showed that the three antigenic clusters presented a sequential distribution in genetic space, with the genetic distance gradually increasing from AC1 through AC2 to AC3, aligning with the topology of the phylogenetic tree. However, the distribution of antigenic clusters (Fig. 1 c, Extended Data Table 4) showed significantly different characteristics, whereby AC3 was located between AC1 and AC2 in antigenic space, with similar antigenic distances to both clusters. These results demonstrate that the relationship between genetic and antigenic evolution of H5 influenza viruses is not strictly linear or directly corresponding. Transition mechanisms between adjacent antigenic clusters During antigenic evolution from AC1 to AC2, the eight specific mutations (SM: mutations that occur with a frequency of > 70% in one clade while having frequencies of 70% in two or more clades) resulted in an approximately 10-fold increase in immune escape from AC1-immunized serum (Fig. 2 a, b, Extended Data Fig. 3 , Extended Data Table 5). Further analysis revealed that amino acid substitutions at six key positions (6M) (N88R, Q131L, S139P, D199N, K205N, and N289H) among the CM collectively mediated this antigenic transition (Fig. 2 a). The combined mutant strain (AC1 + 6M) with mutations at these six positions based on AC1 showed approximately 30-fold enhanced escape from AC1-immunized serum, comparable to AC2 (Fig. 2 c). The single mutations K205N and N289H escaped neutralization by AC1-immunized serum more than 10-fold (Fig. 2 c), illustrating the importance of these two positions in the evolution of AC1 toward AC2. At the same time, the combined mutant strain (AC2-6M) with reversion mutations at these six positions based on AC2 showed comparable immune escape levels to AC1 when tested with AC2-immunized serum, further supporting this conclusion (Fig. 2 d). During antigenic evolution from AC2 to AC3, AC3 underwent a partial reversion of antigenicity and lay between AC1 and AC2 in antigenic space. This intermediate positioning in antigenic space suggested the presence of reverse mutations in AC3. Based on this hypothesis, we found that the same six positions 88, 131, 139, 199, 205, and 289 play key roles in antigenic evolution from AC2 to AC3. Among these positions, three positions showed persistent mutations (88R > 88S, 199N > 199S, and 205N > 205D), while the other three showed reversible mutations (131L > 131Q, 139P > 139S, and 289H > 289N). Forward validation showed that the combined mutant strain (AC2 + 6'M) with mutations at these six positions based on AC2 exhibited comparable immune escape levels to AC3 (Fig. 2 a, e) when tested against AC2-immunized serum. The reverse validation using an AC3-based mutant showed consistent results, further confirming the significance of these six mutations (Fig. 2 f). Additionally, a single point mutation, H289N, was sufficient to confer an approximately 50-fold immune escape from AC2-immunized serum, suggesting that position 289 plays a critical role in the antigenic transition from AC2 to AC3 (Fig. 2 e). Our systematic analysis identified six critical positions (88, 131, 139, 199, 205, and 289) as key determinants of two major antigenic transitions in H5 viral evolution. These six positions are distributed across multiple antigenic epitopes (Fig. 2 g, Extended Data Fig. 4 ) and exhibit two mutation patterns: persistent mutations at positions 88 (N > R > S), 199 (D > N > S), and 205 (K > N > D), contrasted with reversible mutations at positions 131 (Q > L > Q), 139 (S > P > S), and 289 (N > H > N) (Fig. 2 h, Extended Data Fig. 5a). This dual mutation pattern explains the position of AC3 between AC1 and AC2 in antigenic space. Mechanisms underlying antigenic differences among clades within AC1 Through phylogenetic analysis, AC1 and AC2 antigenic clusters were found to contain multiple evolutionary clades that could be divided into two distinct groups based on their neutralizing breadth. Group I vaccine strains showed broad cross-neutralization against all clades within their respective antigenic clusters, including clades 0 ~ 9 and 2.3.4* in AC1 (Fig. 3 a), as well as clades 2.3.4.4b and 2.3.4.4* in AC2 (Fig. 3 b). By contrast, Group II vaccine strains displayed clade-restricted neutralization, only neutralizing viruses within their own phylogenetic lineages, specifically the three AC1 clades 2.1*, 2.2*, and 2.3.2* (Fig. 3 c). Furthermore, we identified key mutations driving antigenic differences within AC1. Using the A/Goose/Guangdong/1/96 (GD/96) strain as the evolutionary origin, we found that the three specific mutations (SM1) in clade 2.1* did not cause significant immune escape, whereas the fifteen common mutations (CM1) resulted in an approximately 10-fold increase of immune escape (Fig. 3 d, e, Extended Data Fig. 3 , Extended Data Table 5). Similarly, the antigenic specificity of clades 2.2* and 2.3.2* was related to the common mutations CM2 and CM3, respectively, resulting in an approximately 10-fold reduction in neutralization titers (Fig. 3 f, g). Further analysis revealed that the antigenic specificity of clade 2.1* is determined by 7 mutations (K205R, N61D, N140D, D142E, E228K, S157P, and A172T), that of clade 2.2* by 8 mutations (N61D, D110N, N140D, D142E, H154Q, S171N, K205R, and E228K), and that of clade 2.3.2* by 10 mutations (K205R, N140D, D110N, H154Q, S171N, R69K, S145L, S149A, E243D, and L285V) (Fig. 3 e, f, g, h, Extended Data Fig. 5b, Extended Data Fig. 6). Moreover, we found that the single mutation A172T alone resulted in a 10-fold reduction in neutralization titers, whereas other individual mutations did not cause significant antigenic changes (Fig. 3 i). These results indicate that the antigenic specificity of these clades is primarily determined by synergistic effects of mutations at multiple positions. Structural analysis (Fig. 3 j, Extended Data Fig. 4 ) revealed that these key mutations were primarily concentrated in five epitopes, especially epitopes A (positions 140, 142, 149, 154, 157) and B (positions 171, 172, 205), suggesting that these regions are hotspots for antigenic variation in H5 viruses. Epidemiology of clade 2.3.4.4b and protective efficacy of existing vaccines Epidemiological data showed that AC1 was predominant before 2010, with its five clades (0 ~ 9, 2.1*, 2.2*, 2.3.2*, and 2.3.4*) emerging successively. Around 2010, AC2 emerged and gradually replaced AC1, with clade 2.3.4.4b becoming predominant. AC3, which emerged around 2015, underwent a period of expansion before gradually declining in prevalence (Fig. 4 a). Although human cases have been reported in all three antigenic clusters, there is no direct correspondence between the prevalence scale and the proportion of human infections. For example, while clade 2.3.4.4b in AC2 accounted for more than 90% of the total prevalence after 2020, the number of human cases was not significantly higher than in clade 2.3.2.1c of AC1 and 2.3.4.4h of AC3 (Fig. 4 a), which suggests that we need to be equally vigilant for all three antigenic clusters. Clade 2.3.4.4b belongs to AC2, which is currently of greatest interest. It evolved from the clade 2.3.4* of AC1, and has been paired with different NA genotypes. It has been found to infect avian, and mammals including humans (Fig. 4 b). Therefore, we further analyzed the ability of different vaccine strains to neutralize the currently prevalent clades of H5, including 2.3.4.4b, 2.3.4.4h, 2.3.2.1a, and 2.3.2.1c (Fig. 4 c, Extended Data Fig. 8, Extended Data Fig. 9). The results showed that sera from animals immunized with either AC2 vaccine strains (except VI20) or the bovine-origin human isolate TE24 (A/Texas/37/2024) exhibited high neutralizing activity against clade 2.3.4.4b viruses. Moreover, there was no significant difference in protection against different subtypes (H5N1, H5N6, H5N8) within clade 2.3.4.4b or against strains from different hosts (avian, human, and other mammals) (Fig. 4 d, e). These results suggested that the current recommended vaccine strains in the AC2 antigenic cluster can effectively protect against clade 2.3.4.4b. Discussion In this study, we constructed a comprehensive antigenic map spanning the evolutionary history of H5 influenza viruses for the first time. Although the H5 viruses evolved into multiple genetic clades, they can be clearly divided into three major antigenic clusters based on their antigenic properties. Each antigenic cluster contains multiple genetic clades that can be divided into two distinct groups based on their neutralizing breadth. Group I vaccine strains showed broad cross-neutralization against all clades within their respective antigenic clusters, while Group II vaccine strains displayed clade-restricted neutralization. These findings help us precisely characterize the antigenic evolution patterns of H5 viruses. Specifically, we identified a unique non-linear transition pattern among the three antigenic clusters (AC1, AC2, and AC3), with mutations at HA positions 88, 131, 139, 199, 205, and 289 driving these antigenic transitions. The persistent mutations at positions 88, 199, and 205, combined with the reversible mutations at positions 131, 139, and 289, explain why AC3 is located between AC1 and AC2 in antigenic space, in contrast to their sequential distribution in genetic space. The effect of these key positions on antigenicity is supported by other studies. For example, Li et al. [ 20 ] found that positions 88, 156, 205, 208, 239 and 289 play key roles in clade 2.3.4.4 antigenic drift, especially combined mutations of 205 and 208. Our study revealed that mutations at position 205 (epitope B, head region) and position 289 (epitope C, near neck region) could lead to significant immune escape. This pattern differs from typical human influenza viruses, where immune escape mutations are mainly concentrated in the head region [ 21 – 24 ] . This difference may be attributed to the fact that H5 viruses mainly circulate in avian populations, where they experience significantly less immune selection pressure compared to human influenza viruses. It is noteworthy that if H5 viruses were to acquire the human-to-human transmission ability, the mutation patterns may change significantly. Multiple genetic clades were identified within H5 antigenic clusters. Within AC1, vaccine strains from clades 0 ~ 9 and 2.3.4* showed broad neutralization ability against all clades within AC1, whereas vaccine strains from clades 2.1*, 2.2*, and 2.3.2* provided protection only against their respective clades. The antigenic specificity of these clades was driven by synergistic effects of multiple mutations, including N61D, R69K, D110N, N140D, D142E, S145L, S149A, H154Q, S157P, K205R, S171N, A172T, E228K, E243D, and L285V. Our findings are consistent with previous studies. Zhang et al. [ 2 ] identified six key positions 120, 126, 141, 156, 185, and 189 (corresponding to positions 136, 142, 157, 172, 201, and 205 in our numbering system). Similarly, Koel et al. [ 25 ] showed that antigenic changes of H5N1 clade 2.1 were mainly influenced by positions 129, 133, 151, 183, 185, and 189 (corresponding to positions 145, 149, 167, 199, 201 and 205 in our numbering system). Additionally, Li et al. [ 20 ] identified positions 156 and 205 as crucial antigenic determinant sites. Notably, position 205 was identified as a key site in many studies. Here, we found that position 205 underwent K > N > D substitutions during the evolution of AC1 > AC2 > AC3, and remained as R in three antigen-specific clades within AC1, highlighting its central role in H5 antigenic evolution. Within the AC2 antigenic cluster, clades 2.3.4.4b and 2.3.4.4* (including 2.3.4.4, 2.3.4.4c, 2.3.4.4e, and 2.3.4.4g) share antigenic similarity. Notably, the 2.3.4.4* clade was primarily found in the H5N6 subtype and eventually evolved into clade 2.3.4.4h (AC3) with H5N6 specificity, which is prevalent primarily in China. Based on a comprehensive analysis of antigenic and genetic characteristics, we propose that 2.3.4.4h be treated as a distinct evolutionary clade, distinguished from other 2.3.4.4 subclades. The risk clade 2.3.4.4b, which is currently causing global concern, belongs to the AC2 antigenic cluster. In response to the threat posed by this clade, the Centers for Disease Control and Prevention (CDC) has developed two candidate vaccine strains (CVVs): A/Astrakhan/3212/2020 (AS20) and A/American Wigeon/South Carolina/USDA-000345-001/2021 (CR21). Recent studies demonstrated that A/Texas/37/2024 (TE24), isolated from a farm worker in Texas, showed cross-reactivity with the CVVs and was effectively neutralized by ferret antisera induced by CVVs [ 26 ] . Our study also confirmed that sera from TE24-immunized guinea pigs neutralized clade 2.3.4.4b at a level comparable to AS20 and CR21. In addition, other vaccine strains within the AC2 antigenic cluster also showed effective neutralization against clade 2.3.4.4b viruses. However, novel mutations may emerge if this clade establishes sustained human-to-human transmission. Therefore, it is essential to continuously monitor the evolutionary dynamics of clade 2.3.4.4b and adjust the vaccine strain selection strategy in a timely manner. Notably, besides clade 2.3.4.4b from the AC2 antigenic cluster, clade 2.3.2.1c of AC1 and clade 2.3.4.4h of AC3 are also circulating and are capable of causing human infections. To counter the threat posed by these active H5 clades, we recommend establishing comprehensive surveillance systems while developing broadly protective H5 vaccines. Conclusions In this study, we constructed a comprehensive antigenic map of H5 influenza viruses, systematically elucidated their antigenic evolution patterns, and identified key amino acid substitutions driving antigenic changes. Our findings demonstrate that the antigenic evolution of H5 viruses can be divided into three major clusters (AC1, AC2, and AC3). In contrast to their sequential genetic evolution, AC3 lies between AC1 and AC2 in the antigenic space. This inconsistency is primarily driven by two mutation patterns at six key positions: persistent mutations at positions 88, 199, and 205, combined with reversible mutations at positions 131, 139, and 289. These findings advance our understanding of antigenic evolution mechanisms in H5 influenza viruses and provide direct guidance for optimizing vaccine strain selection, developing broad-spectrum vaccines, and preparing for potential pandemics. Methods Cells MDCK ( Canis familiaris , kidney, RRID: CVCL_0422) and 293T ( Homo sapiens , embryonic kidney, RRID: CVCL_0063) cell lines were obtained from the American Type Culture Collection. All cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM, high glucose; HyClone, Cat#SH30243.01) with 100 U/ml of penicillin-streptomycin solution (GIBCO, Cat#15140163), and 10% fetal bovine serum (TransGen Biotech, Cat#FS201) at 37°C in a humidified atmosphere with 5% CO 2 . Vaccine and representative strain selection A total of 136 representative strains were used in this study, including 24 vaccine strains (Extended Data Table 1), one bovine-origin human isolate (A/Texas/37/2024; abbreviated as TE24) and 111 other strains (Extended Data Table 2). The vaccine strains were recommended by the WHO for 2024–2025 ( https://www.who.int/teams/global-influenza-programme/ vaccines/who-recommendations/zoonotic-influenza-viruses-and-candidate-vaccine-viruses). The 111 sequences were selected as follows. As of 29 May 2024, 17,591, 2,623 and 3,571 HA protein sequences were downloaded from the Global Initiative on Sharing All Influenza Data ( GISAID) database for H5N1, H5N6 and H5N8, respectively. Taking H5N1 subtypes as an example, sequences from the human host were deduplicated using a similarity threshold of 0.98, resulting in 40 sequences; sequences from other mammalian hosts (excluding humans) were deduplicated using a similarity threshold of 0.99, resulting in 24 sequences; and sequences from avian hosts were deduplicated using a similarity threshold of 0.96, resulting in 12 sequences. Therefore, a total of 76 sequences were selected as representative H5N1 strains. Using the same strategy, 27 and 8 sequences were selected as representative strains for H5N6 and H5N8, respectively. In total, 111 H5 influenza virus strains, 24 vaccine strains, and TE24 were selected for subsequent pseudotyped virus construction. These representative strains covered all clades of H5 viruses and showed even distribution across the phylogenetic tree of H5, demonstrating their representativeness (Extended Data Fig. 1 ). Phylogenetic tree construction Multiple sequence alignment was performed using MAFFT v7.505 with the A/Goose/Guangdong/1/96 (H5N1) (GD/96) strain as the reference sequence. A maximum likelihood phylogenetic tree was constructed with FastTree v2.1.11 using the optimal amino acid substitution model. For evolutionary parameter estimation, divergence time and evolutionary rate were estimated within a Bayesian framework using BEAST v2.7.7 with an uncorrelated relaxed molecular clock model assuming a lognormal distribution. The Blosum62 substitution model was employed with gamma-distributed rate heterogeneity and a proportion of invariant sites. The MCMC chain was run for 50 million generations with sampling every 1,000 generations. The MCMC results were analyzed using Tracer v1.7.2 to ensure effective sample sizes greater than 200. Finally, a maximum clade credibility (MCC) tree was generated using TreeAnnotator with 20% burn-in and node heights set to posterior mean values. Site-directed mutagenesis The HA and NA protein sequences listed in Extended Data Table 2 were downloaded from GISAID. PcDNA3.1-HA and pcDNA3.1-NA recombinant plasmids were constructed by inserting the codon-optimized HA and NA sequences of H5 viruses into pcDNA3.1. The entire sequence was synthesized on the backbone plasmid pcDNA3.1(+) using General Biological System (Anhui, China). The pcDNA3.1-HA plasmid was used as the template to generate the plasmid harboring specific mutations of HA. Following site-directed mutagenesis PCR, the template chain was digested using Dpn I restriction endonuclease (NEB, USA). Afterwards, the PCR product was directly used to transform E. coli DH5a competent cells, after which single colonies were selected and the construct sequenced. Pseudotyped virus production On day 1, 293T cells were resuspended to a concentration of 5 ~ 7×10 5 cell/ml and seeded into a T75 culture flask (15 mL cell suspension per flask). Cells were incubated overnight at 37°C in a humidified incubator with 5% CO₂. On day 2, 293T cells were co-transfected with the HA plasmid, NA plasmid and HIV backbone plasmid (pSG3Δenv-FlucΔnef) at a mass ratio of 1:1:2 using transfection reagent Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA). These cells were incubated at 37°C with 5% CO 2 for 6 hours, after which the medium was replaced with fresh DMEM supplemented with 1% fetal bovine serum (FBS). At 48 hours post-transfection, the virus-containing supernatant was collected, filtered through a 0.45-µm pore-size polyethersulfone membrane (Millipore, Cat# SLHP033RB), aliquoted into 2 mL cryovials, and stored at − 80°C until further use. Production of immunized sera Animal experiments were conducted in strict accordance with the institutional animal care and use guidelines of the Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College (IMBCAMS, Yunnan, China). The experimental protocol received formal approval from the IMBCAMS Animal Ethics Committee (Approval No. DWSP20240616). Twenty-five experimental groups (n = 3 female guinea pigs per group; body weight 200–220 g) received intramuscular electroporation-mediated delivery of 200 µg pcDNA3.1-HA plasmid constructs (25 distinct variants, one plasmid per group) on days 0, 14, and 28. Immunization was repeated three times at two-week intervals, and serum samples were obtained two weeks after the third immunization. Serum samples were stored at ˗20°C, then thawed and heat-inactivated at 56°C for 30 min before use. Neutralization assay Immunized sera were diluted to an appropriate initial concentration and then subjected to a three-fold serial dilution. Subsequently, 100 µL of each serum dilution was added into a 96-well plate. The resulting dilutions were mixed with 50µL of pseudotyped viruses at a concentration of 1300 TCID50/ml and incubated at 37°C for 1 h. Afterwards, MDCK cells were added into the plates (2×10 4 cells/100 µL per well). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 48 h, after which the chemiluminescence signals were detected using the Britelite plus reporter gene assay system (PerkinElmer, Ensight). The pseudovirus neutralization titer was calculated using the Reed-Muench method in PerkinElmer Ensight software. The results are based on 3 replicates unless specified otherwise. Construction of antigenic and genetic maps The antigenic map was constructed based on pseudotyped virus neutralization titer data. Firstly, the raw data were log-transformed and normalized to convert the exponential differences between titers into a linear relationship. The T-distributed stochastic neighbor embedding algorithm was chosen for dimensionality reduction to maintain the local structural relationships of the data to more accurately show the complex antigenic differences among viral strains. In cluster analysis, hierarchical clustering was performed using Ward's method to form compact clusters by minimizing the within-cluster variance, which allowed us to discover virus groups with similar antigenicity. The genetic map was constructed based on the HA amino acid sequences of H5 viruses. After performing multiple sequence alignment, genetic diversity was quantified by calculating the proportion of variant sites between sequences. The same downscaling and clustering strategy was subsequently used to ensure methodological consistency between antigenic and genetic analyses. All data analyses were performed using Python, relying on the Biopython, scipy, and sklearn scientific computing libraries. Data analysis and processing GraphPad Prism 8 (GraphPad Software Inc, San Diego, CA, USA) was used for statistical analysis. Values were shown as geometric means with geometric standard deviations (SD). Declarations Data and Code Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. This paper does not include any original code. Any additional information required to reanalyze the data can also be obtained via email. Acknowledgments We gratefully acknowledge the authors from the originating and submitting laboratories where genetic sequence data were generated and shared via GISAID, enabling this research. This work was supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2022-I2M-3-001), Science and Technology Leading Talent Program of Yunnan Province (202405AB350002), National Key Research and Development Program of China (2023YFC2307900), State Key Laboratory Special Fund (2060204), National Natural Science Foundation of China (82372225), CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-061), CAMS Innovation Fund for Medical Sciences (CIFMS)(2023-PT330-01, 2023-I2M-2-005), CAMS Innovation Fund for Medical Sciences (2022-I2M-2-004), NCTIB Fund for R&D Platform for Cell and Gene Therapy and High-performance Computing Platform of Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College. Author contributions Q.L., A.W. and Y.W. conceived, designed, and supervised the experiments; M.Z., Z.L. and Q.H. immunized the animals; L.Q., M.Z., Y.M. and C.B. selected and constructed the representative pseudotyped virus library; M.Z., Z.L., J.C., J.T., H.L. and R.B. performed the neutralization assays; L.Q., M.Z. and J.L. analyzed the experimental data; X.D, and W.H. were supervision; M.Z. and L.Q. wrote the manuscript; All authors approved the final manuscript. Conflicts of Interest All authors declare no competing interests. References Gao F., Wang Q., Qiu C., et al. Pandemic preparedness of effective vaccines for the outbreak of newly H5N1 highly pathogenic avian influenza virus [J]. Virologica Sinica 39 , 981-985 (2024). Zhang Y., Cui P., Shi J., et al. A broad-spectrum vaccine candidate against H5 viruses bearing different sub-clade 2.3.4.4 HA genes [J]. NPJ Vaccines 9 , 152 (2024). Tian J., Bai X., Li M., et al. Highly Pathogenic Avian Influenza Virus (H5N1) Clade 2.3.4.4b Introduced by Wild Birds, China, 2021 [J]. Emerg Infect Dis 29 , 1367-1375 (2023). Koopmans M. P. G., Barton Behravesh C., Cunningham A. A., et al. The panzootic spread of highly pathogenic avian influenza H5N1 sublineage 2.3.4.4b: a critical appraisal of One Health preparedness and prevention [J]. The Lancet Infectious Diseases 24 , e774-e781 (2024). Xie R., Edwards K. M., Wille M., et al. The episodic resurgence of highly pathogenic avian influenza H5 virus [J]. Nature 622 , 810-817 (2023). Peacock T. P., Moncla L., Dudas G., et al. The global H5N1 influenza panzootic in mammals [J]. Nature 637 , 304-313 (2025). Plaza P. I., Gamarra-Toledo V., Rodríguez Euguí J., et al. Pacific and Atlantic sea lion mortality caused by highly pathogenic Avian Influenza A(H5N1) in South America [J]. Travel Med Infect Dis 59 , 102712 (2024). Alkie T. N., Cox S., Embury-Hyatt C., et al. Characterization of neurotropic HPAI H5N1 viruses with novel genome constellations and mammalian adaptive mutations in free-living mesocarnivores in Canada [J]. Emerg Microbes Infect 12 , 2186608 (2023). Uyeki T. M., Milton S., Abdul Hamid C., et al. Highly Pathogenic Avian Influenza A(H5N1) Virus Infection in a Dairy Farm Worker [J]. N Engl J Med 390 , 2028-2029 (2024). Good M. R., Fernández-Quintero M. L., Ji W., et al. A single mutation in dairy cow-associated H5N1 viruses increases receptor binding breadth [J]. Nat Commun 15 , 10768 (2024). CDC. First H5 Bird Flu Death Reported in United States. https://www.cdc.gov/media/releases/2025/m0106-h5-birdflu-death.html [Z]. (2025) WHO. Zoonotic influenza: candidate vaccine viruses and potency testing reagents.https://www.who.int/teams/global-influenza-programme/vaccines/who-recommendations/zoonotic-influenza-viruses-and-candidate-vaccine-viruses [Z]. (2024) Khurana S., King L. R., Manischewitz J., et al. Licensed H5N1 vaccines generate cross-neutralizing antibodies against highly pathogenic H5N1 clade 2.3.4.4b influenza virus [J]. Nature Medicine 30 , 2771-2776 (2024). Khurana S., Chearwae W., Castellino F., et al. Vaccines with MF59 Adjuvant Expand the Antibody Repertoire to Target Protective Sites of Pandemic Avian H5N1 Influenza Virus [J]. Sci Transl Med 2 , 15ra15-15ra15 (2010). Beigel J. H., Voell J., Huang C.-Y., et al. Safety and Immunogenicity of Multiple and Higher Doses of an Inactivated Influenza A/H5N1 Vaccine [J]. The Journal of Infectious Diseases 200 , 501-508 (2009). Chen L. M., Donis R. O., Suarez D. L., et al. Biosafety risk assessment for production of candidate vaccine viruses to protect humans from zoonotic highly pathogenic avian influenza viruses [J]. Influenza Other Respir Viruses 14 , 215-225 (2020). Okoli G. N., Racovitan F., Abdulwahid T., et al. Variable seasonal influenza vaccine effectiveness across geographical regions, age groups and levels of vaccine antigenic similarity with circulating virus strains: A systematic review and meta-analysis of the evidence from test-negative design studies after the 2009/10 influenza pandemic [J]. Vaccine 39 , 1225-1240 (2021). Uyeki T. M., Hui D. S., Zambon M., et al. Influenza [J]. The Lancet 400 , 693-706 (2022). Carnell G. W., Ferrara F., Grehan K., et al. Pseudotype-based neutralization assays for influenza: a systematic analysis [J]. Front Immunol 6 , 161 (2015). Li J., Gu M., Liu K., et al. Amino acid substitutions in antigenic region B of hemagglutinin play a critical role in the antigenic drift of subclade 2.3.4.4 highly pathogenic H5NX influenza viruses [J]. Transbound Emerg Dis 67 , 263-275 (2020). Wiley D. C., Wilson I. A., Skehel J. J. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation [J]. Nature 289 , 373-378 (1981). Broecker F., Liu S. T. H., Sun W., et al. Immunodominance of Antigenic Site B in the Hemagglutinin of the Current H3N2 Influenza Virus in Humans and Mice [J]. J Virol 92 , e01100-18 (2018). Popova L., Smith K., West A. H., et al. Immunodominance of antigenic site B over site A of hemagglutinin of recent H3N2 influenza viruses [J]. PLoS One 7 , e41895 (2012). Wu N. C., Otwinowski J., Thompson A. J., et al. Major antigenic site B of human influenza H3N2 viruses has an evolving local fitness landscape [J]. Nat Commun 11 , 1233 (2020). Koel B. F., Van Der Vliet S., Burke D. F., et al. Antigenic variation of clade 2.1 H5N1 virus is determined by a few amino acid substitutions immediately adjacent to the receptor binding site [J]. mBio 5 , e01070-01014 (2014). Garg S., Reed C., Davis C. T., et al. Outbreak of Highly Pathogenic Avian Influenza A(H5N1) Viruses in U.S. Dairy Cattle and Detection of Two Human Cases - United States, 2024 [J]. MMWR Morb Mortal Wkly Rep 73 , 501-505 (2024). Additional Declarations There is NO Competing Interest. Supplementary Files Extendeddatafiguresandtables.docx ExtendeddataTable1.xlsx Extended Data Table1 ExtendeddataTable2.xlsx Extended Data Table2 ExtendeddataTable3.xlsx Extended Data Table3 ExtendeddataTable4.xlsx Extended Data Table4 ExtendeddataTable5.xlsx Extended Data Table5 Cite Share Download PDF Status: Published Journal Publication published 28 Nov, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6040842","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":417708304,"identity":"dabcffdf-8569-4061-8d66-fd97a725db8a","order_by":0,"name":"Qianqian 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11:35:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6040842/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6040842/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-65730-y","type":"published","date":"2025-11-28T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77230102,"identity":"a7efb497-386d-49e2-a6cb-2a2ceca7f285","added_by":"auto","created_at":"2025-02-26 12:18:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":954423,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntigenic relationships of H5 influenza viruses.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e molecular clock evolutionary tree of representative H5 viruses and heatmap of neutralization titers of corresponding pseudotyped viruses. In the phylogenetic tree, the blue, red and green areas indicate antigenic cluster AC1, AC2 and AC3, respectively. The representative strains are represented by circles, and the vaccine strains are represented by triangles with black edges. 2.1* comprises clade 2.1 and its descendants. 2.2* comprises clade 2.2 and its descendants. 2.3.2* comprises clade 2.3.2 and its descendants. 2.3.4* comprises 2.3.4 clade and its descendants, excluding 2.3.4.4. 2.3.4.4* comprises clades 2.3.4.4, 2.3.4.4c, 2.3.4.4e, and 2.3.4.4g. The vertical coordinates in the heatmap indicate the abbreviation of each vaccine strain and clade. Values in the heatmap indicate the logarithm (log10) of neutralization titer ratio between representative strain and vaccine strain. \u003cstrong\u003eb-c, \u003c/strong\u003eGenetic map (b) and antigenic map (c) of H5 viruses. Arrows indicate the evolutionary direction.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6040842/v1/9903425a3e9bb6f3cc79e040.png"},{"id":77231752,"identity":"ee739d2f-735f-4ec5-a916-44fe00418de8","added_by":"auto","created_at":"2025-02-26 12:34:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":740605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKey amino acid substitutions driving the transitions between adjacent antigenic clusters of H5.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003eDetailed information of the mutations subjected to experimental validation. \u003cstrong\u003eb,\u003c/strong\u003eRoles of the common mutations (CM) as well as the specific mutations (SM) in the evolution of AC1 toward AC2. \u003cstrong\u003ec,\u003c/strong\u003e Forward validation of six combined mutations (6M: N88R, Q131L, S139P, D199N, K205N, N289H) and single-position mutations involved in the antigenic evolution of AC1 toward AC2. \u003cstrong\u003ed,\u003c/strong\u003eReverse validation of six combined mutations involved in the antigenic evolution of AC1 toward AC2. \u003cstrong\u003ee,\u003c/strong\u003e Forward validation of six combined mutations (6’M: R88S, L131Q, P139S, N199S, N205D, H289N) and single-position mutations involved in the antigenic evolution of AC2 toward AC3.\u003cstrong\u003e f,\u003c/strong\u003eReverse validation of six combined mutations involved in the antigenic evolution of AC2 toward AC3. \u003cstrong\u003eg,\u003c/strong\u003e Epitopic distribution of positions 88, 131, 139, 199, 205, and 289 on the surface of the H5 HA protein, using the H3 epitope classification as a reference.\u003cstrong\u003e h, \u003c/strong\u003eEvolutionary trajectories of positions 88, 131, 139, 199, 205, and 289. Dashed lines separate AC1, AC2 and AC3, and different colors represent different amino acids.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6040842/v1/fb9abeaeefdf79ab9dba7ca5.png"},{"id":77230109,"identity":"b3371a77-53bd-4155-a1c5-078d91549fad","added_by":"auto","created_at":"2025-02-26 12:18:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1032804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCauses of antigenic differences among clades within AC1. a, \u003c/strong\u003eNeutralizing capacity of vaccine strains from clades 0~9 and 2.3.4* against all clades within AC1. \u003cstrong\u003eb, \u003c/strong\u003eNeutralizing capacity of vaccine strains from clades 2.3.4.4b and 2.3.4.4* against all clades within AC2. \u003cstrong\u003ec,\u003c/strong\u003eNeutralizing capacity of vaccine strains from clades 2.1*, 2.2*, and 2.3.2* against all clades within AC1. \u003cstrong\u003ed, \u003c/strong\u003eDetailed information of the mutations subjected to experimental validation. \u003cstrong\u003ee, \u003c/strong\u003eContributions of the common mutation CM1, the specific mutation SM1 as well as 7 mutations (7M: K205R, N61D, N140D, D142E, E228K, S157P, A172T) to antigenic differences between 2.1* and GD/96. \u003cstrong\u003ef,\u003c/strong\u003e Contributions of the common mutation CM2, the specific mutation SM2 as well as 8 mutations (8M: K205R, N61D, N140D, D142E, E228K, D110N, H154Q, S171N) to antigenic differences between 2.2* and GD/96. \u003cstrong\u003eg, \u003c/strong\u003eContributions of the common mutation CM3, the specific mutation SM3 as well as 10 mutations (10M: K205R, N140D, D110N, H154Q, S171N, R69K, S145L, S149A, E243D, L285V) to antigenic differences between 2.3.2* and GD/96. \u003cstrong\u003eh,\u003c/strong\u003e Topological structure among clades within AC1 and evolutionary trajectories of clades 2.1*, 2.2* and 2.3.2*.\u003cstrong\u003e i,\u003c/strong\u003e Immune escape analysis of single mutations N61D, R69K, D110N, N140D, D142E, S145L, S149A, H154Q, S157P, S171N, A172T, K205R, E228K, E243D, and L285V based on GD/96.\u003cstrong\u003e j, \u003c/strong\u003eEpitopic distribution of positions 61, 69, 110, 140, 142, 145, 149, 154, 157, 171, 172, 205, 228, 243, and 285 on the surface of the H5 HA protein, using the H3 epitope classification as a reference.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6040842/v1/46789f636bb0496194b0eb8a.png"},{"id":77230108,"identity":"5e7eeb98-1bed-43c9-be99-5cfe332e9d48","added_by":"auto","created_at":"2025-02-26 12:18:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":902070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEpidemiological analysis of clade 2.3.4.4b and evaluation of vaccine strain effectiveness against it.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e Temporal distribution of H5 influenza virus clades and human infection cases. \u003cstrong\u003eb,\u003c/strong\u003e Molecular clock evolutionary tree of clade 2.3.4.4b, showing NA subtypes and host distribution. \u003cstrong\u003ec,\u003c/strong\u003e Neutralizing capacity of vaccine strains from different antigenic clusters against clade 2.3.4.4b. Vaccine strains from different clades are shown in different colors. Circles in the columns indicate the strains in each clade.\u003cstrong\u003e d-e, \u003c/strong\u003eNeutralization evaluation of vaccine strains in AC2 against clade 2.3.4.4b viruses with different NA subtypes and from different hosts.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6040842/v1/d008648d324ddb4c00ce0f74.png"},{"id":97039819,"identity":"4e266985-eaf8-4e85-9098-7f74a2949146","added_by":"auto","created_at":"2025-11-29 08:07:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5024071,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6040842/v1/e2479d22-99fe-4c45-84da-1bf97b6f9bf8.pdf"},{"id":77230111,"identity":"f6b06c33-e530-43ae-9697-670c5e796c95","added_by":"auto","created_at":"2025-02-26 12:18:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2802888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Extendeddatafiguresandtables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6040842/v1/0f53fb6c4a46d682a5cdeab3.docx"},{"id":77231447,"identity":"9354708d-af93-4652-b5fc-14bd63ad9b1a","added_by":"auto","created_at":"2025-02-26 12:26:15","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11896,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Table1\u003c/p\u003e","description":"","filename":"ExtendeddataTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6040842/v1/01a821aa0b0e797d75f5f3fb.xlsx"},{"id":77231450,"identity":"46b46e52-76b3-469c-8ae0-62c4c3ce6962","added_by":"auto","created_at":"2025-02-26 12:26:15","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18985,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Table2\u003c/p\u003e","description":"","filename":"ExtendeddataTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6040842/v1/50dff481f23a3dd99d9b27d5.xlsx"},{"id":77230104,"identity":"2f796bab-c03b-42d9-8d34-342e87035401","added_by":"auto","created_at":"2025-02-26 12:18:15","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":106982,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Table3\u003c/p\u003e","description":"","filename":"ExtendeddataTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6040842/v1/510b91e99e21ae9e431cab73.xlsx"},{"id":77230110,"identity":"70afe20e-75e0-4500-9e5e-ea3933ada26c","added_by":"auto","created_at":"2025-02-26 12:18:16","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":220143,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Table4\u003c/p\u003e","description":"","filename":"ExtendeddataTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6040842/v1/02256ce9e8e5a8d34c070ce8.xlsx"},{"id":77231448,"identity":"2096d891-d0ed-493d-a54d-8129648c9cf2","added_by":"auto","created_at":"2025-02-26 12:26:15","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14800,"visible":true,"origin":"","legend":"\u003cp\u003eExtended Data Table5\u003c/p\u003e","description":"","filename":"ExtendeddataTable5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6040842/v1/7a59a4f05d0ad5b2615538ec.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Dominant substitutions underlying the antigenic evolution of H5 influenza virus","fulltext":[{"header":"Main","content":"\u003cp\u003eSince the emergence of the highly pathogenic avian influenza (HPAI) H5N1 virus A/Goose/Guangdong/1/1996(GD/96), this subtype has rapidly spread through wild bird populations in Europe, Africa, North America, and Asia, with several lineages evolving\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Clade 2.3.4.4b, first detected in wild birds in Europe and Asia, caused multiple spillover events among poultry and mink at the end of 2021\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, followed by subsequent spread to wild mammals such as water rails, sea lions, red foxes and striped skunks \u003csup\u003e[\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. In late March 2024, the United States Department of Agriculture (USDA) reported an outbreak of H5N1 virus in dairy cattle. More concerningly, a case of H5N1 infection in a dairy farm worker was reported in Texas in the same month\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Since then, clade 2.3.4.4b has expanded to 15 states, resulting in dozens of infections and one death in humans \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. The expanding pattern of zoonotic transmission underscores the risk that continued viral evolution could enable sustained human-to-human transmission and potentially precipitate an influenza pandemic.\u003c/p\u003e \u003cp\u003eIn response to the potential threat of pandemic H5 viruses, nearly 30 vaccine stockpiles have been established globally, including A/Vietnam/1203/2004 (clade 1), A/Indonesia/05/2005 (clade 2.1), and more recently A/Astrakhan/3212/2020 (clade 2.3.4.4b) \u003csup\u003e[\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. However, the protective efficacy of these stockpile vaccines against currently prevalent strains has yet to be evaluated. Given that the protective efficacy of vaccines is largely determined by the degree of antigenic match between vaccine strains and circulating viruses \u003csup\u003e[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, delineating the antigenic relationships of H5 viruses and identifying the key amino acid substitutions driving their antigenic evolution is essential for assessing the effectiveness of existing vaccines and informing future vaccine strain selection.\u003c/p\u003e \u003cp\u003eTo date, the antigenic evolution pattern of H5 virus and its key drivers remain poorly understood. The antigenic evolution from the first H5 isolate (GD/96) to present, especially the antigenic relationships between early clades and the currently prevalent clade 2.3.4.4b, urgently needs elucidation. Traditionally, viral antigenicity studies relied on hemagglutination inhibition (HI) assays, but experiments are extremely challenging due to the high pathogenicity of H5 viruses. By contrast, pseudotyped virus systems, which can be operated in biosafety level 2 laboratories, allow rapid generation of experimental data by incorporating H5 virus hemagglutination (HA) and neuraminidase (NA) genes\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on this advantage, we constructed an H5 pseudotyped virus library containing different clades and obtained sera from guinea pigs immunized with vaccine-recommended strains. Through systematic neutralization assays, we constructed a comprehensive antigenic map spanning the evolutionary history of H5 influenza viruses, revealed their unique antigenic evolutionary pattern and identified the key amino acid substitutions driving transitions between adjacent antigenic clusters. Additionally, we assessed the neutralizing capacity of stockpile vaccine-induced serum against prevalent clade 2.3.4.4b viruses. These findings not only advance our understanding of H5 influenza virus antigenic evolution, but also provide insights for vaccine strain selection and broad-spectrum vaccine development.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAntigenic evolution of H5 influenza virus\u003c/h2\u003e \u003cp\u003eFollowing evolutionary analysis and intensive sampling of three H5 influenza subtypes (H5N1, H5N6, and H5N8), 136 representative strains were selected to establish the H5 pseudotyped virus library (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Extended Data Table\u0026nbsp;1, Extended Data Table\u0026nbsp;2). High-throughput neutralization assays were conducted using serum from guinea pigs immunized with 25 vaccine strains to systematically characterize the antigenic properties of H5 viruses. Based on the neutralization heatmap of H5 vaccine-immunized serum against representative strains, we identified three antigenic clusters of H5 viruses that do not cross-neutralize each other: AC1, AC2, and AC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Similar patterns were observed across different H5 subtypes, including H5N1, H5N6, and H5N8 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In terms of genetic evolution, AC1 comprises five clades (0\u0026thinsp;~\u0026thinsp;9, 2.1*, 2.2*, 2.3.2*, 2.3.4*), AC2 comprises two clades (2.3.4.4b and 2.3.4.4*), and AC3 corresponds to clade 2.3.4.4h.\u003c/p\u003e \u003cp\u003eThe genetic map (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Extended Data Table\u0026nbsp;3) showed that the three antigenic clusters presented a sequential distribution in genetic space, with the genetic distance gradually increasing from AC1 through AC2 to AC3, aligning with the topology of the phylogenetic tree. However, the distribution of antigenic clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, Extended Data Table\u0026nbsp;4) showed significantly different characteristics, whereby AC3 was located between AC1 and AC2 in antigenic space, with similar antigenic distances to both clusters. These results demonstrate that the relationship between genetic and antigenic evolution of H5 influenza viruses is not strictly linear or directly corresponding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTransition mechanisms between adjacent antigenic clusters\u003c/h3\u003e\n\u003cp\u003eDuring antigenic evolution from AC1 to AC2, the eight specific mutations (SM: mutations that occur with a frequency of \u0026gt;\u0026thinsp;70% in one clade while having frequencies of \u0026lt;\u0026thinsp;30% in all other clades) of AC2 failed to induce significant immune escape, whereas the sixteen common mutations (CM: mutations that occur with a frequency of \u0026gt;\u0026thinsp;70% in two or more clades) resulted in an approximately 10-fold increase in immune escape from AC1-immunized serum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Extended Data Table\u0026nbsp;5). Further analysis revealed that amino acid substitutions at six key positions (6M) (N88R, Q131L, S139P, D199N, K205N, and N289H) among the CM collectively mediated this antigenic transition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The combined mutant strain (AC1\u0026thinsp;+\u0026thinsp;6M) with mutations at these six positions based on AC1 showed approximately 30-fold enhanced escape from AC1-immunized serum, comparable to AC2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The single mutations K205N and N289H escaped neutralization by AC1-immunized serum more than 10-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), illustrating the importance of these two positions in the evolution of AC1 toward AC2. At the same time, the combined mutant strain (AC2-6M) with reversion mutations at these six positions based on AC2 showed comparable immune escape levels to AC1 when tested with AC2-immunized serum, further supporting this conclusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eDuring antigenic evolution from AC2 to AC3, AC3 underwent a partial reversion of antigenicity and lay between AC1 and AC2 in antigenic space. This intermediate positioning in antigenic space suggested the presence of reverse mutations in AC3. Based on this hypothesis, we found that the same six positions 88, 131, 139, 199, 205, and 289 play key roles in antigenic evolution from AC2 to AC3. Among these positions, three positions showed persistent mutations (88R\u0026thinsp;\u0026gt;\u0026thinsp;88S, 199N\u0026thinsp;\u0026gt;\u0026thinsp;199S, and 205N\u0026thinsp;\u0026gt;\u0026thinsp;205D), while the other three showed reversible mutations (131L\u0026thinsp;\u0026gt;\u0026thinsp;131Q, 139P\u0026thinsp;\u0026gt;\u0026thinsp;139S, and 289H\u0026thinsp;\u0026gt;\u0026thinsp;289N). Forward validation showed that the combined mutant strain (AC2\u0026thinsp;+\u0026thinsp;6'M) with mutations at these six positions based on AC2 exhibited comparable immune escape levels to AC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, e) when tested against AC2-immunized serum. The reverse validation using an AC3-based mutant showed consistent results, further confirming the significance of these six mutations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Additionally, a single point mutation, H289N, was sufficient to confer an approximately 50-fold immune escape from AC2-immunized serum, suggesting that position 289 plays a critical role in the antigenic transition from AC2 to AC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eOur systematic analysis identified six critical positions (88, 131, 139, 199, 205, and 289) as key determinants of two major antigenic transitions in H5 viral evolution. These six positions are distributed across multiple antigenic epitopes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and exhibit two mutation patterns: persistent mutations at positions 88 (N\u0026thinsp;\u0026gt;\u0026thinsp;R\u0026thinsp;\u0026gt;\u0026thinsp;S), 199 (D\u0026thinsp;\u0026gt;\u0026thinsp;N\u0026thinsp;\u0026gt;\u0026thinsp;S), and 205 (K\u0026thinsp;\u0026gt;\u0026thinsp;N\u0026thinsp;\u0026gt;\u0026thinsp;D), contrasted with reversible mutations at positions 131 (Q\u0026thinsp;\u0026gt;\u0026thinsp;L\u0026thinsp;\u0026gt;\u0026thinsp;Q), 139 (S\u0026thinsp;\u0026gt;\u0026thinsp;P\u0026thinsp;\u0026gt;\u0026thinsp;S), and 289 (N\u0026thinsp;\u0026gt;\u0026thinsp;H\u0026thinsp;\u0026gt;\u0026thinsp;N) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, Extended Data Fig.\u0026nbsp;5a). This dual mutation pattern explains the position of AC3 between AC1 and AC2 in antigenic space.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMechanisms underlying antigenic differences among clades within AC1\u003c/h3\u003e\n\u003cp\u003eThrough phylogenetic analysis, AC1 and AC2 antigenic clusters were found to contain multiple evolutionary clades that could be divided into two distinct groups based on their neutralizing breadth. Group I vaccine strains showed broad cross-neutralization against all clades within their respective antigenic clusters, including clades 0\u0026thinsp;~\u0026thinsp;9 and 2.3.4* in AC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), as well as clades 2.3.4.4b and 2.3.4.4* in AC2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). By contrast, Group II vaccine strains displayed clade-restricted neutralization, only neutralizing viruses within their own phylogenetic lineages, specifically the three AC1 clades 2.1*, 2.2*, and 2.3.2* (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eFurthermore, we identified key mutations driving antigenic differences within AC1. Using the A/Goose/Guangdong/1/96 (GD/96) strain as the evolutionary origin, we found that the three specific mutations (SM1) in clade 2.1* did not cause significant immune escape, whereas the fifteen common mutations (CM1) resulted in an approximately 10-fold increase of immune escape (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Extended Data Table\u0026nbsp;5). Similarly, the antigenic specificity of clades 2.2* and 2.3.2* was related to the common mutations CM2 and CM3, respectively, resulting in an approximately 10-fold reduction in neutralization titers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g). Further analysis revealed that the antigenic specificity of clade 2.1* is determined by 7 mutations (K205R, N61D, N140D, D142E, E228K, S157P, and A172T), that of clade 2.2* by 8 mutations (N61D, D110N, N140D, D142E, H154Q, S171N, K205R, and E228K), and that of clade 2.3.2* by 10 mutations (K205R, N140D, D110N, H154Q, S171N, R69K, S145L, S149A, E243D, and L285V) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f, g, h, Extended Data Fig.\u0026nbsp;5b, Extended Data Fig.\u0026nbsp;6). Moreover, we found that the single mutation A172T alone resulted in a 10-fold reduction in neutralization titers, whereas other individual mutations did not cause significant antigenic changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). These results indicate that the antigenic specificity of these clades is primarily determined by synergistic effects of mutations at multiple positions. Structural analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) revealed that these key mutations were primarily concentrated in five epitopes, especially epitopes A (positions 140, 142, 149, 154, 157) and B (positions 171, 172, 205), suggesting that these regions are hotspots for antigenic variation in H5 viruses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEpidemiology of clade 2.3.4.4b and protective efficacy of existing vaccines\u003c/h3\u003e\n\u003cp\u003eEpidemiological data showed that AC1 was predominant before 2010, with its five clades (0\u0026thinsp;~\u0026thinsp;9, 2.1*, 2.2*, 2.3.2*, and 2.3.4*) emerging successively. Around 2010, AC2 emerged and gradually replaced AC1, with clade 2.3.4.4b becoming predominant. AC3, which emerged around 2015, underwent a period of expansion before gradually declining in prevalence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Although human cases have been reported in all three antigenic clusters, there is no direct correspondence between the prevalence scale and the proportion of human infections. For example, while clade 2.3.4.4b in AC2 accounted for more than 90% of the total prevalence after 2020, the number of human cases was not significantly higher than in clade 2.3.2.1c of AC1 and 2.3.4.4h of AC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), which suggests that we need to be equally vigilant for all three antigenic clusters.\u003c/p\u003e \u003cp\u003eClade 2.3.4.4b belongs to AC2, which is currently of greatest interest. It evolved from the clade 2.3.4* of AC1, and has been paired with different NA genotypes. It has been found to infect avian, and mammals including humans (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Therefore, we further analyzed the ability of different vaccine strains to neutralize the currently prevalent clades of H5, including 2.3.4.4b, 2.3.4.4h, 2.3.2.1a, and 2.3.2.1c (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, Extended Data Fig.\u0026nbsp;8, Extended Data Fig.\u0026nbsp;9). The results showed that sera from animals immunized with either AC2 vaccine strains (except VI20) or the bovine-origin human isolate TE24 (A/Texas/37/2024) exhibited high neutralizing activity against clade 2.3.4.4b viruses. Moreover, there was no significant difference in protection against different subtypes (H5N1, H5N6, H5N8) within clade 2.3.4.4b or against strains from different hosts (avian, human, and other mammals) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). These results suggested that the current recommended vaccine strains in the AC2 antigenic cluster can effectively protect against clade 2.3.4.4b.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we constructed a comprehensive antigenic map spanning the evolutionary history of H5 influenza viruses for the first time. Although the H5 viruses evolved into multiple genetic clades, they can be clearly divided into three major antigenic clusters based on their antigenic properties. Each antigenic cluster contains multiple genetic clades that can be divided into two distinct groups based on their neutralizing breadth. Group I vaccine strains showed broad cross-neutralization against all clades within their respective antigenic clusters, while Group II vaccine strains displayed clade-restricted neutralization. These findings help us precisely characterize the antigenic evolution patterns of H5 viruses.\u003c/p\u003e \u003cp\u003eSpecifically, we identified a unique non-linear transition pattern among the three antigenic clusters (AC1, AC2, and AC3), with mutations at HA positions 88, 131, 139, 199, 205, and 289 driving these antigenic transitions. The persistent mutations at positions 88, 199, and 205, combined with the reversible mutations at positions 131, 139, and 289, explain why AC3 is located between AC1 and AC2 in antigenic space, in contrast to their sequential distribution in genetic space. The effect of these key positions on antigenicity is supported by other studies. For example, Li et al. \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e found that positions 88, 156, 205, 208, 239 and 289 play key roles in clade 2.3.4.4 antigenic drift, especially combined mutations of 205 and 208. Our study revealed that mutations at position 205 (epitope B, head region) and position 289 (epitope C, near neck region) could lead to significant immune escape. This pattern differs from typical human influenza viruses, where immune escape mutations are mainly concentrated in the head region\u003csup\u003e[\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. This difference may be attributed to the fact that H5 viruses mainly circulate in avian populations, where they experience significantly less immune selection pressure compared to human influenza viruses. It is noteworthy that if H5 viruses were to acquire the human-to-human transmission ability, the mutation patterns may change significantly.\u003c/p\u003e \u003cp\u003eMultiple genetic clades were identified within H5 antigenic clusters. Within AC1, vaccine strains from clades 0\u0026thinsp;~\u0026thinsp;9 and 2.3.4* showed broad neutralization ability against all clades within AC1, whereas vaccine strains from clades 2.1*, 2.2*, and 2.3.2* provided protection only against their respective clades. The antigenic specificity of these clades was driven by synergistic effects of multiple mutations, including N61D, R69K, D110N, N140D, D142E, S145L, S149A, H154Q, S157P, K205R, S171N, A172T, E228K, E243D, and L285V. Our findings are consistent with previous studies. Zhang et al.\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e identified six key positions 120, 126, 141, 156, 185, and 189 (corresponding to positions 136, 142, 157, 172, 201, and 205 in our numbering system). Similarly, Koel et al. \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e showed that antigenic changes of H5N1 clade 2.1 were mainly influenced by positions 129, 133, 151, 183, 185, and 189 (corresponding to positions 145, 149, 167, 199, 201 and 205 in our numbering system). Additionally, Li et al.\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e identified positions 156 and 205 as crucial antigenic determinant sites. Notably, position 205 was identified as a key site in many studies. Here, we found that position 205 underwent K\u0026thinsp;\u0026gt;\u0026thinsp;N\u0026thinsp;\u0026gt;\u0026thinsp;D substitutions during the evolution of AC1\u0026thinsp;\u0026gt;\u0026thinsp;AC2\u0026thinsp;\u0026gt;\u0026thinsp;AC3, and remained as R in three antigen-specific clades within AC1, highlighting its central role in H5 antigenic evolution. Within the AC2 antigenic cluster, clades 2.3.4.4b and 2.3.4.4* (including 2.3.4.4, 2.3.4.4c, 2.3.4.4e, and 2.3.4.4g) share antigenic similarity. Notably, the 2.3.4.4* clade was primarily found in the H5N6 subtype and eventually evolved into clade 2.3.4.4h (AC3) with H5N6 specificity, which is prevalent primarily in China. Based on a comprehensive analysis of antigenic and genetic characteristics, we propose that 2.3.4.4h be treated as a distinct evolutionary clade, distinguished from other 2.3.4.4 subclades.\u003c/p\u003e \u003cp\u003eThe risk clade 2.3.4.4b, which is currently causing global concern, belongs to the AC2 antigenic cluster. In response to the threat posed by this clade, the Centers for Disease Control and Prevention (CDC) has developed two candidate vaccine strains (CVVs): A/Astrakhan/3212/2020 (AS20) and A/American Wigeon/South Carolina/USDA-000345-001/2021 (CR21). Recent studies demonstrated that A/Texas/37/2024 (TE24), isolated from a farm worker in Texas, showed cross-reactivity with the CVVs and was effectively neutralized by ferret antisera induced by CVVs \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Our study also confirmed that sera from TE24-immunized guinea pigs neutralized clade 2.3.4.4b at a level comparable to AS20 and CR21. In addition, other vaccine strains within the AC2 antigenic cluster also showed effective neutralization against clade 2.3.4.4b viruses. However, novel mutations may emerge if this clade establishes sustained human-to-human transmission. Therefore, it is essential to continuously monitor the evolutionary dynamics of clade 2.3.4.4b and adjust the vaccine strain selection strategy in a timely manner. Notably, besides clade 2.3.4.4b from the AC2 antigenic cluster, clade 2.3.2.1c of AC1 and clade 2.3.4.4h of AC3 are also circulating and are capable of causing human infections. To counter the threat posed by these active H5 clades, we recommend establishing comprehensive surveillance systems while developing broadly protective H5 vaccines.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we constructed a comprehensive antigenic map of H5 influenza viruses, systematically elucidated their antigenic evolution patterns, and identified key amino acid substitutions driving antigenic changes. Our findings demonstrate that the antigenic evolution of H5 viruses can be divided into three major clusters (AC1, AC2, and AC3). In contrast to their sequential genetic evolution, AC3 lies between AC1 and AC2 in the antigenic space. This inconsistency is primarily driven by two mutation patterns at six key positions: persistent mutations at positions 88, 199, and 205, combined with reversible mutations at positions 131, 139, and 289. These findings advance our understanding of antigenic evolution mechanisms in H5 influenza viruses and provide direct guidance for optimizing vaccine strain selection, developing broad-spectrum vaccines, and preparing for potential pandemics.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCells\u003c/h2\u003e \u003cp\u003eMDCK (\u003cem\u003eCanis familiaris\u003c/em\u003e, kidney, RRID: CVCL_0422) and 293T (\u003cem\u003eHomo sapiens\u003c/em\u003e, embryonic kidney, RRID: CVCL_0063) cell lines were obtained from the American Type Culture Collection. All cell lines were cultured in Dulbecco\u0026rsquo;s modified Eagle medium (DMEM, high glucose; HyClone, Cat#SH30243.01) with 100 U/ml of penicillin-streptomycin solution (GIBCO, Cat#15140163), and 10% fetal bovine serum (TransGen Biotech, Cat#FS201) at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eVaccine and representative strain selection\u003c/h2\u003e \u003cp\u003eA total of 136 representative strains were used in this study, including 24 vaccine strains (Extended Data Table\u0026nbsp;1), one bovine-origin human isolate (A/Texas/37/2024; abbreviated as TE24) and 111 other strains (Extended Data Table\u0026nbsp;2). The vaccine strains were recommended by the WHO for 2024\u0026ndash;2025 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/teams/global-influenza-programme/\u003c/span\u003e\u003cspan address=\"https://www.who.int/teams/global-influenza-programme/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e vaccines/who-recommendations/zoonotic-influenza-viruses-and-candidate-vaccine-viruses). The 111 sequences were selected as follows. As of 29 May 2024, 17,591, 2,623 and 3,571 HA protein sequences were downloaded from the Global Initiative on Sharing All Influenza Data \u003cb\u003e(\u003c/b\u003eGISAID) database for H5N1, H5N6 and H5N8, respectively. Taking H5N1 subtypes as an example, sequences from the human host were deduplicated using a similarity threshold of 0.98, resulting in 40 sequences; sequences from other mammalian hosts (excluding humans) were deduplicated using a similarity threshold of 0.99, resulting in 24 sequences; and sequences from avian hosts were deduplicated using a similarity threshold of 0.96, resulting in 12 sequences. Therefore, a total of 76 sequences were selected as representative H5N1 strains. Using the same strategy, 27 and 8 sequences were selected as representative strains for H5N6 and H5N8, respectively. In total, 111 H5 influenza virus strains, 24 vaccine strains, and TE24 were selected for subsequent pseudotyped virus construction. These representative strains covered all clades of H5 viruses and showed even distribution across the phylogenetic tree of H5, demonstrating their representativeness (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic tree construction\u003c/h2\u003e \u003cp\u003eMultiple sequence alignment was performed using MAFFT v7.505 with the A/Goose/Guangdong/1/96 (H5N1) (GD/96) strain as the reference sequence. A maximum likelihood phylogenetic tree was constructed with FastTree v2.1.11 using the optimal amino acid substitution model. For evolutionary parameter estimation, divergence time and evolutionary rate were estimated within a Bayesian framework using BEAST v2.7.7 with an uncorrelated relaxed molecular clock model assuming a lognormal distribution. The Blosum62 substitution model was employed with gamma-distributed rate heterogeneity and a proportion of invariant sites. The MCMC chain was run for 50\u0026nbsp;million generations with sampling every 1,000 generations. The MCMC results were analyzed using Tracer v1.7.2 to ensure effective sample sizes greater than 200. Finally, a maximum clade credibility (MCC) tree was generated using TreeAnnotator with 20% burn-in and node heights set to posterior mean values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSite-directed mutagenesis\u003c/h2\u003e \u003cp\u003eThe HA and NA protein sequences listed in Extended Data Table\u0026nbsp;2 were downloaded from GISAID. PcDNA3.1-HA and pcDNA3.1-NA recombinant plasmids were constructed by inserting the codon-optimized HA and NA sequences of H5 viruses into pcDNA3.1. The entire sequence was synthesized on the backbone plasmid pcDNA3.1(+) using General Biological System (Anhui, China). The pcDNA3.1-HA plasmid was used as the template to generate the plasmid harboring specific mutations of HA. Following site-directed mutagenesis PCR, the template chain was digested using \u003cem\u003eDpn\u003c/em\u003eI restriction endonuclease (NEB, USA). Afterwards, the PCR product was directly used to transform \u003cem\u003eE. coli\u003c/em\u003e DH5a competent cells, after which single colonies were selected and the construct sequenced.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePseudotyped virus production\u003c/h2\u003e \u003cp\u003eOn day 1, 293T cells were resuspended to a concentration of 5\u0026thinsp;~\u0026thinsp;7\u0026times;10\u003csup\u003e5\u003c/sup\u003e cell/ml and seeded into a T75 culture flask (15 mL cell suspension per flask). Cells were incubated overnight at 37\u0026deg;C in a humidified incubator with 5% CO₂. On day 2, 293T cells were co-transfected with the HA plasmid, NA plasmid and HIV backbone plasmid (pSG3Δenv-FlucΔnef) at a mass ratio of 1:1:2 using transfection reagent Lipofectamine\u0026trade; 3000 (Invitrogen, Carlsbad, CA, USA). These cells were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 6 hours, after which the medium was replaced with fresh DMEM supplemented with 1% fetal bovine serum (FBS). At 48 hours post-transfection, the virus-containing supernatant was collected, filtered through a 0.45-\u0026micro;m pore-size polyethersulfone membrane (Millipore, Cat# SLHP033RB), aliquoted into 2 mL cryovials, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProduction of immunized sera\u003c/h2\u003e \u003cp\u003eAnimal experiments were conducted in strict accordance with the institutional animal care and use guidelines of the Institute of Medical Biology, Chinese Academy of Medical Sciences \u0026amp; Peking Union Medical College (IMBCAMS, Yunnan, China). The experimental protocol received formal approval from the IMBCAMS Animal Ethics Committee (Approval No. DWSP20240616). Twenty-five experimental groups (n\u0026thinsp;=\u0026thinsp;3 female guinea pigs per group; body weight 200\u0026ndash;220 g) received intramuscular electroporation-mediated delivery of 200 \u0026micro;g pcDNA3.1-HA plasmid constructs (25 distinct variants, one plasmid per group) on days 0, 14, and 28. Immunization was repeated three times at two-week intervals, and serum samples were obtained two weeks after the third immunization. Serum samples were stored at ˗20\u0026deg;C, then thawed and heat-inactivated at 56\u0026deg;C for 30 min before use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNeutralization assay\u003c/h2\u003e \u003cp\u003eImmunized sera were diluted to an appropriate initial concentration and then subjected to a three-fold serial dilution. Subsequently, 100 \u0026micro;L of each serum dilution was added into a 96-well plate. The resulting dilutions were mixed with 50\u0026micro;L of pseudotyped viruses at a concentration of 1300 TCID50/ml and incubated at 37\u0026deg;C for 1 h. Afterwards, MDCK cells were added into the plates (2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/100 \u0026micro;L per well). The cells were incubated at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e for 48 h, after which the chemiluminescence signals were detected using the Britelite plus reporter gene assay system (PerkinElmer, Ensight). The pseudovirus neutralization titer was calculated using the Reed-Muench method in PerkinElmer Ensight software. The results are based on 3 replicates unless specified otherwise.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of antigenic and genetic maps\u003c/h2\u003e \u003cp\u003eThe antigenic map was constructed based on pseudotyped virus neutralization titer data. Firstly, the raw data were log-transformed and normalized to convert the exponential differences between titers into a linear relationship. The T-distributed stochastic neighbor embedding algorithm was chosen for dimensionality reduction to maintain the local structural relationships of the data to more accurately show the complex antigenic differences among viral strains. In cluster analysis, hierarchical clustering was performed using Ward's method to form compact clusters by minimizing the within-cluster variance, which allowed us to discover virus groups with similar antigenicity. The genetic map was constructed based on the HA amino acid sequences of H5 viruses. After performing multiple sequence alignment, genetic diversity was quantified by calculating the proportion of variant sites between sequences. The same downscaling and clustering strategy was subsequently used to ensure methodological consistency between antigenic and genetic analyses. All data analyses were performed using Python, relying on the Biopython, scipy, and sklearn scientific computing libraries.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eData analysis and processing\u003c/h2\u003e \u003cp\u003eGraphPad Prism 8 (GraphPad Software Inc, San Diego, CA, USA) was used for statistical analysis. Values were shown as geometric means with geometric standard deviations (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eData and Code Availability\u003c/h2\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request. This paper does not include any original code. Any additional information required to reanalyze the data can also be obtained via email.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe gratefully acknowledge the authors from the originating and submitting laboratories where genetic sequence data were generated and shared via GISAID, enabling this research. This work was supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2022-I2M-3-001), Science and Technology Leading Talent Program of Yunnan Province (202405AB350002), National Key Research and Development Program of China (2023YFC2307900), State Key Laboratory Special Fund (2060204), National Natural Science Foundation of China (82372225), CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-061), CAMS Innovation Fund for Medical Sciences (CIFMS)(2023-PT330-01, 2023-I2M-2-005), CAMS Innovation Fund for Medical Sciences (2022-I2M-2-004), NCTIB Fund for R\u0026amp;D Platform for Cell and Gene Therapy and High-performance Computing Platform of Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences \u0026amp; Peking Union Medical College.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eQ.L., A.W. and Y.W. conceived, designed, and supervised the experiments; M.Z., Z.L. and Q.H. immunized the animals; L.Q., M.Z., Y.M. and C.B. selected and constructed the representative pseudotyped virus library; M.Z., Z.L., J.C., J.T., H.L. and R.B. performed the neutralization assays; L.Q., M.Z. and J.L. analyzed the experimental data; X.D, and W.H. were supervision; M.Z. and L.Q. wrote the manuscript; All authors approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eConflicts of Interest\u003c/h2\u003e\n\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGao F., Wang Q., Qiu C., et al. Pandemic preparedness of effective vaccines for the outbreak of newly H5N1 highly pathogenic avian influenza virus [J]. Virologica Sinica \u003cstrong\u003e39\u003c/strong\u003e, 981-985 (2024).\u003c/li\u003e\n\u003cli\u003eZhang Y., Cui P., Shi J., et al. A broad-spectrum vaccine candidate against H5 viruses bearing different sub-clade 2.3.4.4 HA genes [J]. NPJ Vaccines \u003cstrong\u003e9\u003c/strong\u003e, 152 (2024).\u003c/li\u003e\n\u003cli\u003eTian J., Bai X., Li M., et al. Highly Pathogenic Avian Influenza Virus (H5N1) Clade 2.3.4.4b Introduced by Wild Birds, China, 2021 [J]. Emerg Infect Dis \u003cstrong\u003e29\u003c/strong\u003e, 1367-1375 (2023).\u003c/li\u003e\n\u003cli\u003eKoopmans M. P. G., Barton Behravesh C., Cunningham A. A., et al. The panzootic spread of highly pathogenic avian influenza H5N1 sublineage 2.3.4.4b: a critical appraisal of One Health preparedness and prevention [J]. The Lancet Infectious Diseases \u003cstrong\u003e24\u003c/strong\u003e, e774-e781 (2024).\u003c/li\u003e\n\u003cli\u003eXie R., Edwards K. M., Wille M., et al. The episodic resurgence of highly pathogenic avian influenza H5 virus [J]. Nature \u003cstrong\u003e622\u003c/strong\u003e, 810-817 (2023).\u003c/li\u003e\n\u003cli\u003ePeacock T. P., Moncla L., Dudas G., et al. The global H5N1 influenza panzootic in mammals [J]. Nature \u003cstrong\u003e637\u003c/strong\u003e, 304-313 (2025).\u003c/li\u003e\n\u003cli\u003ePlaza P. I., Gamarra-Toledo V., Rodr\u0026iacute;guez Eugu\u0026iacute; J., et al. Pacific and Atlantic sea lion mortality caused by highly pathogenic Avian Influenza A(H5N1) in South America [J]. Travel Med Infect Dis \u003cstrong\u003e59\u003c/strong\u003e, 102712 (2024).\u003c/li\u003e\n\u003cli\u003eAlkie T. N., Cox S., Embury-Hyatt C., et al. Characterization of neurotropic HPAI H5N1 viruses with novel genome constellations and mammalian adaptive mutations in free-living mesocarnivores in Canada [J]. Emerg Microbes Infect \u003cstrong\u003e12\u003c/strong\u003e, 2186608 (2023).\u003c/li\u003e\n\u003cli\u003eUyeki T. M., Milton S., Abdul Hamid C., et al. Highly Pathogenic Avian Influenza A(H5N1) Virus Infection in a Dairy Farm Worker [J]. N Engl J Med \u003cstrong\u003e390\u003c/strong\u003e, 2028-2029 (2024).\u003c/li\u003e\n\u003cli\u003eGood M. R., Fern\u0026aacute;ndez-Quintero M. L., Ji W., et al. A single mutation in dairy cow-associated H5N1 viruses increases receptor binding breadth [J]. Nat Commun \u003cstrong\u003e15\u003c/strong\u003e, 10768 (2024).\u003c/li\u003e\n\u003cli\u003eCDC. First H5 Bird Flu Death Reported in United States. https://www.cdc.gov/media/releases/2025/m0106-h5-birdflu-death.html [Z]. (2025)\u003c/li\u003e\n\u003cli\u003eWHO. Zoonotic influenza: candidate vaccine viruses and potency testing reagents.https://www.who.int/teams/global-influenza-programme/vaccines/who-recommendations/zoonotic-influenza-viruses-and-candidate-vaccine-viruses [Z]. (2024)\u003c/li\u003e\n\u003cli\u003eKhurana S., King L. R., Manischewitz J., et al. Licensed H5N1 vaccines generate cross-neutralizing antibodies against highly pathogenic H5N1 clade 2.3.4.4b influenza virus [J]. Nature Medicine \u003cstrong\u003e30\u003c/strong\u003e, 2771-2776 (2024).\u003c/li\u003e\n\u003cli\u003eKhurana S., Chearwae W., Castellino F., et al. Vaccines with MF59 Adjuvant Expand the Antibody Repertoire to Target Protective Sites of Pandemic Avian H5N1 Influenza Virus [J]. Sci Transl Med\u003cstrong\u003e 2\u003c/strong\u003e, 15ra15-15ra15 (2010).\u003c/li\u003e\n\u003cli\u003eBeigel J. H., Voell J., Huang C.-Y., et al. Safety and Immunogenicity of Multiple and Higher Doses of an Inactivated Influenza A/H5N1 Vaccine [J]. The Journal of Infectious Diseases \u003cstrong\u003e200\u003c/strong\u003e, 501-508 (2009).\u003c/li\u003e\n\u003cli\u003eChen L. M., Donis R. O., Suarez D. L., et al. Biosafety risk assessment for production of candidate vaccine viruses to protect humans from zoonotic highly pathogenic avian influenza viruses [J]. Influenza Other Respir Viruses \u003cstrong\u003e14\u003c/strong\u003e, 215-225 (2020).\u003c/li\u003e\n\u003cli\u003eOkoli G. N., Racovitan F., Abdulwahid T., et al. Variable seasonal influenza vaccine effectiveness across geographical regions, age groups and levels of vaccine antigenic similarity with circulating virus strains: A systematic review and meta-analysis of the evidence from test-negative design studies after the 2009/10 influenza pandemic [J]. Vaccine \u003cstrong\u003e39\u003c/strong\u003e, 1225-1240 (2021).\u003c/li\u003e\n\u003cli\u003eUyeki T. M., Hui D. S., Zambon M., et al. Influenza [J]. The Lancet \u003cstrong\u003e400\u003c/strong\u003e, 693-706 (2022).\u003c/li\u003e\n\u003cli\u003eCarnell G. W., Ferrara F., Grehan K., et al. Pseudotype-based neutralization assays for influenza: a systematic analysis [J]. Front Immunol \u003cstrong\u003e6\u003c/strong\u003e, 161 (2015).\u003c/li\u003e\n\u003cli\u003eLi J., Gu M., Liu K., et al. Amino acid substitutions in antigenic region B of hemagglutinin play a critical role in the antigenic drift of subclade 2.3.4.4 highly pathogenic H5NX influenza viruses [J]. Transbound Emerg Dis\u003cstrong\u003e 67\u003c/strong\u003e, 263-275 (2020).\u003c/li\u003e\n\u003cli\u003eWiley D. C., Wilson I. A., Skehel J. J. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation [J]. Nature \u003cstrong\u003e289\u003c/strong\u003e, 373-378 (1981).\u003c/li\u003e\n\u003cli\u003eBroecker F., Liu S. T. H., Sun W., et al. Immunodominance of Antigenic Site B in the Hemagglutinin of the Current H3N2 Influenza Virus in Humans and Mice [J]. J Virol \u003cstrong\u003e92\u003c/strong\u003e, e01100-18 (2018).\u003c/li\u003e\n\u003cli\u003ePopova L., Smith K., West A. H., et al. Immunodominance of antigenic site B over site A of hemagglutinin of recent H3N2 influenza viruses [J]. PLoS One \u003cstrong\u003e7\u003c/strong\u003e, e41895 (2012).\u003c/li\u003e\n\u003cli\u003eWu N. C., Otwinowski J., Thompson A. J., et al. Major antigenic site B of human influenza H3N2 viruses has an evolving local fitness landscape [J]. Nat Commun \u003cstrong\u003e11\u003c/strong\u003e, 1233 (2020).\u003c/li\u003e\n\u003cli\u003eKoel B. F., Van Der Vliet S., Burke D. F., et al. Antigenic variation of clade 2.1 H5N1 virus is determined by a few amino acid substitutions immediately adjacent to the receptor binding site [J]. mBio \u003cstrong\u003e5\u003c/strong\u003e, e01070-01014 (2014).\u003c/li\u003e\n\u003cli\u003eGarg S., Reed C., Davis C. T., et al. Outbreak of Highly Pathogenic Avian Influenza A(H5N1) Viruses in U.S. Dairy Cattle and Detection of Two Human Cases - United States, 2024 [J]. MMWR Morb Mortal Wkly Rep \u003cstrong\u003e73\u003c/strong\u003e, 501-505 (2024).\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6040842/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6040842/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHighly pathogenic avian influenza (HPAI) H5 viruses have recently been documented in mammals including humans, posing a major threat to global public health. To prevent a potential H5 pandemic, it is critical to elucidate the antigenic evolutionary pattern and identify key drivers underlying its evolution. In this study, we constructed a comprehensive antigenic map of H5 influenza viruses spanning their evolutionary history for the first time, revealing three distinct antigenic clusters (AC1, AC2, and AC3) with no cross-neutralization. In contrast to its sequential genetic evolution, AC3 lies between AC1 and AC2 in antigenic space. This divergence stems from two distinct mutation patterns at six key amino acid positions: (1) persistent mutations at positions 88 (N\u0026thinsp;\u0026gt;\u0026thinsp;R\u0026thinsp;\u0026gt;\u0026thinsp;S), 199 (D\u0026thinsp;\u0026gt;\u0026thinsp;N\u0026thinsp;\u0026gt;\u0026thinsp;S), and 205 (K\u0026thinsp;\u0026gt;\u0026thinsp;N\u0026thinsp;\u0026gt;\u0026thinsp;D), and (2) reversible mutations at positions 131 (Q\u0026thinsp;\u0026gt;\u0026thinsp;L\u0026thinsp;\u0026gt;\u0026thinsp;Q), 139 (S\u0026thinsp;\u0026gt;\u0026thinsp;P\u0026thinsp;\u0026gt;\u0026thinsp;S), and 289 (N\u0026thinsp;\u0026gt;\u0026thinsp;H\u0026thinsp;\u0026gt;\u0026thinsp;N). Moreover, single mutations at positions 205 and 289 can lead to significant immune escape. The risk clade of current interest, 2.3.4.4b belongs to AC2 and remains sensitive to current AC2-targeted vaccine strains. Additionally, clades 2.3.2.1c of AC1 and 2.3.4.4h of AC3 are also prevalent and capable of human infection, necessitating continuous surveillance of their epidemiological dynamics. These findings not only reveal the antigenic evolution mechanism of H5 influenza unseen in other influenza viruses, but also provide important guidance for vaccine strain selection and broad-spectrum vaccine development.\u003c/p\u003e","manuscriptTitle":"Dominant substitutions underlying the antigenic evolution of H5 influenza virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-26 12:18:11","doi":"10.21203/rs.3.rs-6040842/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3cd8dd07-4274-4424-8e18-68eefcca6943","owner":[],"postedDate":"February 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44535739,"name":"Biological sciences/Evolution"},{"id":44535740,"name":"Biological sciences/Genetics"}],"tags":[],"updatedAt":"2025-11-29T08:07:25+00:00","versionOfRecord":{"articleIdentity":"rs-6040842","link":"https://doi.org/10.1038/s41467-025-65730-y","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-11-28 05:00:00","publishedOnDateReadable":"November 28th, 2025"},"versionCreatedAt":"2025-02-26 12:18:11","video":"","vorDoi":"10.1038/s41467-025-65730-y","vorDoiUrl":"https://doi.org/10.1038/s41467-025-65730-y","workflowStages":[]},"version":"v1","identity":"rs-6040842","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6040842","identity":"rs-6040842","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}