Search

Swine influenza virus invades the host through the respiratory mucosa, which severely restricts the development of the pig breeding industry. To construct monomeric and trimeric vaccines, we developed recombinant Escherichia coli Nissle 1917 (EcN) strains that express the receptor binding site (RBS) of the hemagglutinin (HA) antigen from H1N1 swine influenza virus. After the mucosal immunization of mice, we found that probiotics activated CD40 and CD86 in DCs and increased the levels of IL-4 and IFN-γ secretion by T cells. Furthermore, the probiotics improved the function of the mucosal immune system, increased the level of SIgA, level of IgG and number of B220 + IgA + , and activated germinal center B cells. The challenge experiment revealed that the probiotics alleviated weight loss, reduced pathological injury to the lungs, and protected the mice from virus infection. We also observed that the serum neutralizing antibodies of immunized piglets significantly increased, which reduced the shedding frequency of swine influenza virus in the nose of the piglets and reduced the pathological damage by activating the T cell immune response in infected piglets. Thus, the constructed probiotics are promising candidates for effective non-traditional swine influenza vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Two biological types of influenza virus are known and distinguished by the structure of the surface glycoprotein, hemagglutinin (HA). The noninfectious virions contain the uncleaved HA0 (80 kDa), whereas the infectious type have a cleaved form of two subunits, HA1 (55 kDa) and HA2 (25 kDa). The point cleavage of HA0→HA1+HA2 by host proteases regulates the virion integrity to maintain the functionality of the intravirion axis HA→M2→M1→RNP. The HA0 containing virions are more resistant than the HA1/HA2 virions to the 75 °C temperature used in pasteurizing milk and foods. The noninfectious HA0 virions treated at 75 °C were able to retain infectious potential, which was activated by trypsin; in contrast, the infectious HA1/HA2 virions lost infectivity irreversibly under pasteurization. The data suggest that (i) influenza viruses retain their infectious potential in the external environment by means of noninfectious virions containing the uncleaved HA0 and (ii) a stronger pasteurization regimen in terms of temperature and duration of thermal treatment is recommended to inactivate such potentially infectious virions in food products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier Inc.)

The global threat of pandemics highlights the urgency of developing innovative vaccine strategies. Viral spike proteins are the primary antigens recognized by the immune system and serve as key targets for vaccine development. This study reports the production of full-length Influenza A virus surface glycoprotein, hemagglutinin (HA), and its incorporation into Nanodiscs (NDs). HA was expressed in insect cells and purified using detergents, maintaining its functional integrity. Characterisation by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) confirmed that HA could be incorporated into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) NDs as a single trimer. SEC-MALS was instrumental in analysing the composition of NDs, which included HA, membrane scaffold proteins, lipids, and glycans. These findings provide a robust framework for the production and reconstitution of glycoproteins in NDs, and offers valuable insights into the study of multi-component nanoparticles using MALS. Our work highlights the potential of NDs for studying viral glycoproteins and advances the development of well-defined recombinant ND-based vaccines., Competing Interests: Declaration of competing interest The project was funded by the Dutch Research Council with financial contributions from Batavia Biosciences B.V. and ZoBio B.V., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

During the 2022-2023 winter season in South Korea, a novel clade 2.3.4.4b H5N1 HPAIV was first detected in wild birds, which then subsequently caused multiple outbreaks in poultry farms and wild birds. This study aimed to investigate the genetic characteristics of H5N1 HPAIVs isolated during the 2022-2023, along with their pathogenicity and transmissibility in chickens and ducks. The clade 2.3.4.4b H5N1 HPAIV viruses caused outbreaks in 75 poultry farms and detected in 174 wild bird cases. Phylogenetic analysis of hemagglutinin genes revealed that the South Korean H5N1 HPAIV isolates were closely related to Eurasian and American HPAIVs isolated between 2022 and 2023. In total, 21 diverse genotypes (22G0-22G20) were identified in virus isolates from poultry and wild birds, among which 22G7 was the dominant genotype. The 22G1 genotype (A/duck/Korea/H493/2022(H5N1)) caused high virulence and pathogenicity, with a 100 % mortality rate in specific-pathogen-free chickens. Ducks inoculated with genotypes 22G1 or 22G7 (A/duck/Korea/H537/2022(H5N1)) showed neurological signs, with 60 %-80 % mortality rate. In the contact groups of ducks, 100 % of transmissibility was observed. Notably, in the 22G7-inoculated group, viral shedding via the cloacal route was longer, and viral replication in the cecal tonsil was higher than that in the 22G1-inoculated group, which may have contributed to the dominancy of the 22G7 genotype. Therefore, better understanding of the genetic and pathogenic features of HPAI viruses is important for effective virus control in the field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier B.V.)

Influenza poses a serious threat to both individual and public health. This study aimed to investigate the virological and epidemiological characteristics of influenza infections and to explore the genetic diversity of the circulating influenza viruses. In total, 1886 nasopharyngeal specimens from patients with acute respiratory illnesses were tested against 13 respiratory viruses using a multiplex real-time PCR. Whole-genome sequencing, phylogenetic, and amino acid analyses of representative influenza strains were performed. At least one respiratory virus was detected in 869 (46.1%) patients; 87 (4.6%) were co-infected with two or three viruses. Influenza A(H1N1)pdm09 was the most prevalent virus (16.1%), followed by rhinoviruses (8.1%) and RSV (6.7%). Hemagglutinin (HA) genes of the 74 influenza A(H1N1)pdm09 viruses were categorized in subclades C.1.8, C.1.9, and C.1 within clade 5a.2a and D1, D.2, and D.3 within clade 5a.2a.1. The A(H3N2) viruses analyzed belonged to clade 2a.3a.1, subclades J.2 and J.1. The sequenced B/Victoria lineage viruses fell into clade V1A.3a.2, subclades C.5.6 and C.5.7. Amino acid substitutions in most viral proteins were identified compared with the vaccine strains, including in the HA antigenic sites. This study demonstrated the dominant distribution of the influenza A(H1N1)pdm09 virus among the respiratory viruses studied and the genetic diversity of the circulating influenza viruses.

There is a need to improve the effectiveness of seasonal influenza vaccines. Influenza vaccines based on recombinant hemagglutinin offer advantages over traditional approaches. We asked whether Advax-CpG55.2 or alum-CpG55.2 adjuvants could enhance the immunogenicity of a quadrivalent influenza vaccine (QIV) comprising recombinant full-length native hemagglutinins (HA0) produced in Sf9 insect cells. Adult C57BL/6 mice were immunized intramuscularly twice 10 days apart with 4 μg of QIV alone or with adjuvant. QIV induced only modest levels of anti-influenza IgG1 whereas the adjuvanted formulations induced significantly higher levels of IgG1 plus IgG2c. When challenged with the H1N1pdm strain, A/South Australia/348/2023, adjuvanted QIV immunized mice showed minimal illness, whereas QIV alone immunized mice all succumbed to infection. Advax-CpG55.2 adjuvanted QIV similarly increased influenza-specific IgG against all four vaccine strains in Cynomolgus macaques. Adjuvanted recombinant hemagglutinin approaches offer a promising alternative to existing seasonal influenza vaccine platforms., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: YHO, UV and NP are affiliated with Vaxine Pty Ltd which holds the rights to Advax™ and CpG55.2™ adjuvants., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Despite annual vaccines, Influenza B viruses (IBVs) continue to cause severe infections and have a significant impact and burden on the healthcare system. Improving IBV vaccine effectiveness is a key focus, with various strategies under investigation. In this research, we used a computational design to select wildtype sequences for a multivalent viral-vectored vaccine (rAd-Tri-Vic) targeting the Victoria-like (Vic) hemagglutinin (HA) protein. In mouse models, the vaccine induced strong antibody and T cell responses, providing complete protection against both lineage-specific and cross-lineage (Yamagata-like) lethal challenges. The immune responses remained robust for up to six months, which demonstrated sustained protection. These results highlight the potential of HA-targeted multivalent vaccines to enhance the IBV efficacy and address protection against antigenically diverse IBV strains.

Following two human infections with the H10N3 avian influenza virus (AIV) in 2021 and 2022, a third case was discovered in Yunnan, China, in 2024, raising concerns about the potential for future pandemics. Recent studies have indicated that novel H10N3 viruses are highly pathogenic in mice and can be transmitted between guinea pigs via respiratory droplets without prior adaptation. However, the biological characteristics of novel H10N3 in poultry have not been fully elucidated. Our findings revealed that H10 subtype AIVs are predominantly prevalent in waterfowl. Notably, H10N8 and H10N3 viruses that have infected humans were primarily isolated from chickens. For the first time, double basic hemagglutinin cleavage sites (motif PEIKQGR↓GL) were identified in novel H10N3 AIVs, which exhibit enhanced replication in chickens, and can be transmitted between chickens through direct contact and respiratory droplets. Animal experimental studies demonstrated that ducks are also susceptible to H10N3 viruses and that the virus is transmissible through direct contact, suggesting a greater risk of transmission and recombination. Serological studies conducted among poultry workers suggest that while the human population was largely naïve to H10N3 infection, sporadic and undetected human infections did occur, indicating a potential increasing trend. These data further emphasize the growing threat to public health posed by zoonotic H10N3 subtype AIVs.IMPORTANCEExposure to poultry in live poultry markets (LPMs) is strongly associated with human infection with avian influenza viruses (AIVs), with chickens being the most common species found in these markets in China. The prevalence of AIVs in chickens, therefore, increases the risk of human infection. Notably, the main host of the novel H10N3 virus has shifted from waterfowl to chickens, and the virus can be transmitted between chickens via respiratory droplets, posing a potential risk of a pandemic within poultry populations. The novel H10N3 virus also remains sensitive to ducks and can be transmitted through direct contact, which means a greater risk of transmission and recombination. Significantly, the human population remains largely naïve to H10N3 infection, but sporadic seropositivity among poultry workers indicates previous exposure to H10 subtype AIVs. Therefore, a comprehensive surveillance of the novel H10N3 viruses in poultry is imperative. Effective control of the virus within poultry populations could significantly reduce the risk of emerging human infections., Competing Interests: The authors declare no conflict of interest.

H1N1 influenza viruses are responsible for both seasonal and pandemic influenza. The continual antigenic shift and drift of these viruses highlight the urgent need for a universal influenza vaccine to elicit broadly neutralizing antibodies (bnAbs). Identification and characterization of bnAbs elicited in natural infection and immunization to influenza virus hemagglutinin (HA) can provide insights for development of a universal influenza vaccine. Here, we structurally and biophysically characterize four antibodies that bind to a conserved region on the HA membrane-proximal region known as the anchor epitope. Despite some diversity in their V H and V K genes, the antibodies interact with the HA through germline-encoded residues in HCDR2 and LCDR3. Somatic mutations on HCDR3 also contribute hydrophobic interactions with the conserved HA epitope. This convergent binding mode provides extensive neutralization breadth against H1N1 viruses and suggests possible countermeasures against H1N1 viruses., Competing Interests: Competing interests: The authors declare the following competing interests. Florian Krammer is listed as a co-inventor on patent applications filed by The Icahn School of Medicine at Mount Sinai relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines, influenza virus vaccines, and influenza virus therapeutics. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, Castlevax, to develop SARS-CoV-2 vaccines. Florian Krammer is a co-founder and scientific advisory board member of Castlevax. Florian Krammer has consulted for Merck, CureVac, Seqirus, GSK, and Pfizer and is currently consulting for 3rd Rock Ventures, Sanofi, Gritstone and Avimex. The Krammer laboratory is also collaborating with Dynavax on influenza vaccine development. Patrick Wilson and Jenna Guthmiller are listed as co-inventors on a patent application filed by the University of Chicago relating to anchor epitope targeting antibodies. The remaining Authors have no competing interests to declare., (© 2025. The Author(s).)

Hemagglutinin (HA), a variable viral surface protein, is essential for influenza vaccine development. Annually, traditional trivalent vaccines containing influenza A/H1N1, A/H3N2 and B viruses are administered globally, which are not very effective for the mutations in HA protein. The aim of this study was to design a multi-epitope vaccine containing epitopes of the HA protein of H1N1, H3N2 and B viruses using immunoinformatics methods. The HA protein epitope prediction was performed using Immune Epitope Database. Toxicity, antigenicity and conservancy of the epitopes were evaluated using ToxinPred, VaxiJen and Epitope Conservancy Analysis tools, respectively. Then, nontoxic, antigenic and high conserved epitopes with high prediction scores were selected. Their binding affinity was evaluated against human and mouse MHC class I and II molecules using the HPEPDOCK tool. Physicochemical properties and post-translational modifications were evaluated using ProtParam, SOLpro and MusiteDeep tools, respectively. Top selected epitopes were joined using linkers to produce the best effective recombinant trivalent vaccine candidate to elicit cellular and humoral immune responses in mouse and human host models. These sequences were modeled and verified. By evaluating the results of various analyses of all models and the most similarity to the native HA protein, model 5 was selected as the best model. Finally, in silico cloning of this model as vaccine candidate was performed in pET21. This study was a computer-aided analysis for a multi-epitope trivalent recombinant vaccine candidate against influenza viruses. The efficiency of our best model of vaccine candidates should be validated using in vitro and in vivo studies. Communicated by Ramaswamy H. Sarma.

Understanding how immune history influences influenza immunity is essential for developing effective vaccines and therapeutic strategies. This study examines the antigenic imprinting of influenza hemagglutinin (HA) and neuraminidase (NA) using a mouse model with sequential infections by H1N1 virus strains exhibiting substantial antigenic differences in HA. In our pre-2009 influenza infection model, we observed that mice with more extensive infection histories produced higher levels of functional NA-inhibiting antibodies (NAI). However, following further infection with the 2009 pandemic H1N1 strain, these mice demonstrated a reduced NAI to the challenged virus. Interestingly, prior exposure to older strains resulted in a lower HA antibody response (neutralization and HAI) to the challenged virus in both pre- and post-2009 scenarios, potentially due to faster viral clearance facilitated by immune memory recall. Overall, our findings reveal distinct trajectories in HA and NA immune responses, suggesting that immune imprinting can differentially impact these proteins based on the extent of antigenic variation in influenza viruses., Importance: Influenza viruses continue to pose a significant threat to human health, with vaccine effectiveness remaining a persistent challenge. Individual immune history is a crucial factor that can influence antibody responses to subsequent influenza exposures. While many studies have explored how pre-existing antibodies shape the induction of anti-HA antibodies following influenza virus infections or vaccinations, the impact on anti-NA antibodies has been less extensively studied. Using a mouse model, our study demonstrates that within pre-2009 H1N1 strains, an extensive immune history negatively impacted anti-HA antibody responses but enhanced anti-NA antibody responses. However, in response to the 2009 pandemic H1N1 strain, which experienced an antigenic shift, both anti-HA and anti-NA antibody responses were hindered by antibodies from prior pre-2009 H1N1 virus infections. These findings provide important insights into how antigenic imprinting affects both anti-HA and anti-NA antibody responses and underscore the need to consider immune history in developing more effective influenza vaccination strategies., Competing Interests: N.C.W. serves as a consultant for HeliXon.

Background: The rapid mutation of avian influenza virus (AIV) poses a significant threat to both the poultry industry and public health. Herein, we have successfully developed an mRNA-LNPs candidate vaccine for H5 subtype highly pathogenic avian influenza and evaluated its immunogenicity and protective efficacy., Results: In experiments on BALB/c mice, the vaccine candidate elicited strong humoral and a certain cellular immune responses and protected mice from the heterologous AIV challenge. Antibody and splenocyte passive transfer assays in mice suggested that antibodies played a crucial role in providing protection. Experiments involving SPF chickens have revealed that two doses of the 5 µg vaccine candidate in this study provided 100% complete protection against homologous strains, but only 50% complete protection against heterologous strains. Even immunization with two doses of the 15 µg vaccine candidate resulted in 90% complete protection against heterologous strains. To enhance the immune efficacy of the candidate vaccine, we designed 6 sequences with different secondary structures and screened out the candidate sequence with the highest expression (SY2-HA mRNA). Experiments on SPF chickens showed that two doses of 5 µg SY2-HA mRNA-LNP vaccine provided 100% complete protection against homologous and heterologous H5N1 AIV strains. Immunization tests with the SY2-HA mRNA-LNP vaccine were repeated in the SPF chicken model, inducing antibody production levels that are consistent with previous tests and providing 100% complete protection against both homologous and heterologous strains of the virus, indicating that the vaccine has a stable immune efficacy., Conclusions: The vaccine developed in this study provides complete protection against divergent H5N1 AIV strains in chickens, offering a promising approach for the future development of mRNA vaccines against multivalent avian influenza subtypes., Competing Interests: Declarations. Ethics approval and consent to participate: All mouse and chicken experiments with the H5N1 avian influenza viruses challenge were conducted in animal bio-safety level 3 facilities of South China Agricultural University (SCAU) (CNAS BL0011). All animals involved in the experiments were approved by the Institutional Animal Care and Use Committee of SCAU and were treated according to the guideline (2017A002). Consent for publication: All authors agree for publication. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The effectiveness of poultry vaccination in preventing the transmission of highly pathogenic avian influenza viruses (AIVs) has been debated, and its impact on wild birds remains uncertain. Here, we reconstruct the movements of H5 subtype AIV lineages among vaccinated poultry, unvaccinated poultry, and wild birds, worldwide, from 1996 to 2023. We find that there is a time lag in viral transmission among different host populations and that movements from wild birds to unvaccinated poultry were more frequent than those from wild birds to vaccinated poultry. Furthermore, our findings suggest that the HA (hemagglutinin) gene of the AIV lineage that circulated predominately in Chinese poultry experienced greater nonsynonymous divergence and adaptive fixation than other lineages. Our results indicate that the epidemiological, ecological, and evolutionary consequences of widespread AIV vaccination in poultry may be linked in complex ways and that much work is needed to better understand how such interventions may affect AIV transmission to, within, and from wild birds.

The Eurasian avian-like (EA) H1N1 swine influenza virus (SIV) possesses the capacity to instigate the next influenza pandemic, owing to its heightened affinity for the human-type α-2,6 sialic acid (SA) receptor. Nevertheless, the molecular mechanisms underlying the switch in receptor binding preferences of EA H1N1 SIV remain elusive. In this study, we conduct a comprehensive genome-wide CRISPR/Cas9 knockout screen utilizing EA H1N1 SIV in porcine kidney cells. Knocking out the enzyme gamma glutamyl carboxylase (GGCX) reduces virus replication in vitro and in vivo by inhibiting the carboxylation modification of viral haemagglutinin (HA) and the adhesion of progeny viruses, ultimately impeding the replication of EA H1N1 SIV. Furthermore, GGCX is revealed to be the determinant of the D225E substitution of EA H1N1 SIV, and GGCX-medicated carboxylation modification of HA 225E contributes to the receptor binding adaption of EA H1N1 SIV to the α-2,6 SA receptor. Taken together, our CRISPR screen has elucidated a novel function of GGCX in the support of EA H1N1 SIV adaption for binding to α-2,6 SA receptor. Consequently, GGCX emerges as a prospective antiviral target against the infection and transmission of EA H1N1 SIV., Competing Interests: Competing interests: The authors declare no competing interest., (© 2025. The Author(s).)

The ongoing circulation of influenza A H5N1 in the United States has raised concerns of a pandemic caused by highly pathogenic avian influenza. Although the United States has stockpiled and is prepared to produce millions of vaccine doses to address an H5N1 pandemic, currently circulating H5N1 viruses contain multiple mutations within the immunodominant head domain of hemagglutinin (HA) compared to the antigens used in stockpiled vaccines. It is unclear if these stockpiled vaccines will need to be updated to match the contemporary H5N1 strains. Here we show that a replicating RNA vaccine expressing the HA of an H5N1 isolated from a US dairy cow confers complete protection against homologous lethal challenge in mice. A repRNA encoding the HA of a clade 1 H5 from 2004 (A/Vietnam/1203/2004) as utilized by some stockpiled vaccines, confers only partial protection. Our data highlight the utility of nucleic acid vaccines to be rapidly updated to match emergent viruses of concern while demonstrating that contemporary bovine H5N1 viruses can evade immunity elicited by historical HA antigens., Competing Interests: Competing interests: J.H.E., E.T.S., N.W., S.P., K.H., E.H., and K.G. have an equity interest in HDT Bio. J.H.E. is an inventor on patents (US Patent nos. 11,458,209; 11,433,142; 11,752,218; 11,648,321; and 11,654,200) and patent applications (PCT/US22/76787, PCT/US23/60225, and PCT/US2024/010326) pertaining to the LION formulation and repRNA compositions described in the studies. Funders had no role in study design, data interpretation or decision to publish. The remaining authors declare no competing interests., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Supersecondary structure code (SSSC), which is represented as a conformation term using the letters "H," "S," "T," and "D" for each amino acid peptide unit, can be utilized to look up supersecondary structure motifs like a helix-hairpin-helix (HhH) motif as character strings from the Protein Data Bank (PDB) structure files. The deep neural network-based conformational variability prediction system of protein structures (SSSCPreds) can simultaneously predict locations of protein flexibility or rigidity and the shapes of those regions with high accuracy. The sequence flexibility/rigidity map obtained from SSSCPreds has the prediction accuracy enough to discuss the correlation with the sequence-to-phenotype ones by mutations. In this chapter, the protocol of conformational variability prediction methods using SSSC, including the analysis of influenza virus hemagglutinins with amino acid mutations, is described. The conformational variability pattern of hemagglutinins for human influenza A H1N1pdm extremely resembles that of avian influenza A H5N1, except for the furin cleavage site of H5N1. The transition of virus variants is visually understandable from the maps, including the sharply increased flexibility with the insight into the recent unseasonal influenza epidemics in Japan. The prediction accuracy of conformational variability is low for proteins at pH 5 with very few measurement conditions, so this is the limitation of the conformational variability prediction method., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Clade 2.3.2.1 of the H5N1 avian influenza virus (AIV) evolved into several subclades. However, the effect of glycosylation on the biological characteristics of hemagglutinin (HA) and/or neuraminidase (NA) from H5N1 AIVs remains unclear. Here, we determined that the global prevalence of clade 2.3.2.1 H5N1 AIVs with deglycosylated residue 158 on HA (HA158-) and glycosylated residue 88 on NA (NA88+) were predominant via multiple sequence analysis. The deglycosylation of residue on NA 88 (NA88-) was observed in clade 2.3.2.1a (new) and clade 2.3.2.1e H5N1 AIVs. Interestingly, NA88- was coupled with the acquisition of 158 glycosylation sites on HA (HA158+) in clade 2.3.2.1e H5N1 AIVs from China, and clade 2.3.2.1a (new) H5N1 AIVs exhibiting the HA158-NA88- pattern were predominant in Bangladesh. Meanwhile, the temporal distribution of strain HA158+ NA88- was highly consistent with the implementation of Re-6 vaccine in China. The recombinant H5N1 AIVs constructed using a reverse genetic system showed that the acquisition of the HA158 glycosylation site facilitated viral evasion from Re-6 antisera, and the virus lacking glycosylation sites at HA158 and NA88 resulted in reduced NA activity, replication in mammalian cells, and pathogenicity in both chickens and mice compared to that of the viruses with alternative glycosylation patterns. Therefore, the acquisition of HA158+ in clade 2.3.2.1e H5N1 AIVs enables evasion of Re-6 vaccination pressure, and the virulence of clade 2.3.2.1 H5N1 AIVs is modulated by the absence of glycosylation sites at HA158 and NA88. Our finding highlighted the importance of epidemiological surveillance and timely updating vaccines of H5 AIVs., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study explores a dual expression vector system for delivering prokaryotic and eukaryotic antigens to improve conventional vaccination strategies. To enhance immune protection against H9N2 avian influenza virus (AIV), which threatens poultry and humans, we used the previously constructed pJHL270 and pJHL305 plasmids with the Ptrc and CMV promoters to stimulate MHC class II and I responses through exogenous and endogenous antigenic presentation. Salmonella Gallinarum (SG), a delivery vector, was engineered to have defective lipopolysaccharide structures through lon, pagL, and rfaL deletion. It demonstrated a safety profile with lower induction of inflammatory cytokines than the wild-type strain. Bioinformatics tools predicted that the HA1 and M2e sequences, which were designed as consensus sequences of South Korean strains (2000-2021), would have high antigenicity and favorable structures. In vitro expression of the vaccine constructs was validated by western blotting. Birds immunized with attenuated SG harboring pJHL270 (JOL3025) or pJHL305 (JOL3027) containing HA-M2e showed significant increases in serum IgY and mucosal IgA antibodies, indicating strong humoral and mucosal immune responses, comparable with inactivated commercial vaccine. Post-immunization, we found a substantial rise in the hemagglutination inhibition titer, suggesting effective prevention of viral attachment and robust cell-mediated immunity, with a 1.96-fold and 2.80-fold increase in CD4 + and CD8 + T cells, respectively, for JOL3025 and a 1.75-fold and 2.49-fold increase for JOL3027. Furthermore, MHC class I and II expression increased 1.35-fold and 1.63-fold, for JOL3025, and 1.61-fold and 1.68-fold, respectively, for JOL3027. The IL-4 and IFN-γ levels were elevated, indicating a balanced Th-1 and Th-2 response. Post-challenge, birds immunized with vaccine candidates or the commercial vaccine exhibited minimal to no clinical signs, reduced lesions, lower lung viral titers, and negligible impacts on egg production compared to controls. In conclusion, both plasmids successfully delivered HA1-M2e immunogens through the engineered SG strains, eliciting strong humoral, mucosal, and cell-mediated immune responses and co-stimulating MHC class I and II antigen presentation pathways to provide effective protection against H9N2 AIV with minimal adverse effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The seasonal human influenza virus undergoes rapid evolution, leading to significant changes in circulating viral strains from year to year. These changes are typically driven by adaptive mutations, particularly in the antigenic epitopes, the regions of the viral surface protein hemagglutinin targeted by human antibodies. Here, we describe a consistent set of methods for data-driven predictive analysis of viral evolution. Our pipeline integrates four types of data: (1) sequence data of viral isolates collected on a worldwide scale, (2) epidemiological data on incidences, (3) antigenic characterization of circulating viruses, and (4) intrinsic viral phenotypes. From the combined analysis of these data, we obtain estimates of relative fitness for circulating strains and predictions of clade frequencies for periods of up to 1 year. Furthermore, we obtain comparative estimates of protection against future viral populations for candidate vaccine strains, providing a basis for pre-emptive vaccine strain selection. Continuously updated predictions obtained from the prediction pipeline for influenza and SARS-CoV-2 are available at https://previr.app ., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In recent years, infection cases of H5 subtype highly pathogenic avian influenza viruses (HPAIVs) in wild mammals have increased globally. To obtain recent epidemiological information regarding influenza A virus (IAV) infection in raccoons (Procyon lotor), the prevalence of anti-IAV antibodies in sera was analyzed among raccoons captured in Tokachi District, Hokkaido, Japan, from 2019 to 2023. Screening of serum samples using enzyme-linked immunosorbent assay and agar gel precipitation test detected anti-IAV antibodies in 5 of 114 (4.4 %) raccoons. All positive sera were from raccoons captured from 2022 to 2023. The hemagglutination inhibition test revealed that all five serum samples contained anti-H5 subtype HPAIV antibodies, and one also contained anti-H1 subtype antibodies. The neuraminidase inhibition test revealed that all five sera contained anti-N1 subtype antibodies, and one also contained anti-N8 subtype antibodies. In the virus neutralization test, these five sera showed stronger neutralization activity against the H5 subtype clade 2.3.4.4b HPAIV strain recently circulating worldwide compared to the old H5 HPAIV strain isolated in Japan in 2007. These findings suggested that raccoons could be involved in the circulation of H5 HPAIVs in nature., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

H9N2 avian influenza virus is a significant poultry pathogen that provides internal genes for multiple zoonotic subtypes of avian influenza, presenting a severe threat to public health. The isolation rate of H9N2 in poultry has increased annually in recent years. In this study, a recombinant Herpesvirus of Turkeys (HVT) vaccine expressing H9-HA was constructed using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. In the construction of HVT-EGFP-HA recombinant virus, nonhomologous end joining (NHEJ) is a much more efficient strategy compare to Homology-directed recombination (HDR). HVT-HA demonstrated stability and consistent replication with the parent strain. Subcutaneous injection and in-ovo injection of HVT-HA induced different levels of immune response. Compared to in-ovo injection of HVT-HA, subcutaneous injection induced significantly higher neutralizing serum antibodies. This finding is supported by the significantly higher CD4 + T cell response in Peripheral blood mononuclear cell Peripheral blood mononuclear cell (PBMC) in the subcutaneous injection group. However, in-ovo injection of HVT-HA resulted in significantly higher neutralizing antibodies in the Harderian glands. In addition, it significantly inhibited viral shedding after intranasal exposure to H9N2. This phenomenon could be attributed to the mucosal immunity present in the Hadrian gland. Thus, our findings indicate that the in-ovo injection of the HVT-HA recombinant vaccine is a promising method to inhibit the transmission of H9N2 via the upper respiratory tract in chickens., Competing Interests: DISCLOSURES The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiaowen Liu reports financial support was provided by National Key Research and Development Project of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Avian influenza viruses are ubiquitous in the Anatinae subfamily of aquatic birds and occasionally spill over to poultry. Infection with low pathogenicity avian influenza viruses generally leads to subclinical or mild clinical disease. In contrast, highly pathogenic avian influenza viruses emerge from low pathogenic forms and can cause severe disease associated with extraordinarily high mortality rates. Here, we describe the natural history of avian influenza virus, with a focus on H5Nx and H7Nx subtypes, and the emergence of highly pathogenic forms; we review the biology of AIV; we examine cleavage of haemagglutinin by host cell enzymes with a particular emphasis on the biochemical properties of the proprotein convertases, and trypsin and trypsin-like proteases; we describe mechanisms implicated in the functional evolution of the haemagglutinin cleavage site motif that leads to emergence of HPAIVs; and finally, we discuss the diversity of H5 and H7 haemagglutinin cleavage site sequence motifs. It is crucial to understand the molecular attributes that drive the emergence and evolution of HPAIVs with pandemic potential to inform risk assessments and mitigate the threat of HPAIVs to poultry and human populations., (© 2024 Commonwealth of Australia. Reviews in Medical Virology published by John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Avian influenza virus (AIV) subtype H9N2 still poses a great threat to the poultry farming industry and public health worldwide, and the development of a new influenza vaccine that is safe and conservative and able to address influenza virus mutations is highly promising for application. HA2, the neck of the HA protein, and M2e, the extracellular N-terminal structural domain of the M2 protein, are conserved and effective protective antigens. In this study, the HA2 sequences were fused with three M2e copies (H9N2, H1N1 and H5N1) to the norovirus VP1 protein via the SpyTag-SpyCatcher platform to form self-assembled nanoparticles and display antigenic proteins on its surface, yielding pYL262. The chicken dendritic cells (DCs) targeting the nanobody phage-54 were then fused to HA2-3M2e to yield pYL327. Finally, a synthesized 20-repeat CpG adjuvant gene fragment was inserted into pYL327, resulting in the plasmid pYL331. All the constructed plasmids were then transformed into the sifA gene-deficient Salmonella vector χYL56 for oral immunization. The results showed that sifA-deficient Salmonella could efficiently increase antigen-specific mucosal sIgA antibody titers, especially in alveolar lavage samples, whereas the presence of the phage-54 nanobody could dramatically increase intracellular IFN-γ mRNA levels, indicating its ability to enhance the Th1-type immune response. Finally, the presence of the CpG adjuvant clearly increased T-cell proliferation and promoted DC activation, with elevated splenic TLR21 levels observed. Strikingly, after oral immunization with χYL56 (pYL331), chickens were protected against challenge with the G57 genotype H9N2 virus, which presented similar or even better levels of virus shedding and body weight gain compared with the commercial inactivated vaccine, providing a new option for controlling H9N2 virus infection in the future., Competing Interests: Conflict of interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier Inc.)