The H9N2 subtype avian influenza virus (AIV) hemagglutinin (HA) protein is a major immunogen in which HA1 is a genetic variant and HA2 is relatively conserved.  Identifying broad-spectrum antigen epitopes targeting HA1 is crucial for vaccine design and detection.  Based on the phylogenetic and serological analyses, we identified 2 antigenic groups and 3 representative viruses: A/chicken/Jiangsu/JY040218C/2019, A/pigeon/Jiangsu/JY020616/2019, and A/chicken/Jiangsu/WX090312/2018.  An overlapping peptide library was synthesized using HA1 amino acid sequences of the viruses as templates.  Through peptide scanning of the sera against different strains of H9N2 subtype AIV, we identified peptides from 4 regions (H9-2/3, H9-20/21, H9-26, and H9-29/30/31) that demonstrated broad-spectrum reactivity.  Immunological assay results demonstrated that H9-21 (²¹⁹RIFKPLIGPRPLVNGLMGRI²³⁹), H9-26 (²⁶⁹SGESHGRILKTDLKMGSCTV²⁸⁹), and H9-30 (³⁰⁹YAFGNCPKYI GVKSLKLAVG³²⁹) effectively induced antibody generation and conferred partial protective efficacy against the parent virus JY040218C.  The results of lymphocyte proliferation and ELISpot assays indicated that peptides H9-15 (¹⁵⁹MRWLTQKNNAYPTQDAQYTN¹⁷⁹), H9-22 (²²⁹PLVNGLMGRINYYWSVLKP G²⁴⁹), and H9-23 (²³⁹NYYWSVLKPGQTLRIKSDGN²⁵⁹) could effectively stimulate the expression of interferon-gamma in peripheral blood lymphocytes of chickens immunized against different strains of H9N2 AIV.  Collectively, 5 novel cell epitopes H9-15, H9-22, H9-23, H9-26, and H9-30, including the best B cell epitope H9-26 and the best T cells epitope H9-22, were identified that could be targeted for vaccine design or detection approaches against H9N2 AIVs.

H7N9 subtype avian influenza virus poses a great challenge for poultry industry.  Newcastle disease virus (NDV)-vectored H7N9 avian influenza vaccines (NDV_vecH7N9) are effective in disease control because they are protective and allow mass administration.  Of note, these vaccines elicit undetectable H7N9-specific hemagglutination-inhibition (HI) but high IgG antibodies in chickens.  However, the molecular basis and protective mechanism underlying this particular antibody immunity remain unclear.  Herein, immunization with an NDV_vecH7N9 induced low anti-H7N9 HI and virus neutralization titers but high levels of hemagglutinin (HA)-binding IgG antibodies in chickens.  Three residues (S150, G151 and S152) in HA of H7N9 virus were identified as the dominant epitopes recognized by the NDV_vecH7N9 immune serum.  Passively transferred NDV_vecH7N9 immune serum conferred complete protection against H7N9 virus infection in chickens.  The NDV_vecH7N9 immune serum can mediate a potent lysis of HA-expressing and H7N9 virus-infected cells and significantly suppress H7N9 virus infectivity.  These activities of the serum were significantly impaired after heat-inactivation or treatment with complement inhibitor, suggesting the engagement of the complement system.  Moreover, mutations in the 150-SGS-152 sites in HA resulted in significant reductions in cell lysis and virus neutralization mediated by the NDV_vecH7N9 immune serum, indicating the requirement of antibody-antigen binding for complement activity.  Therefore, antibodies induced by the NDV_vecH7N9 can activate antibody-dependent complement-mediated lysis of H7N9 virus-infected cells and complement-mediated neutralization of H7N9 virus.  Our findings unveiled a novel role of the complement in protection conferred by the NDV_vecH7N9, highlighting a potential benefit of engaging the complement system in H7N9 vaccine design.

The hemagglutinin (HA) protein of the H9N2 subtype avian influenza virus (AIV) undergoes frequent antigenic drift, which compromises the efficacy of existing inactivated vaccines. We have identified 12 key HA residues responsible for antigenic differences between the 2 major H9N2 antigenic groups; however, their role in eliciting broad cross-reactive immunity remains undefined. In this study, we systematically evaluated the impact of single- and multi-residue mutations in HA antigenic regions A, B1, B2, and E on viral antigenicity using antigenic cartography and monoclonal antibody profiling. 4 recombinant viruses—R118-A, R118-AE, R118-B1, and R118-AB1E—demonstrated broadened antigenic reactivity and were selected for further analysis. Among them, R118-A elicited immune sera with high hemagglutination inhibition and microneutralization titers against a diverse panel of H9N2 strains and exhibited broad antigenic coverage on antigenic cartography. In chicken challenge experiments, immunization with R118-A conferred cross-protection against group 1 (B4.4+B4.6) and group 2 (B4.7) H9N2 viruses, underscoring the critical role of site A modifications in broadening vaccine protection. These findings offer theoretical support and practical strategies for the rational design of next-generation H9N2 vaccines with improved cross-protective efficacy.

Highly pathogenic avian influenza viruses (AIV) primarily circulate within poultry populations. However, continuous evolution and mutation accumulation drive antigenic drift and may enable the virus to evade host immunity and cross the species barrier. To identify residues associated with antigenic changes and virulence in the H5N1 virus under immune selection pressure, SPF chickens, SPF chicken embryos, and chicken embryo fibroblast cells were used as model to serially passage the SY (Re-5 like) virus in the presence of homologous chicken antiserum. Progeny viruses escaped the neutralizing capacity of the antiserum were sequenced. A total of twelve amino acid mutation sites were identified in the HA, PB2, and PB1 proteins. The results showed that in the HA of the H5N1 virus, both K205N and K205T mutation patterns resulted in a significant reduction in HI titers and microneutralization titers when tested with chicken antisera. The K32M and E69K mutations in PB2, along with the M246I mutation in PB1 could effectively attenuate viral pathogenicity in mice, whereas the S155N mutation in PB2 significantly enhanced it. Notably, under the immune pressure, the S155N mutation in PB2 delayed the emergence of K205N substitution in HA. This in vivo and in vitro method for selecting immune-escape mutants provides a valuable tool for predicting emerging antigenic variants and mammalian adaptive mutations, as well as elucidating the co-evolution dynamics between surface and internal genes in H5N1 viruses.