Manman Dai

H5N6 highly pathogenic avian influenza virus (HPAIV) poses a serious threat to both poultry and public health due to its ability to cross species barriers.

H9N2 avian influenza virus (AIV) poses a persistent threat as inactivated vaccines (InV) often fail to prevent viral shedding. To address this, we developed a recombinant turkey herpesvirus (HVT-BNT) expressing conserved B and T cell epitopes from H9N2 AIV to enhance both humoral and cellular immunity. HVT-BNT exhibited genetic stability over 15 serial passages and growth kinetics comparable to the parental strain in vitro. We evaluated immunization strategies of HVT-BNT combined with InV in 1-day-old chicks and 18-day-old embryos via subcutaneous (HVT-BNT+InV) or in ovo (HVT-BNT-ovo+InV) routes, respectively. Compared to InV alone, HVT-BNT+InV elicited significantly higher HI and neutralizing antibody titers, elevated IgG and IgM levels, and increased proportions of CD8⁺ T cells. Similarly, the HVT-BNT-ovo+InV group exhibited a trend of higher values in these indicators. Notably, while the InV group displayed no significant differences in key immune cytokines compared to the control group, the combined immunization groups exhibited significant upregulation of IFN-α, IFN-β, IFN-γ, IL-2, IL-5, IL-6, IL-10, and IL-13. Furthermore, ELISPOT assays confirmed enhanced IFN-γ secretion in response to conserved AIV peptides (NP_380-393, NP_455-463, and NS1_98-106) in the combined immunization groups. Following heterologous H9N2 AIV challenge, oropharyngeal positivity rates in the combined immunization groups were lower than those in the InV group at 5 DPI. Similarly, cloacal positivity rates were more reduced in the combined groups compared to the InV group at 3 and 5 DPI. By 7 DPI, viral shedding was completely cleared in both combined immunization groups, whereas the InV group continued to shed virus via the oropharyngeal route. These findings demonstrate that the HVT-BNT-based vaccination strategy effectively enhances both humoral and cellular immune responses, providing superior early protection. While the in ovo strategy provides a viable hatchery intervention, the subcutaneous route exhibits the superior immune activation and protection compared with conventional InV alone.

Since 2020, the clade 2.3.4.4b highly pathogenic avian influenza (HPAI) H5N1 viruses have caused unprecedented outbreaks in wild birds and domestic poultry globally, resulting in significant ecological damage and economic losses due to the disease and enforced stamp-out control. In addition to the avian hosts, the H5N1 viruses have expanded their host range to infect many mammalian species, potentially increasing the zoonotic risk. Here, we review the current knowns and unknowns of clade 2.3.4.4b HPAI H5N1 viruses, and we highlight common challenges in prevention. By integrating our knowledge of viral evolution and ecology, we aim to identify discrepancies and knowledge gaps for a more comprehensive understanding of the virus. Ultimately, this review will serve as a theoretical foundation for researchers involved in related avian influenza virus studies, aiding in improved control and prevention of H5N1 viruses.

The in vitro culture of T cells is a critical method for studying immune responses, viral infections, and potential therapeutic strategies. However, no established protocols for culturing avian T cells in vitro have been reported to date. In this study, we present a protocol for the first time, utilizing the highly pathogenic avian influenza (HPAIV) H5N1 virus as a model. Here, 4-week-old ducks were infected with the virus, and memory T lymphocytes were isolated 28 days post-infection to culture H5N1-specific duck T lymphocytes, enabling the investigation of the specific immune response to the H5N1 virus. Key steps in the protocol include isolating memory peripheral blood mononuclear cells (PBMCs) from infected ducks, activating antigen-presenting cells (APCs) with an optimal multiplicity of infection (MOI = 5) for 6 h, and preparing T cell culture media supplemented with recombinant IL-2 and other specific additives. T-cell proliferation, cytokine secretion, and cytotoxic activity were closely monitored throughout the process. Additionally, we established an intracellular cytokine staining protocol to quantify IFN-γ secretion in duck T cells. This involved generating a hybridoma cell line expressing IgG3κ antibodies specific to duck IFN-γ, followed by successful antibody purification. The purified antibody was diluted 1:10 and applied in flow cytometry to precisely measure IFN-γ secretion. This method offers a reliable tool for evaluating duck T cell responses and lays the foundation for future investigations into avian viral immunology.

The in vitro culture of T cells is a critical method for studying immune responses, viral infections, and potential therapeutic strategies. However, no established protocols for culturing avian T cells in vitro have been reported to date. In this study, we present a protocol for the first time, utilizing the highly pathogenic avian influenza (HPAIV) H5N1 virus as a model. Here, 4-week-old ducks were infected with the virus, and memory T lymphocytes were isolated 28 days post-infection to culture H5N1-specific duck T lymphocytes, enabling the investigation of the specific immune response to the H5N1 virus. Key steps in the protocol include isolating memory peripheral blood mononuclear cells (PBMCs) from infected ducks, activating antigen-presenting cells (APCs) with an optimal multiplicity of infection (MOI = 5) for 6 h, and preparing T cell culture media supplemented with recombinant IL-2 and other specific additives. T-cell proliferation, cytokine secretion, and cytotoxic activity were closely monitored throughout the process. Additionally, we established an intracellular cytokine staining protocol to quantify IFN-γ secretion in duck T cells. This involved generating a hybridoma cell line expressing IgG3κ antibodies specific to duck IFN-γ, followed by successful antibody purification. The purified antibody was diluted 1:10 and applied in flow cytometry to precisely measure IFN-γ secretion. This method offers a reliable tool for evaluating duck T cell responses and lays the foundation for future investigations into avian viral immunology.

TCRγδ+ T cells are a major lymphocyte population of chickens, but their response or contribution to immunity against avian influenza virus (AIV) remains unknown. Here, we report an increase in the proportion and activation state of TCRγδ+CD8α+ T cells in the PBMCs of 3 chicken lines (MHC homozygous H-B2 and H-B21 lines and outbred G-WL line) with the strongest responses observed in the more resistant H-B2 chickens. H9N2 AIV infection induced mRNA upregulation of interferon (IFN)-γ and cytotoxicity-associated molecules, including, Granzyme A, Granzyme K, and perforin in sorted TCRγδ+CD8α+ T cells. Moreover, in ex vivo cultured TCRγδ+CD8α+ T cells in response to H9N2 AIV infected splenocytes, strongly indicates the activation of these cells’ cytolytic potential via detection of transcription levels of cytotoxic genes with quantitative reverse transcription polymerase chain reaction (qRT-PCR), and IFN-γ protein level with ELISPOT and an intracellular cytokine staining assays. Most importantly, in vivo depletion of γδ T cells led to reduced H9N2 AIV control, which was particularly evident in the early phase of infection. Taken together, these results indicate that strong TCRγδ+CD8α+T cell response plays a critical role in protecting chicken against H9N2 AIV infection.

Interferon-γ (IFN-γ), a member of the Type II IFN family, is a crucial cytokine in the immune system and serves as an important indicator of immune response. Intracellular cytokine staining (ICS) is a technique used to analyze the production of cytokines within individual cells, and it has a wide range of applications in the fields of immunological monitoring, vaccine trials, and the study of infectious diseases. This study aimed to prepare monoclonal antibodies against duck IFN-γ protein and to establish an ICS protocol for detecting the duck IFN-γ protein. The duIFN-γ-His or duIFN-γ-Fc gene was cloned into the pEE12.4 expression vector and expressed as a recombinant protein of size 20.2 KDa or 54.9 KDa in 293F cells. The purified recombinant proteins were inoculated into BALB/c mice to generate splenic lymphocytes capable of secreting anti-duIFN-γ antibodies, and hybridoma cells were obtained after fusion with SP2/0 cells. A new hybridoma cell line named 24H4, which stably secreted IgG3 κ subtype antibody against duck IFN-γ, was established. This monoclonal antibody (mAb) was identified by Western blot to recognize duck IFN-γ antibodies, and the indirect ELISA results showed that its ability to recognize IFN-γ protein reached 0.001 μg/mL. The established ICS method was used to stain PBMCs after Concanavalin A (ConA) stimulation, and duck IFN-γ protein was successfully detected by flow cytometry, indicating that the ICS method was successful. In this study, we provide a crucial tool for subsequent research on duck cellular immune responses by using the monoclonal antibody 24H4.

: Interferon-γ (IFN-γ), a member of the type II IFN family, is a crucial cytokine in the immune system and serves as an important indicator of immune response. Intracellular Cytokine Staining (ICS) is a technique used to analyze the production of cytokines within individual cells, and it has a wide range of applications in the fields of immunological monitoring, vaccine trials, and the study of infectious diseases. This study aimed to prepare monoclonal antibodies against duck IFN-γ protein and to establish an ICS protocol for detecting the duck IFN-γ protein. The duIFN-γ-His or duIFN-γ-Fc gene was cloned into the pEE12.4 expression vector and expressed as a recombinant protein of size 20.2 KDa or 54.9 KDa in 293F cells. The purified recombinant proteins were inoculated into BALB/c mice to generate splenic lymphocytes capable of secreting anti-duIFN-γ antibodies, and hybridoma cells were obtained after fusion with SP2/0 cells. A new hybridoma cell line named 24H4, which stably secreted IgG3 κ subtype antibody against duck IFN-γ. This monoclonal antibody (mAb) was identified by Western Blot to recognize duck IFN-γ antibodies, and the indirect ELISA results showed that its ability to recognize IFN-γ protein reached 0.001 μg/ml. The established ICS method was used to stain PBMCs after ConA stimulation, and duck IFN-γ protein was successfully detected by flow cytometry, indicating that the ICS method was successfully established. This study we use the monoclonal antibody, 24H4, provides a crucial tool for subsequent research on duck cellular immune responses.