HVT-based multiepitope vaccine administered subcutaneously or <em>in ovo</em> boosts protection of inactivated H9N2 vaccine against heterologous virus

Zimin Xie; Yingyi Chen; Shanyao Du; Xiumei Huang; Yuhuan Shao; Jiangwu Huang; Ming Liao; Manman Dai

doi:10.1016/j.virs.2026.04.008

April 2026

Citation: Zimin Xie, Yingyi Chen, Shanyao Du, Xiumei Huang, Yuhuan Shao, Jiangwu Huang, Ming Liao, Manman Dai. HVT-based multiepitope vaccine administered subcutaneously or in ovo boosts protection of inactivated H9N2 vaccine against heterologous virus .VIROLOGICA SINICA, 2026, 41(2) : 455-465. http://dx.doi.org/10.1016/j.virs.2026.04.008

HVT-based multiepitope vaccine administered subcutaneously or in ovo boosts protection of inactivated H9N2 vaccine against heterologous virus

Zimin Xie ^a ,
Yingyi Chen ^a ,
Shanyao Du ^a ,
Xiumei Huang ^a ,
Yuhuan Shao ^a ,
Jiangwu Huang ^a ,
Ming Liao ^{a,b
,,} ,
Manman Dai ^{a,b
,,}

a. Guangdong Laboratory for Lingnan Modern Agriculture, National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
b. UK-China Centre of Excellence for Research on Avian Diseases, Guangzhou 510642, China

Corresponding author: Ming Liao, mliao@scau.edu.cn
Manman Dai, daimanman1229@scau.edu.cn
Received Date: 16 January 2026
Accepted Date: 22 April 2026
Available online: 27 April 2026

Abstract

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.
- H9N2 AIV
- , HVT
- , In ovo immunization
- , Multiepitope vaccine
- , Cellular immunity

Electronic Supplementary Material

10.1016j.virs.2026.04.008-ESM.docx
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