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

. doi: 10.1016/j.virs.2026.04.008

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

基于火鸡疱疹病毒载体的多表位疫苗经皮下和胚内接种增强H9N2灭活疫苗对异源病毒的保护效力

谢梓民 ^a ,
陈滢仪 ^a ,
杜珊瑶 ^a ,
黄秀梅 ^a ,
邵玉欢 ^a ,
黄江武 ^a ,
廖明 ^a,b,, ,
代曼曼 ^a,b,,

a 华南农业大学兽医学院;
b 中英禽疾病研究中心

通讯作者： 廖明, mliao@scau.edu.cn; 代曼曼, daimanman1229@scau.edu.cn
收稿日期： 2026-01-16
录用日期： 2026-04-22

摘要

H9N2 亚型禽流感病毒（AIV）持续构成威胁，因为灭活疫苗（InV）通常无法阻止病毒脱落。为了解决这个问题，该团队研发了一种重组火鸡疱疹病毒（HVT-BNT），该病毒表达H9N2 AIV保守的B和T细胞表位，以增强体液和细胞免疫。研究表明，HVT-BNT在15次连续传代中表现出遗传稳定性，其生长动力学与亲本菌株在体外的复制能力相当。研究团队在1日龄雏鸡和18日龄鸡胚中分别通过皮下（HVT-BNT+InV）和胚内（HVT-BNT-ovo+InV）途径评估了HVT-BNT与InV联合的免疫策略。与单独的InV相比，HVT-BNT+InV显著提高了HI和中和抗体滴度、IgG和IgM水平以及CD8+T细胞的比例。同样，HVT-BNT-ovo+InV组在这些指标上也表现出相同的趋势。值得注意的是，与对照组相比，InV组在关键免疫细胞因子方面没有显著差异，而联合免疫组表现出IFN-α、IFN-β、IFN-γ、IL-2、IL-5、IL-6、IL-10和IL-13的显著上调。此外，ELISPOT检测证实，在联合免疫组中，保守的AIV肽（NP380-393、NP455-463和NS198-106）能显著增强IFN-γ的分泌。在异源高剂量（10⁷ EID₅₀）H9N2禽流感病毒（AIV）攻毒后，联合免疫组的咽拭子阳性率在攻毒后5天（5 DPI）时低于InV组。同样，在3 DPI和5 DPI时，与InV组相比，联合免疫组的泄殖腔阳性率更低。到7 DPI时，两个联合免疫组的病毒脱落被完全清除，而InV组仍有通过口咽途径排出病毒的鸡只。这些发现表明，基于HVT-BNT的疫苗接种策略有效地增强了宿主的体液和细胞免疫反应，提供了更好地早期保护。虽然胚内策略提供了一种可行的干预，但1日龄皮下途径表现出更优的免疫激活和保护作用。
- H9N2禽流感病毒
- , 火鸡疱疹病毒
- , 卵内免疫
- , 多表位疫苗
- , 细胞免疫

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

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

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