Co-expression of IBDV VP2 and H9N2 HA by recombinant HVT induces high protection against both pathogens in chickens

Abstract

Infectious bursal disease virus (IBDV) and H9N2 avian influenza virus (AIV) are significant global threats to poultry health and production. While IBDV induces severe immunosuppression, undermining host defense and vaccine efficacy, H9N2 AIV is characterized by widespread prevalence, persistent shedding, and substantial economic losses. Conventional inactivated vaccines often fail to elicit robust cellular immunity and necessitate multiple booster doses, underscoring the urgent requirement for advanced multivalent vaccination platforms. To address this, we developed a recombinant herpesvirus of turkey (rHVT BAC-VP2-HA) using a bacterial artificial chromosome (BAC) vector system, engineered to co-express the major protective antigen VP2 of IBDV and the hemagglutinin (HA) of H9N2 AIV. Genetic stability and in vitro characterization confirmed that the recombinant exhibited replication kinetics and plaque morphology comparable to parental HVT, with stable antigen expression. In SPF chickens, rHVT BAC-VP2-HA induced strong humoral immune responses against both target antigens, comparable to those elicited by a commercial inactivated vaccine. Crucially, the recombinant virus significantly enhanced cellular immunity, evidenced by markedly elevated CD3+CD8+ T cell responses. Upon challenge, the recombinant conferred high clinical protection (86%) against virulent IBDV, significantly ameliorating bursal pathology and reducing viral loads. Notably, it provided complete (100%) protection against H9N2 AIV, effectively abolishing viral shedding and suppressing viral replication in respiratory tissues. These results demonstrate that rHVT BAC-VP2-HA is a safe and efficacious candidate capable of eliciting humoral and cellular immune responses, offering a promising strategy for the integrated control of major poultry diseases. ImportanceInfectious bursal disease virus (IBDV) and H9N2 avian influenza virus (AIV) are major pathogens that frequently co-circulate in poultry, where IBDV-induced immunosuppression compromises the efficacy of vaccination against other infectious diseases. Conventional inactivated vaccines primarily induce humoral immunity and are often insufficient to prevent viral shedding or provide broad protection against multiple pathogens. In this study, we developed a recombinant herpesvirus of turkeys (HVT) vaccine co-expressing the IBDV VP2 and H9N2 HA antigens and demonstrated that it induces both robust antibody responses and enhanced CD8+ T cell immunity. Notably, this vaccine not only provided effective protection against IBDV but also completely prevented viral shedding following H9N2 challenge. These findings highlight the advantage of HVT-vectored multivalent vaccines in eliciting balanced immune responses and controlling virus transmission, providing important insights for the development of next-generation vaccines against immunosuppressive and respiratory viral co-infections in poultry.