Vaccination is the most effective preventative measure against economically devastating poultry diseases, such as infectious bursal disease (IBD), H9 subtype avian influenza (AI), and Marek's disease. In recent decades, various vaccination strategies have been investigated and developed. Among these, multivalent and combination vaccines, which confer protection against multiple diseases in a single dose, have emerged as a significant advancement in veterinary medicine. This study demonstrates that the attenuated Marek's disease virus serotype 1 (MDV-1) vaccine strain, rMSΔMeq, known for its safety and robust carrier immunogenicity, can serve as a vector for the heterologous expression of multiple antigenic proteins. We constructed the recombinant virus rMDV-VP2-HA by inserting the H9 subtype avian influenza virus (AIV) hemagglutinin (HA) expression cassette into the UL41 region and the infectious bursal disease virus (IBDV) VP2 expression cassette into the US2 region. The growth characteristics of this recombinant virus were consistent with those of the parental virus, and it stably expressed the HA and VP2 genes. Vaccination with rMDV-VP2-HA induces high titers of IBDV-neutralizing antibodies and hemagglutinin inhibition (HI) antibodies and elicits strong cellular immune responses. Specifically, vaccination with rMDV-VP2-HA enhances interferon-gamma (IFN-γ) expression in response to stimulation with HA and VP2 proteins. Meanwhile, cytokines associated with both Th1 and Th2 responses were also upregulated. Remarkably, chickens vaccinated with rMDV-VP2-HA achieved complete protection against very virulent IBDV, H9 subtype AIV, and very virulent MDV. These findings underscore the exceptional practical potential of rMDV-VP2-HA as the first MDV-1 vector-based multivalent vaccine candidate for combating these poultry diseases.IMPORTANCECommercial vaccines for infectious bursal disease (IBD) and H9 subtype avian influenza (AI) require multiple doses, increasing costs and causing stress in chicken flocks. Additionally, their efficacy is frequently compromised by maternal antibody interference. This underscores the urgent need for a multivalent, multi-component, and single-dose vaccines capable of streamlining immunization protocols, overcoming maternal antibody interference, and providing lifelong immunity. Our research demonstrates, for the first time, serotype 1 Marek's disease virus (MDV-1) can stably express multiple exogenous genes. More significantly, the rMDV-VP2-HA elicits robust humoral and cellular immune responses, achieving complete protection against H9 subtype AIV, very virulent IBDV, and very virulent MDV with a single immunization. These findings contribute to enhancing the efficiency of disease prevention and confirm that the MDV-1 is an ideal vector for developing multivalent vaccines, achieving the goal of "multiple protections with a single shot." This advancement represents a significant progression in the prevention and control of economically important poultry diseases.

Keywords: H9 subtype AIV; HA; IBDV; MDV-1; VP2; vaccine.