Co-immunization with two recombinant Newcastle disease viruses expressing ILTV gB and H9N2 AIV HA confers protective efficacy against three avian pathogens

Newcastle disease virus (NDV) has been well established as a versatile vector platform for heterologous antigen delivery in poultry vaccine development. However, the prospects for combined application of two recombinant NDV (rNDV) vaccines remain poorly understood. In previous studies, we generated two rNDVs based on the thermostable TS09-C strain, expressing glycoprotein B (gB) of infectious laryngotracheitis virus (ILTV) and hemagglutinin (HA) of the H9N2 subtype avian influenza virus (H9N2 AIV). In the present study, we evaluated the potential interference between the two rNDVs and using both in vitro and in vivo models. When two rNDVs co-infected BHK-21 and CEK cells, their viral replication kinetics and expression levels of heterologous antigens (gB and HA) were comparable to those in the respective single-virus infection groups. This finding indicates that the two rNDVs do not exhibit significant interference in vitro. SPF chickens co-immunized with the two rNDVs via intranasal and intraocular routes elicited effective H9N2-, ILTV-, and NDV-specific antibody responses comparable to those in the single-virus immunization group. Compared with control chickens, co-immunized chickens displayed milder clinical signs and reduced viral shedding following challenge with H9N2 AIV, ILTV, and NDV. Based on these findings, spray co-immunization was further tested in commercial chickens and proven to confer effective protection against challenges with NDV, H9N2 AIV, and ILTV. In conclusion, this study confirms the feasibility of co-immunization using the two rNDVs, thereby providing a highly viable strategy for the simultaneous control of multiple avian pathogens.