Heterologous prime-boost immunization with herpesvirus of turkeys vectored and inactivated vaccines elicits an early and long-term immunity against a lethal H5N1 virus in chickens

Effective vaccination strategies should provide both rapid onset and sustained protection against pathogens. Conventional inactivated H5N1 highly pathogenic avian influenza (HPAI) vaccines in layer chickens typically require 2-3 weeks to establish robust immunity and multiple booster doses for sustained protection, posing significant challenges in intensive poultry production systems. This study comprehensively evaluated a heterologous prime-boost immunization strategy comprising a single dose of recombinant herpesvirus of turkeys (HVT) vector vaccine administered at one day of age, followed by a single dose of inactivated vaccine at 2 weeks of age. We constructed a recombinant HVT (rHVT-HA-Re6) expressing the hemagglutinin (HA) gene of an H5N1 virus. The protective efficacy was evaluated in specific-pathogen-free (SPF) layer chickens administered a heterologous prime-boost regimen: a single dose (approximately 5000 PFU) of rHVT-HA-Re6 vector vaccine administered at 1 day of age, followed by a single dose (0.3 mL) of inactivated H5-Re6 vaccine at 14 days of age. This regimen's efficacy was compared with that of single-vaccine regimens (either rHVT-HA-Re6 or H5-Re6 alone) administered at corresponding time points. The heterologous prime-boost regimen elicited detectable hemagglutination-inhibiting and neutralizing antibody titers by 3 weeks of age, and sustained significantly higher HI titers from 4 to 28 weeks of age compared with groups receiving single-vaccine regimens. After challenge with the lethal H5N1 virus at 3 weeks of age, all the vaccinated birds in this group survived and virus shedding was only detected in one of the ten birds. From 4 to 28 weeks of age, the vaccinated chickens in this group were completely protected against the lethal virus challenge. These results demonstrated that heterologous prime-boost vaccination with rHVT-HA-Re6 and H5-Re6 vaccines could provide protection as early as one week post-inactivated vaccine administration and long-term protection against H5N1 avian influenza virus infections, which could be an alternative vaccine regimen for the control of HPAI in layer chickens.