Highly pathogenic avian influenza (HPAI), infectious bronchitis (IB), and Newcastle disease (ND) are highly contagious and economically important diseases in poultry. Vaccination is the major strategy which is implemented to combat highly pathogenic avian influenza virus (HPAIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV), worldwide. However, among these viruses, some NDV strains are naturally avirulent and have been used as highly safe vaccines for more than 60 years. Live attenuated IBV vaccines that are produced by passaging virulent strains in eggs have safety concerns and are genetically unstable. Inactivated IBV and HPAIV vaccines also are less efficacious and affordable. Therefore, development of alternative vaccines against IBV and HPAIV is highly needed. In this multistep study, we have employed NDV vector and other novel avian paramyxovirus (APMV) vectors to develop improved IBV and HPAIV vaccines.

Firstly, we conducted a study to investigate the contributions of the S1, S2, and S proteins of IBV in protection against virulent IBV, and to develop a safe and efficacious recombinant NDV-vectored IBV vaccine. We generated recombinant (rNDV) strain LaSota viruses expressing S1, S2 or S protein of IBV using reverse genetics. We evaluated the protective efficacies of rNDVs against virulent IBV and NDV challenges. Our results showed that the S protein, which contains the S1 and S2 neutralizing epitopes in correct confirmation is the best protective antigen of IBV. These results suggest that the rNDV expressing the S protein of IBV is a safe and effective bivalent vaccine candidate for both IBV and NDV.

Secondly, besides rNDV strain LaSota vector, we employed a novel chimeric rNDV/avian paramyxovirus serotype-2 (rNDV/APMV-2) vector that replicates less efficiently and a modified NDV strain LaSota (rLaSota-527) vector that replicates more efficiently to develop a likely improved viral vectored vaccine against IBV. We generated rNDV/APMV-2 or rLaSota-527 virus expressing the best protective protein of IBV (S protein), which was found in the first study. The protective efficacies of rNDV/APMV-2 or rLaSota-527 virus expressing the S protein was evaluated against IBV in chickens. Our results showed that immunization of chickens with either chimeric rNDV/APMV-2 expressing the S protein, which is a better candidate for in ovo vaccination, or rLaSota virus expressing the S protein provided protection against IBV. Most importantly, compared to prime-boost vaccination or vaccination with rLaSota-527 virus expressing the S protein, single immunization of chickens with rLaSota virus expressing the S protein induced better immune responses against IBV.

Thirdly, we conducted a study to evaluate the contributions of HA1 and HA2 subunits of HPAIV HA protein in the induction of neutralizing antibodies and protection in chickens, using rNDV strain LaSota vector. Our results showed that the HA1 and HA2 subunits when expressed separately, neither provided protection nor induced neutralizing antibodies. To be effective the HA protein must be incorporated into a vaccine as an intact protein. These results also highlight the importance of using chickens in HPAIV vaccine studies as they are susceptible natural hosts.

Finally, we employed APMV-3 strain Netherlands as a vaccine vector, for its high efficiency replication in multiorgans of host, to generate an improved vaccine against HPAIV. Our results showed that immunization of chickens with either rAPMV-3 expressing the HA protein (rAPMV-3/HA) or rNDV expressing the HA protein (rNDV/HA) provided complete protection against HPAIV challenge. However, the immunization of chickens with rAPMV-3/HA induced higher levels of neutralizing antibodies than that induced by rNDV/HA. These results suggest that mass-vaccination with a rAPMV-3/HA might provide better protection against H5N1 HPAIV in field conditions.

In conclusion, the individual subunits of the S protein of IBV or the HA protein of HPAIV when expressed separately, neither provided protection nor induced neutralizing antibodies. To provide protective efficacy, the intact HA or S protein must be incorporated into vaccine. The rNDV expressing the S protein is a safe and efficacious bivalent vaccine against IBV and NDV. Other than rNDV strain LaSota, rNDV/APMV-2 and rAPMV-3 are promising vaccine vectors for development vaccines against IBV and HPAIV, respectively.