Immunogenicity and protective efficacy of recombinant fusion proteins containing spike protein of infectious bronchitis virus and hemagglutinin of H3N2 influenza virus in chickens

Abstract

Infectious bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens and vaccination is the main method for disease control. The S1 protein, which contains several virus neutralization epitopes, is considered to be a target site of vaccine development. However, although protective immune responses could be induced by recombinant S1 protein, the protection rate in chickens was still low (<50%). Here, we generated fused S1 proteins with HA2 protein (rS1-HA2) or transmembrane domain and cytoplasmic tail (rS1-H3(TM)) from hemagglutinin of H3N2 influenza virus. After immunization, animals vaccinated with fusion proteins rS1-HA2 and rS1-H3(TM) demonstrated stronger robust humoral and cellular immune responses than that of rS1 and inactivated M41 vaccine. The protection rates of groups immunized with rS1-HA2 (87%) were significantly higher than the groups inoculated with rS1 (47%) and inactivated M41 vaccine (53%). And chickens injected with rS1-H3(TM) had similar level of protection (73%) comparing to chickens vaccinated with rS1 (47%) (P=0.07). Our data suggest that S1 protein fused to the HA2 or TM proteins from hemagglutinin of H3N2 influenza virus may provide a new strategy for high efficacy recombinant vaccine development against IBV.

Keywords: Fusion proteins; H3N2; IBV; Immunogenicity; S1 protein.

Fig. 1

Baculovirus construct for production of proteins. (A) Construction of recombinant plasmids. pFBac1 contains polyhedrin promoter (P_PH), SV40 pA, SV40 polyadenylation signal; gentamicin; Tn7R and Tn7L. Different target genes (S1, S1-H3 and S1-HA2) were inserted between P_PH and SV40 pA respectively. (B) Construction of recombinant fusion proteins rS1, rS1-H3(TM) and rS1-HA2. For rS1-H3(TM) protein, rS1 protein was fused to the CT and TM domain of H3N2 HA protein; for rS1-HA2 protein, rS1 protein was fused to the HA2 domain of the HA protein of H3N2.

Fig. 2

Analyses of protein expression of recombinant rS1, rS1-H3(TM), and rS1-HA2 in Sf9 cells. Sf9 cells were infected with recombinant baculoviruses (rBVs). Sf9 cells infected with wild type baculoviruses were used as a negative control (NC). (A) Western blot of recombinant rS1, rS1-H3(TM) and rS1-HA2 proteins. (B) SDS-PAGE of recombinant rS1, rS1-H3(TM) and rS1-HA2 proteins. The expected bands of rS1, rS1-H3(TM), and rS1-HA2 are indicated with arrow.

Fig. 3

Infectious bronchitis virus (IBV)-specific antibody titers in chickens sera. Six groups of chickens (n = 15 chickens per group) were subcutaneously immunized with rS1, rS1-H3(TM), or rS1-HA2, respectively. Inactivated M41 was used as a positive control, rWT (Sf9 cell lysate infected by wild-type baculoviruses) and PBS were used as negative controls. Sera from each chicken were collected and the IBV-specific antibody titers were determined by ELISA. Data is represented by the mean antibody titer +S.D.

Fig. 4

Flow cytometry analysis of T lymphocyte subsets in chickens after immunization with rS1, rS1-H3(TM), rS1-HA2, inactivated M41, rWT (Sf9 cell lysate infected by wild-type baculoviruses) and PBS. This test was performed two weeks after the boosting immunization. Data are presented by means + SD. n = 5 chickens per group.