Characterization of host responses against a recombinant fowlpox virus-vectored vaccine expressing the hemagglutinin antigen of an avian influenza virus

Abstract

There currently are commercial fowlpox virus (FPV)-vectored vaccines for use in chickens, including TROVAC-AIV H5, which expresses the hemagglutinin (HA) antigen of an avian influenza virus and can confer immunity against avian influenza in chickens. Despite the use of recombinant FPV (rFPV) for vaccine delivery, very little is known about the immune responses generated by these viruses in chickens. The present study was designed to investigate host responses to rFPV in vivo and in vitro. In cultured cells infected with TROVAC-AIV H5, there was an early increase in the expression of type I interferons (IFN), Toll-like receptors 3 and 7 (TLR3 and TLR7, respectively), TRIF, and MyD88, which was followed by a decrease in the expression of these genes at later time points. There also was an increase in the expression of interleukin-1beta (IL-1beta), IL-8, and beta-defensin genes at early time points postinfection. In chickens immunized with TROVAC-AIV H5, there was higher expression of IFN-gamma and IL-10 at day 5 postvaccination in spleen of vaccinated birds than in that of control birds. We further investigated the ability of the vaccine to induce immune responses against the HA antigen and discovered that there was a cell-mediated response elicited in vaccinated chickens against this antigen. The findings of this study demonstrate that FPV-vectored vaccines can elicit a repertoire of responses marked by the early expression of TLRs, type I interferons, and proinflammatory cytokines, as well as cytokines associated with adaptive immune responses. This study provides a platform for designing future generations of rFPV-vectored vaccines.

FIG. 1.

Relative expression ratio of cytokines following infection of CEF cells with TROVAC-AIV H5 compared to that of control cells. The relative expression of IFN-α (A), IFN-β (B), IFN-γ (C), IL-1 β (D), and chIL-8 (E) in CEF cells was measured following mock infection or infection with TROVAC-AIV H5 at different time points postinfection in four independent experiments. Target and reference gene expression in the cells was quantified by real-time reverse transcription-PCR, and the expression ratio was calculated as the means of gene expression in samples of TROVAC-AIV H5-treated mice compared to that of the controls. An asterisk indicates significant difference (P < 0.05). Error bars represent standard errors from the means.

FIG. 2.

Relative expression ratio of antimicrobial peptides in CEF cells after TROVAC-AIV H5 infection compared to that of uninfected cells. Relative expression of AvBD4 and AvBD6 in CEF cells was measured after infection with TROVAC-AIV H5 in four independent experiments. The expression of AvBD4 (A) and AvBD6 (B) in TROVAC-AIV H5-treated CEF cells is illustrated. The levels of target and reference gene expression in the cells were quantified by real-time RT-PCR and are presented as expression ratios of target genes to reference genes. An asterisk indicates significant difference (P < 0.05). Error bars represent standard errors from the means.

FIG. 3.

Relative expression ratio of TLRs and TLR adaptors in infected and mock-treated CEF cells. The expression of TLR3 (A), TLR7 (B), TRIF (C), and MyD88 (D) in the mock-treated and TROVAC-AIV H5-infected cells in four biological replicates are illustrated. Target and reference gene expression in the cells were quantified by real-time RT-PCR and are presented as the expression ratio of target genes to reference genes. An asterisk indicates significant difference (P < 0.05). Error bars represent standard errors from the means.

FIG. 4.

Relative expression ratio of cytokines in spleen of vaccinated chickens compared to that of control birds. The expression of IFN-γ (A) and IL-10 (B) was compared between chickens vaccinated with TROVAC (n = 6) and control chickens that were treated with vaccine diluent only (n = 6). Target and reference gene expression in spleen cells was quantified by real-time RT-PCR, and expression ratios were calculated as the means of gene expression in samples of vaccinated group compared to that of controls. An asterisk indicates significant difference (P < 0.05). Error bars represent standard errors from the means.

FIG. 5.

In vitro proliferative responses of splenocytes to recombinant HA antigen. Chickens were vaccinated with TROVAC-AIV H5 or received vaccine diluent as a control. Splenocytes from four vaccinated chickens were treated with HA or medium and are shown as Vac. HA and Vac. medium, respectively. Also, splenocytes from four unvaccinated chickens (as controls) were treated with HA and medium and are shown as Cont. HA and Cont. medium. The differences in proliferative responses among the groups at each time point were tested and considered significant at P ≤ 0.05 (*).

FIG. 6.

Relative expression ratio of cytokines in spleen cells of vaccinated chickens following stimulation with HA antigen compared to that of control cells. The expression levels of IFN-γ (A) and IL-10 (B) were compared between the HA-stimulated group and medium-treated controls in four biological replicates. Target and reference gene expression levels in spleen cells were quantified by real-time RT-PCR, and the expression ratio was calculated as the means of gene expression in samples of HA-treated group compared to that of medium-treated controls. An asterisk indicates significant difference (P < 0.05). Error bars represent standard errors from the means.