Vaccination of mice with recombinant baculovirus expressing spike or nucleocapsid protein of SARS-like coronavirus generates humoral and cellular immune responses

Abstract

Continuous efforts have been made to develop a prophylactic vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, two recombinant baculoviruses, vAc-N and vAc-S, were constructed, which contained the mammalian-cell activate promoter element, human elongation factor 1alpha-subunit (EF-1alpha), the human cytomegalovirus (CMV) immediate-early promoter, and the nucleocapsid (N) or spike (S) gene of bat SARS-like CoV (SL-CoV) under the control of the CMV promoter. Mice were subcutaneously and intraperitoneally injected with recombinant baculovirus, and both humoral and cellular immune responses were induced in the vaccinated groups. The secretion level of IFN-gamma was much higher than that of IL-4 in vAc-N or vAc-S immunized groups, suggesting a strong Th1 bias towards cellular immune responses. Additionally, a marked increase of CD4 T cell immune responses and high levels of anti-SARS-CoV humoral responses were also detected in the vAc-N or vAc-S immunized groups. In contrast, there were significantly weaker cellular immune responses, as well as less antibody production than in the control groups. Our data demonstrates that the recombinant baculovirus can serve as an effective vaccine strategy. In addition, because effective SARS vaccines should act to not only prevent the reemergence of SARS-CoV, but also to provide cross-protection against SL-CoV, findings in this study may have implications for developing such cross-protective vaccines.

Fig. 1

Expression of the N or S protein in recombinant baculovirus-transduced BHK cells (A) BHK cells were transduced with recombinant or control viruses for 24 h and total RNAs were extracted for RT-PCR analysis. Lane 1: non-transduced BHK cells; Lane 2: BHK cells transduced with Ac-GFP; Lane 3: BHK cells transduced with vAc-N or vAc-S. Arrows indicate the specific PCR products. (B) BHK cells were transduced with recombinant viruses for 24 h and raw proteins were extracted for western blot analysis. Lane 1: BHK cells transduced with vAc-N or vAc-S; Lane 2: non-transduced BHK cells; Lane 3: BHK cells transduced with Ac-GFP. Arrows indicate the specific proteins. One out of three experiments is shown.

Fig. 2

Examination of antibody responses in immunized mice. Mice were immunized with vAc-N, vAc-S, Ac-GFP or PBS. Sera of immunized mice from different groups were collected before each immunization. The titers of antibodies interacting with SARS-CoV were measured by ELISA. Data shown are the mean ± S.D. of three experiments from different animals.

Fig. 3

Determination of IFN-γ or IL-4 producing splenocytes in immunized mice. Mouse splenocytes were harvested 10 days after the final immunization. ELISPOT assay was used to determine IFN-γ (A) or IL-4 (B) producing cells. Data shown are the mean ± S.D. of three experiments from different animals.

Fig. 4

Analysis of IFN-γ and IL-2 positive cells in CD4+ or CD8+ splenocytes from immunized mice. Mouse splenocytes were harvested 10 days after the final immunization. IFN-γ and IL-2 positive cells in CD4+ or CD8+ cell populations were analyzed by flow cytometry. Data shown are the mean ± S.D. of three experiments from different animals.