| Respiratory syncytial virus (RSV), which causes bronchiolitis and pneumonia, even death when get infected, is the leading cause of lower respiratory infection in global infants. Generally, the effective way to prevent virus infection is vaccination. So far, there is currently no approved RSV vaccine. Therefore, development of safe and effective vaccines is important for preventing respiratory tract infection disease caused by RSV in young infants.Fusion (F) protein is the main glycoprotein and protective antigen of RSV, and is also used to develop subunit vaccine candidates. In this study, the recombinant baculoviruses expressing RSV full-length and/or truncated F proteins were constructed. Using a mouse model, the immunogenicity and immune responses induced by recombinant baculoviruses were investigated.Firstly, F1 fragment (155-524aa) of F protein was amplified by RT-PCR using RSV genome RNA as template, and cloned into prokaryotic expression vector pET-28a. Recombinant protein was expressed in E. coli BL21 by addition of IPTG. After purification of the recombinant F1 protein, rabbit polyclonal antibodies were prepared by immunizing with the purified protein. ELISA analysis showed that the antibody titer was 106. Immunofluorescence staining analysis showed that the antibodies specifically reacted with the F protein expressed by recombinant baculovirus.Secondly, three recombinant baculoviruses-Bac-tF64?Bac-CF? Bac-CF/tF64-were generated using MultiBac system. Bac-tF64 expressed ectodomain of F with SP and CTD derived from gp64 under the control of p10 promotor in insect cells, thus the F protein displayed on the baculoviral envelope. Bac-CF harbored the F gene under the control of CMV-IE promoter, and the F protein was expected to express endogenously after transduction of mammalian cells. Bac-CF/tF64 encompassed both of the above expression cassettes, so it was pseudotyped with F and could express F in the transduced eukaryotic cells.Thirdly, in a mouse model, dual expressing recombinant baculovirus could elicit high level anti-F antibodies and remarkable cellular immune response. The analysis of F specific antibodies and the type of cytokines showed that the Bac-tF64 immunized group had high Th2 cytokine(IL-4, IL-5, IL-10) levels with IgG2a/IgG1 ratio below 1, and Bac-CF immunized group had high Thl cytokine(IFN-y and TNF-a) levels with IgG2a/IgG1 ratio above 1. However, Bac-CF/tF64 had high Thl and Th2 cytokine levels with IgG2a/IgG1 ratio above 1, indicating the immune response was mixed Th1 and Th2 types, but Thl-baised. In the lungs of mice immunized by Bac-tF64 and Bac-CF, virus titer of 102pfu per lung was detected after RSV challenge, but no virus was detected in Bac-CF/tF64 immunized mice. Analysis of cytokines in lung homogenate showed that Bac-tF64 had high Th2 cytokines concentration, and Bac-CF had high Thl cytokines concentration. But Bac-CF/tF64 had high Thl and Th2 cytokines concentration. Histological pathology analysis showed severe pulmonary inflammation in the lungs of Bac-tF64 immunized mice, while Bac-CF and Bac-CF/tF64 immunized mice showed mild inflamation.Finally, in order to improve the immune effect of the vaccine, the recombinant baculovirus co-expressing innate immune signal molecule VISA was constructed. Results of humoral responses revealed that VISA had no impact on the levels of antibodies(p>0.05). However, VISA could significantly lower the levels of Th2 cytokines (p<0.05), and reduce Th2 response. At the same time, histological pathology analysis showed that VISA could eliminate pulmonary inflammation, indicating the potential of VISA as a genetic adjuvant.These results will contribute to the development of safe and effective RSV vaccines. |
PDF Full Text Request