Balance Regulation Of Immunological Response Of Respiratory Syncytial Virus Recombinant Protein Immunogen

An ideal respiratory syncytial virus(RSV) vaccine should be capable of inducing humoral immune (antibody) , as well as cellular immune (CTLs). Cellular immune mediated by CTL plays an important role in elimination of pathogen, which is particularly important for prevention of RSV. Following RSV infection, humoral immunity is major protective mechanism, but this protection is not fully enough. After infection patients will be repeated infected during relatively short period of time or lifetime. Meanwhile, a safe RSV vaccine should be capable of inducing balanced immune response. However, previous studies in mice indicated that recombinant G protein or G protein fragment immunization resulted in IgG1 antibody and Th2-type response and failed to induce IgG2a, Th1 and MHC I-restricted CD8+T cell response.We engineered a fusion protein G1F/M2, consisting of an antibody epitope G: 125-225(G1) fragment from RSV-A G protein and a CD8+ T cell epitope (M2:81-95) from RSV-M2 protein. High titer of specific antibody and CTL response were induced in BALB/c mice intraperitoneal vaccinated with G1F/M2 formulated in Al(OH)3. And the induction of CD8+ T cells reduced the ratio of IgG1/IgG2a. But the titer of neutralization antibody induced by recombinant protein G1F/M2 was lower than DsbA-G1F1/M2, containing carrier protein DsbA. And despite the lower ratio of IgG1/IgG2a induced by G1F/M2, it was higher than that IgG1/IgG2a induced by RSV virus. The main aim of this study is to further enhance the G1F/M2's ability of inducing humoral immunity and cellular immune, especially enhance MHC I-restricted CD8+T cell response and CTL activity. And then more balanced immune response will be achieved. So on the basis of recombinant protein G1F/M2, new recombinant proteins containing protein transduction domain HIV TAT or heat shock protein HSP70 were constructed to facilitate antigen cross-presentation. And studies of immunogenicity, protective efficacy and safety of recombinant proteins were investigated.Two new recombinant fusion proteins expression vectors were constructed. The inductions of recombinant proteins expression were achieved in prokaryotic expression system. And recombinant antigens were separated and purified by affinity chromatography. By primers design and PCR amplification, the gene of protein transduction domain HIV TAT was connected to the upstream of gene G1F/M2. A new recombinant protein expression vector, termed pET-TAT-G1F/M2, was constructed. Because recombinant fusion protein containing HSP70 and G1F/M2 can not be expressed in prokaryotic expression system, a new recombinant protein which can high-affinity bind to HSP70. By primers design and PCR amplification, peptide Javelin was introduced to the N terminal of G1F/M2. Through the high-affinity interaction between Javelin and HSP70, new recombinant fusion protein Javelin-G1F/M2 can form complex with HSP70. These expression vectors were transformed into prokaryotic expression system to achieve successful fusion protein expression. RSV-specific antigenicity of recombinant fusion protein were characterized by Western-blot. Recombinant fusion proteins were separated and purified by affinity chromatography on Ni2+ chelating Sepharose and refolded by gradient dialysis.HSP70: Javelin-G1F/M2 complex was prepared by incubation of Javelin-G1F/M2 and HSP70 in vitro. The titer of IgG antibody, IgG antibody subtype and neutralization antibody, CTL activities and protective efficacy induced by complex, Javelin-G1F/M2 and Javelin-G1F/M2 formulated in Al(OH)3 were investigated. High titer of IgG antibody and strong CTL responses were induced in BALB/c mice subcutaneous vaccinated with HSP70: Javelin-G1F/M2 complex without additional adjuvant. The titer of IgG antibody and the activity of CTL induced by HSP70: Javelin-G1F/M2 complex were markedly higher than those in mice subcutaneous vaccinated with Javelin-G1F/M2 or intraperitoneal vaccinated with Javelin-G1F/M2 formulated in Al(OH)3. The titer of neutralization antibody induced by HSP70: Javelin-G1F/M2 was equivalent with that induced by Javelin-G1F/M2 formulated in Al(OH)3. Compared with Javelin-G1F/M2 formulated in Al(OH)3, HSP70: Javelin-G1F/M2 complex can induce more balanced Th1/Th2 response. BALB/c mice were challenged with 106 pfu RSV-A at 14 days post immunization. before and after challenge the ratios of IgG1/IgG2a in mice subcutaneous vaccinated with HSP70: Javelin-G1F/M2 complex were lower than that of IgG1/IgG2a in mice subcutaneous vaccinated with Javelin-G1F/M2 or intraperitoneal vaccinated with Javelin-G1F/M2 formulated in Al(OH)3. After challenge, eosinophil infiltration in lungs of mice subcutaneous vaccinated with HSP70: Javelin-G1F/M2 complex was lighter. Virus titration results indicate that HSP70: Javelin-G1F/M2 complex protected the lungs against RSV-A challenge.The titer of IgG antibody, IgG antibody subtype and neutralization antibody, CTL activities and protective efficacy induced by TAT-G1F/M2 and G1F/M2 formulated in Al(OH)3 were investigated. The titer of IgG antibody and neutralization antibody induced by TAT-G1F/M2 was equivalent with that induced by G1F/M2. But the CTL activity of TAT-G1F/M2 group was higher than that of G1F/M2 group. Furthermore, TAT-G1F/M2 could induce lower ratio of IgG1/IgG2a in mice before and post challenge. Similarly, TAT-G1F/M2 could protect mice from RSV infection as well as G1F/M2.Therefore the cross presentation of antigen G1F/M2 can be enhanced by introduction of protein transduction domains HIV TAT or chaperoned by HSP70 in forms of HSP70:G1F/M2 complex. Ehanced cross presentation results in enhanced MHC I-restricted CD8+T cell response. So more balanced Th1/Th2 responses were induced and side effects caused by Th2 response were improved.