Fusion Gene Ag85b Mpt64 <sub> 190-198 </ Sub>-mtb8.4 In The Antituberculosis New Recombinant Bcg And Subunit Vaccines,

Tuberculosis remains a serious world health problem.Infection with the causative agent,Mycobacterium tuberculosis,is a major clinical burden with 8 million new cases and approximately 2 million deaths per year.Ninety percent of the estimated deaths from TB and 95%of the estimated eight million new cases of TB each year occur in developing countries,which comprise 85%of the world's population. Incidence is increasing fastest in African countries that are affected by HIV,followed by Eastern Europe and the former Soviet Union,both of which are plagued by multi-drug resistant strains of TB that present an ominous global threat.Mycobacterium boris bacille Calmette G'eurin(BCG) vaccine,an attenuated strain of M.bovis,is the only vaccine currently available against Mycobacterium tuberculosis and the most widely used vaccine,with over three billion administered doses.BCG protects children efficiently against miliary and meningeal TB,but the protective efficiency against adult pulmonary TB ranges from 0%to 80%based on large,well-controlled field trials.Therefore,it is urgently needed to develop more effective and satisfactory BCG vaccines against TB.This has prompted the search for new,improved TB vaccines.A heap of promising new approaches has been developed during the last two decades.Dozens of vaccine candidates have been tested in recent years in animal models,including subunit protein/peptide vaccines in adjuvants,DNA vaccines,rationally attenuated strains of M.tb,recombinant mycobacterias and live vectors expressing genes coding for immuno-dominant mycobacterial antigens or mycobacterial lipids.And our research is focus on two kinds of tuberculosis vaccines:recombinant BCG and subunit protein vaccine.1.Recombinant BCG:As BCG vaccination is widely accepted within current immunization programmes, is associated with minimal side effects and the vaccine is amenable to genetic manipulation the opportunity exists to modify BCG to render it a more effective vaccine.There are a number of key observations,which provide the basis for a rationale approach to improving BCG.First,BCG lacks a number of genes that are present in parental M.bovis and M. tuberculosis which can insertion in it.Second,genes encoding immunodominant antigens or immunostimulatory cytokines can be over-expression in BCG.Third,the multigenic vaccine approach is attractive because broad immune responses can be generated by simultaneously targeting several antigens.We constructed three recombinant BCG strains expressing Ag85B,mpt64,mtb8.4 ESAT-6 and HSPX:rBCG-Ag85B-Mpt64_(190-198)-Mtb8.4 and rBCG-Ag85B-ESAT6-Rv2031c(HSPX) are both in vector pMV261,while rBCG-Ag85B-ESAT6 is in vector pMV361.First,we compared immune responses between three recombinant BCG vaccines and BCG.After C57BL/6 mice immunized with BCG and rBCG vaccines,we studied the level of antibody response,IgG2a/IgG1 and the amount of IFN-γsecreted,and found that P261-rBCG-Ag85B-Mpt64_(190-198)-Mtb8.4 could induce stronger cellular immunity than the others and enhance Th1 response.Then,we chose P261-rBCG-Ag85B-Mpt64_(190-198)-Mtb8.4 as our candidate vaccine and did some more researches on it.In cellular immune response, P261-rBCG-Ag85B-Mpt64_(190-198)-Mtb8.4 could not only elicited higher IFN-γthan BCG,but also generated more antigen-specific CD4 T cells and CD8 T cells than those vaccinated with BCG.Moreover,P261-rBCG-Ag85B-Mpt64_(190-198)-Mtb8.4 induced TNF-αsecretion in THP-1 cells in vitro and the level was similar with BCG did.The protective efficacy of rBCG was also determined.The results demonstrated that the recombinant BCG strain could confer similar or even better protective efficacy than others.The protective effect of rBCG could relate to a difference in the persistence of the recombinant strain within the host.To investigate this,we determined if expression of the recombinant genes ag85B-mpt64_(190-198)-mtb8.4 influenced the growth of BCG in vivo and in vitro.Mice were infected intravenous with rBCG and BCG and the growth in the lungs,liver and spleen was monitored.In all cases,the bacterial load did not differ significantly between rBCG and BCG, irrespective of the time after infection.Then we compared the growth characteristics of rBCG and BCG:there was no any significant difference in proliferation characteristics between rBCG and BCG.These results suggest that the improved protective efficacy was not due to increased persistence of rBCG in vivo,but was related directly to expression in BCG of the Ag85B-Mpt64_(190-198)-Mtb8.4 fusion protein2.Ag85B-Mpt64_(190-198)-Mtb8.4 in the subunit vaccine of TBSince Ag85B,MPT64_190-198,and Mtb8.4 were confirmed to be the effective protective antigens(or antigen epitopes),they were fused together to construct a new subunit vaccine(AMM).To compare antigen-specific immune response primed by single and fusion protein immunization,humoral immune responses were examined in mice immunized with Ag85B and AMM formulated in Freund's Incomplete Adjuvant (FIA).The results indicate that the humoral immune response against Ag85B,the cellular immune responses elicited by Ag85B and Mpt64_190-198 induced by the fusion protein AMM was stronger than that induced by single Ag85B.The function of adjuvants in subunit vaccine was also determined.AMM fusion with different adjuvants(FIA,chitosan and MPL) vaccinated in mice.And the results showed that chitosan and FIA could help AMM to elicit strong cellular and humoral immune responses.Having these essential elements of a successful vaccine,AMM could be a strong candidate subunit vaccine for further study.