Study Of Recombinant Ag85B-ESAT6Tuberculosis Vaccine

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) is aninfectious disease that leads to substantial morbidity and mortality worldwide. In2010, there were an estimated8.8million incident cases of TB and almost1.5million deaths from TB.The only currently licensed anti-TB vaccine, Mycobacterium bovis bacillusCalmette-Gue′rin (BCG), has been used in humans worldwide since1921. However,collective data indicates the efficacy of BCG is somewhat controversial. AlthoughBCG is effective in protecting from severe forms of childhood TB, principallymiliary disease and meningitis, it fails to prevent adult pulmonary TB epidemic witha wide range of efficacy (0to80%). It is considered that the limited protection ofBCG may at least correlate with the lost of some protective antigens after successiveculture. Unlike most antibody-inducing vaccines, the ones against intracellular M. tbneed, at least in part, strong cellular immune responses for protective immunity.Thus, there is an urgent need to develop improved TB vaccines and effectivevaccination strategies, which are capable of promoting long-term cellular immunity.Recently, several candidate antigens, including Ag85A, Ag85B, TB10.4,ESAT-6, CFP-10, HSP65and Mtb39A, were shown to induce protective responses toM. tuberculosis challenge. A combination of some of these antigens would increasethe number of potentially immunogenic epitopes for a given vaccinated population.The well-known Ag85B-ESAT6fusion protein, which has been shown to provideeffective protection against TB in mice, guinea pigs, and non-human primates, maybe an ideal TB vaccine candidate antigen.Since BCG revaccination does not provide enhanced protection, and can beeven deleterious, heterologous prime-boost immunization strategy represents anideal way to extend BCG-initiated immunity and improve the protective efficacy. Todate, several types of TB vaccines such as protein adjuvant formulations, viral-based,and plasmid DNA vaccines have been used as boosters based on this strategy, andsome have achieved encouraging results. Remarkably, recombinant viral vectored,especially adenovirus-and poxvirus-based TB vaccines, which could induce bothcellular and humoral responses with strong immunogenicity and good safety records,would be extremely promising candidates. Furthermore, the potential of recombinant viral carriers for respiratory mucosal vaccination gives them the priority to defensethis mucosal infectious disease. On the other hand, BCG provides thousands ofantigens and stimulates the immune system for prolonged time periods as a liveattenuated vaccine, in contrast to subunit vaccines based on one or limited antigens.Accordingly, attempts have been made to genetically modify BCG to improve itsimmunogenecity. It became more fascinating that a combination vaccine comprises arecombinant BCG vaccine followed by subsequent viral-vectored boosters.This study contains four parts of prophylactic TB vaccines.Firstly, we constructed the proteins of Ag85B, ESAT6and Ag85B-ESAT6fusion protein. We builded up the immunological evaluation system of TB vaccine.Secondly, we generated BCG and rBCG, and evaluated their immunogenecity.We demonstrate that rBCG maintain over expressing Ag85B without selectivepressure and rBCG triggers increased specific cell-mediated immune responses toAg85B. Different strains of mice possess various responses to BCG.Thirdly, we engineered recombinant bivalent poxvirus-based vaccine,MVA85B-E6, and an adenovirus-based vaccine, AD85B-E6, both of which expressthe fusion protein Ag85B-ESAT6. The mice in the AD85B-E6vaccination group hadincreased antigen-specific IFN-γ-producing CD8T cells, thus highlighting the abilityof adenoviral vectors to trigger CD8T cells. In constrast, subcutaneous vaccinationwith MVA85B-E6induced moderate levels of IFN-γ production; however, itgenerated efficient protection in the lungs even beyond that observed with BCGvaccination. In the present study, CTLs were9-2-specific CD8T cells, which weretriggered primarily by vaccination with AD85B-E6. However, the CTL response andIFN-γ production by lymphocytes in the spleens generated by AD85B-E6vaccination did not significantly contribute to protection in the lungs or even in thespleens. Furthermore, aggravating histological damage and more diffuse tubercles inthe lungs were induced by AD85B-E6vaccination compared with the na ve group.Fourthly, in the Prime-boost experiments, we investigated whether theserecombinant viral vaccines could boost conventional BCG-induced immunity, andprovide enhanced protection. Further, we investigated whether they could be givenas booster vaccines on top of recombinant BCG overexpressing Ag85B as a prime.We show that the protective efficacy of mycobacterial vaccine-primed mice is notimproved by either one dose of viral booster; BCG-primed mice receiving two dosesof boosters afford enhanced protection in the lungs; only the rBCG-primed micereceiving AD85B-E6and subsequent MVA85B-E6exhibits enhanced protection against M. tb H37Rv. Bacterial priming plus AD85B-E6boosting followed byMVA85B-E6always affords better protection than that boosted with MVA85B-E6followed by AD85B-E6in the present study. Bacterial priming followed bysequential AD85B-E6and MVA85B-E6boosting, led to compact granulomas withmuch more lymphocytic infiltration than that induced by bacterial vaccine alone, andthe lymphocytes were less diffused to the surrounding parenchyma.The persistent and recurrent infection offers opportunity for immunotherapy,which is a useful complement to chemotherapy with modulating the host immuneresponse in a more anti-pathogen direction to eliminate the bacteria. Recently, MVAvaccinia virus vector and adenoviral vector, which have strong immunogenicity totrigger cell-mediated immune (CMI) response, have been employed inimmunotherapy in viral infection. In the current study, we tested whetherpost-exposure vaccination with the viral vaccines could protect mice against theongoing M. tb infection. In addition, therapeutic effect of Ag85B-ESAT6/DDA/MPLwas also tested, for whose ability to induce protective CMI in prophylactic modelwas reported previously. Disappointingly, they did not exert immunotherapeuticactivity in the absence of chemotherapy. In contrast, single antibiotic treatment withINH for3weeks, generated a significant reduction in bacterial load and improvedpathology. Nevertheless, this short-term chemotherapy did not achieve aculture-negative state, emphasizing that drug combination and duration of antibiotictherapy are important in tuberculosis treatment.In general, we generated MVA85B-E6and AD85B-E6vaccines. We evaluatedthe immune responses and protective efficacy of bacterial vaccines and viralvaccines, when they used alone or in prime-boost strategy. We also investigatedtheir immunotherapeutic effects. Our results suggest that greater efforts must bemade to identify correct biomarkers of vaccine efficacy. Furthermore, appropriateadministration regimens are important for protection against TB.