Co-expression Of The Tubercle Antigen 85b, Esat-6 And Mice Of Ifn-gamma Recombinant Bcg And Tubercle Antigen 85b, Esat-6 Chimeric Protein Subunit Vaccine Research

Tuberculosis(TB)remains one of the leading infectious disease worldwide,and it is estimated over one-third of the world's population is infected with M.tuberculosis and almost 3 million people die from this disease annually.Moreover,the percentage in whom progressive disease develops increases markedly in recent decades due to the spread of HIV/AIDS and the emergence of multidrug-resistant TB.BCG is the only available vaccine against TB currently,and has been administered more 3 billion times.This vaccine has a protective effect among children,particularly against TB meningitis,however it does not satisfactorily prevent the development of pulmonary TB in adults.Therefore,it is urgently needed to develop more effective vaccines against TB.Novel vaccine candidates include the following:improved recombinant BCG(rBCG)vaccines,which have greater antigenicity or immunogenicity than the parental BCG vaccine and could replace the current BCG vaccine;and subunit vaccines composed of dominant secreted antigens,which could boost the immune response after priming with BCG.This experiment involves two kinds of tuberculosis vaccines:recombinant BCG and chimaeric protein vaccine.1.Recombinant BCG vaccineBCG has some advantages to be used as live delivery vehicles:be nonpathogenic, be capable of expressing and secreting M.tuberculosis protein in native form,be capable of inducing strong humoral and cell-mediated immunity.We constructed three recombinant BCG strains expressing Ag85B,ESAT-6 and mouse-IFN-γ:rBCG-Ag85B(rBCG-A),rBCG-Ag85B-ESAT6(rBCG-AE),rBCG-Ag85B-ESAT6-IFNγ(rBCG-AEI).Ag85B is one major secretory and protective antigen of M.tuberculosis, ESAT-6 is the deleted gene of BCG by continuing attenuation,and IFN-γis one of the most important cytokines.After C57BL/6 mice immunized with BCG and rBCG vaccines,we studied the level of antibody response,IgG2b/IgG1 and the amount of IFN-γsecreted,and found that rBCG-AEI could induce stronger humoral and cell-mediated immunity than the others and enhance Th1 response.The protective efficacy of rBCG was also determined.C57BL/6 mice and guinea pigs were respectively immunized subcutaneously with 5×10~6 and 5×10~4 cfu of BCG/rBCG vaccines.We challenged immunized and control animals with virulent H37Rv strain of M.tuberculosis 8 weeks after immunization,monitored the subsequent course of infection.Mice were killed at 3 weeks,6 weeks and 9weeks to determine the bacterial enumeration and histopathology.The results show that the numbers of cfu(clone forming units)recovered from the mice vaccinated with rBCG-AEI were reduced more compared to that from others.In addition,the mice vaccinated with rBCG-AEI gained weight and other groups lost weight at the different degree at 9 weeks.It was also noted that there was less lung fibrosis in the rBCG-AEI vaccinated mice compared with BCG or other rBCG vaccinated animals.The results demonstrated that the novel recombinant BCG strain could confer similar or even better protective efficacy than others.It suggests that in order to investigate the protective efficacy difference between recombinant BCG and parental conventional BCG,to prolong the observed time after challenge is needed.2.Chimaeric protein subunit vaccineThe M.tuberculosis Ag85B and ESAT-6 antigens that are generally recognized as immunodominant antigens are the major protective components of the tubercle bacillus,and fusion of the two antigens(Ag85B-ESAT-6:A-E)has proved highly effective protection guinea pig against M.tuberculosis.Mapping of T-cell epitopes of Ag85B and ESAT-6 had been reported.Overlapping peptides spanning the sequence of ESAT-6 were used to map two T-cell epitopes on this molecule.One epitope was located in the N-terminal part of the molecule,and the other epitope covered amino acids 51-60.In C57BL/6 mice,a cross-reactive T-cell response was detected against two carboxy -terminal peptides spanning amino acids 241-260 and 261-280 of Ag85B.According the results,we have inserted ESAT-6 into Ag85B from the nucleotides 501-545 and constructed the chimaeric protein:Ag85B_N-ESAT-6 -Ag85B_C(A_N-E-A_C).We have compared the immune responses induced by A_N-E-A_C with the immune responses induced by A-E.An increase in the ratio of IgG2b/IgG1 or preferentially increased secretion of IFN-γin case of A_N-E-A_C immunized mice, when compared with corresponding values from A-E immunized mice,was considered as enhanced Th1 response.Having these essential elements of a successful vaccine,A_N-E-A_C could be a strong candidate for further study.We also put forward a new strategy for designing subunit vaccine.