Screening Of Novel Subunit Vaccine Candidates Against Tuberculosis And Immune Profile Analysis

Tuberculosis(TB)caused by Mycobacterium tuberculosis(M.tb)remains a severe threat worldwide while China is one of the five high-burden countries.TB control is facing major challenges today due to the fact that:1)Bacillus Calmette-Guérin(BCG),the only TB vaccine currently available,cannot prevent the adult from TB,2)lacking of high efficient diagnosis methods 3)the severe emergence of drug-resistant M.tb,and 4)the high economic burden for TB patients and high-epidemic countries.High immunogenicity antigens such as ESAT-6 and CFP-10 are demonstrated to facilitate TB diagnosis as well as subunit vaccine development.In the first part of this thesis,we extended our survey to provide alternative M.tb antigens for the potential diagnostic value.More than 40 new M.tb antigens associated with virulence,latency or metabolisms were purified.Antigen-specific IFN-γseceretion was detected comparatively by enzyme-linked immunospot(ELISPOT)assay in active TB patients and healthy controls.Among them,27 protiens exhibited high immunogenicity in which Rv3119,Rv0733,Rv0432,Rv3006,Rv3914,Rv1908c,Rv0899 and Rv1411c were for the first time implied for their high potential for active TB diagnosis.To further investigate the correlation between clinical manifestation and the immunogenecity of newly identified M.tb antigens,our results demonstrated that Rv0899 induced significantly higher IFN-γresponses in drug resistant pulmonary TB or tuberculous pleurisy patients than conventional pulmonary TB patients(P<0.05)while Rv0899,Rv0432,Rv1411c,Rv0733and Rv3006 promoted significantly higher IFN-γrelease in endobronchial TB(EBTB)patients than pulmonary tuberculosis patients(P<0.05).Anti-TB chemotherapy also affects antigen-specific immune responses in TB patients.We found that specific immune responses to Rv0733 or Rv3006 decreased dramatically after 1-month treatment in follow-up study of TB patients.Owing to the prevalence of latent M.tb infection,latency-associated antigens with high immunogenicity might be good candidates for subunit vaccine development against either latent or active TB.In our study,we have demonstrated that Rv0733 and Rv3006also induced high IFN-γresponses during latent tuberculosis infection(LTBI).Previous studies have shown that Rv0733 has high affinity to MHC I and MHC II molecules while Rv3006 induces high antibody responses in TB patients.We further evaluated the possibility as subunit vaccine candidates through investigating the immune protection of Rv0733 and Rv3006 against M.tb infection in mouse model.Mouse immunized by Rv0733and Rv3006 induced high IFN-γand specific antibody responses.More significantly,Rv0733 and Rv3006 were efficacious in reducing the growth of the baceteria in C57BL/6mice challenged by H37Rv stain with comparable efficacy of BCG immunization.Our study thus suggested that Rv0733 and Rv3006 could be considered as subunit vaccine candidates against TB.IFN-γrelease assay(IGRA)shows great predominance for M.tb infection diagnosis given the available T-SPOT.TB and QuantiFERON-TB Gold In-Tube(QFT-IT)commercial kits.However,with the uncertainty to discriminate between LTBI and ATB and ambiguous results due to low expression level of antigen-specific IFN-γ,it is suggested to search for novel antigen-specific cytokines or chemokines either alone or combined with IFN-γfor the improvement of TB diagnosis accuracy.To verify this possibility,the ESAT-6/CFP-10 peptide pool(E6C10P)used by both T-SPOT.TB and QFT-IT assays were adapted to stimulate PBMCs from either TB patients or healthy controls.A panel of 22 cytokines/chemokines was detected by Luminex multiplex assay.The results indicated that PBMCs isolated from TB patients released significantly higher IFN-γ,MIG,IL-2,IFN-α2 and IP-10 than from healthy controls(P<0.01).Interestingly,IFN-γ,MIG,IL-2,IFN-α2 and IP-10 levels in LTBI individuals were also significantly higher than in healthy controls.ROC analysis indicated that IFN-γ,MIG,IL-2 and IFN-α2 had high diagnostic potentials for both ATB and LTBI in which E6C10P specific IFN-α2 was a novel indicator for M.tb infection.When T cells were depleted from PBMCs by utilizing magnetic anti-CD3 antibody microbeads,E6C10P could no longer induce IL-2 and IP-10 secretion suggesting that E6C10P specific IL-2 and IP-10production was T cell-dependent whereas IFN-γ,MIG and IFN-α2 were partially T cell-dependent.To further investigate the origin of IP-10,results from intracellular staining indicated that E6C10P specific IP-10 was mainly secreted by CD14~+monocytes.With the cut-off value of IP-10 at 945.2 pg/ml corresponding to the maximum likelihood,E6C10P specific IP-10 secretion level had high sensitivity(95.45%)and specificity(99.12%)for ATB.E6C10P specific IP-10 decreased dramatically during anti-tuberculosis treatment.These results indicated that E6C10P specific IP-10 might be a good biomarker candidate for clinical prognosis.Interestingly,about 71.8%LTBI samples secreted E6C10P specific IP-10 lower than945.2 pg/ml.As IGRA such as T-SPOT.TB,ELISPOT or QFT-IT can detected both LTBI and ATB,combination of E6C10P specific IFN-γassay and E6C10P specific IP-10 assay should rule out 71.8%LTBI population from ATB patients,which could enhance the specificity for ATB diagnosis.In conclusion,by using IGRA,we have obtained 8 novel M.tb antigens with high immunogenicity in which lantency-associated Rv0733 and Rv3006 exhibited protection against M.tb infection in C57BL/6 mice.Moreover,ESAT-6/CFP-10-specific MIG,IL-2,IFN-α2 and IP-10 secretion were demonstrated to be supplementary for immunological diagnosis of M.tb infection.Monocyte-derived IP-10 might become new biomarker candidate for TB diagnosis and prognosis as well as LTBI.These results provided new clues for subunit vaccine development against TB and the improvement of TB diagnosis.