Study On T Cell Exhaustion Induced By Persistent Mycobacterium Tuberculosis Antigen And Development Of Tuberculosis Subunit Vaccine Candidate LT70-DPC

Part One: Study on T cell Exhaustion Induced by Persistent Mycobacterium tuberculosis AntigenTuberculosis is a chronic infectious disease, caused by Mycobacterium tuberculosis(M. tuberculosis) infection. Studies have reported that CD4+ T cells, especially the Th1 cells were declined in serious TB patients. We also found that the T cell responses at some fibrous cavitary TB patients with smear-positive were lower than the close contacts and the new cases of TB patients. The phenomena above showed that severe M. tuberculosis infection may cause the dysfunction of T cell, and its mechanism is not known until now.Objective To establish a model of T-cell exhaustion induced by persistent M. tuberculosis antigen, to restore the T-cell exhaustion by hematopoietic stem cells(HSC) or IL-2 and analyze its mechanism.Methods 1, The C57BL/6 mice were primed with BCG(5×106 CFU) and boosted with the combination of M. tuberculosis subunit vaccines ESAT6-Ag85B-MPT64190-198-Mtb8.4-Rv2626c(LT70 for short) 10μg and Mtb10.4-Hsp X(MH for short) 10μg for 10 times every week. 2, Three weeks post the last boosting, the IFN-γ and IL-2 secretions from the spleen T cells were analyzed by ELISPOT and ELISA; the number of CD4+/CD8+ T cells and the expression of programmed death 1(PD-1) on the CD4+/CD8+ T cells were evaluated by Flow Cytometry(FCM); the number of TB10.4 specific CD8+ memory T cells were evaluated by TB10.44-12 pentamer; the protective efficacy against BCG or Pseudomonas aeruginosa(P. aeruginosa) challenge were evaluated; and the number and proliferation of HSC cells were also evaluated by FCM. 3, The T cell exhausted mice were treated with HSC, mesenchymal stem cells(MSC), IL-2, and rapamycin respectively. After treatment, the therapeutic effects were evaluated by detecting the level of IFN-γ and IL-2 secretions; the number of antigen specific CD8+ memory T cells; the expression of PD-1 on CD4+ T cells; the protective efficacy against BCG challenge; the number and proliferation of HSC. 4, IL-2 can restore the T cell exhaustion. We further explored the activation of JAK-STAT or PI3 K signaling pathway following IL-2 treatment, which related to HSC proliferation, especially the expression of JAK-3 and P85 and phosphorylation levels of STAT-5 by immunoblot.Results 1, We establish a model of T-cell exhaustion by primed with BCG and boosted with the combination of M. tuberculosis subunit vaccines LT70 and MH for about 10 times every week. 2, Compared with the mice received transient antigen stimulation, the mice treated with persistent antigen had lower level of IFN-γ and IL-2 secretions(p<0.05); the number of CD4+/CD8+ T cells and TB10.44-12 specific CD8+ memory T cells were significantly lower(p<0.05); the level of inhibitory receptor PD-1 on CD4+ T cells were higher(p<0.05); the protective efficacy against challenging with BCG and P. aeruginosa decreased; the number and proliferation of HSC also decreased(p<0.05). 3, With the treatment of HSC and IL-2, the production of cytokines IFN-γ and IL-2 increased(p<0.05); the number of specific CD8+ TB10.4 memory T cells increased(p<0.05); PD-1 expression level on CD4+ T cells decreased(p<0.01); the bacterial load in HSC group(6.18 ± 0.28 Log10 CFU) and IL-2 group(5.94 ± 0.17 Log10 CFU) were significantly lower than the Tcell exhaustion group without treatment(6.74 ± 0.37 Log10 CFU); and the proliferation of HSC was recovered. 4, By the study of JAK-STAT signaling pathway and PI3 K pathway which were related to HSC proliferation, kinase JAK3 mainly expressed in hematopoietic cells, the phosphorylation of transcription factor STAT-5 was inhibited in T cell exhaustion model and IL-2 could reverse the phosphorylation of STAT-5.Conclusion We established a model of T-cell exhaustion by persistent M. tuberculosis antigen, it partly reflecting the mechanism of T cell dysfunction in M. tuberculosis infection with large bacteriums stimulation. The ability of HSC differentiate into T cells was inhibited, which can be restored by IL-2 and HSC. We found that IL-2 restored the dysfunctional T cells by restoring the proliferation of HSC mainly through activating JAK-STAT pathway.Part Two: Construction and evaluation of a multistage Mycobacterium tuberculosis subunit vaccine candidate LT70M. tuberculosis is the main cause of Tuberculosis; it’s the most serious infectious diseases in the world. With the human immunodeficiency virus(HIV) infection and the drug-resistant strains, TB remains one of the major diseases. The current vaccine, Bacilli Calmette-Guerin(BCG), is a live attenuated Mycobacterium bovis vaccine, now is widely used and the only vaccine in the world. It could protect child from severe TB, can not prevent reactivation of latent TB and is unreliable against the pulmonary TB in adults. Thus, the new TB vaccine research and development must be urgent.Objective To develop an effective subunit vaccine which target tubercle bacilli with different metabolic states and provide effective protective immunity.Methods 1, We fused antigens ESAT6, Ag85 B, peptide 190-198 of MPT64, and Mtb8.4 mainly expressed by proliferating bacteria and latency-associated antigen Rv2626 c together to construct a multistage protein LT70. 2, The protein LT70 expressed in Escherichia coli(E. coli) BL21 strain. We purified it by hydrophobic interaction chromatography and gel filtration method. 3, The immune responses of LT70 in the adjuvant of dimo-thylidioctyl ammonium bromide(DDA) and polyinosinic-polycytidylic acid(Poly I:C) and its protective efficacy against M. tuberculosis infection in C57BL/6 mice were evaluated. Then, the immunization of BCG prime and LT70 boost strategy were also evaluated.Results 1, LT70 was stably produced in Escherichia coli BL21 and could be purified by hydrophobic interaction chromatography on Butyl-FF and molecular sieve chromatography successfully. 2, The mice immunized with LT70 produced higher levels of antigen-specific IFN-γ and antibodies Ig G1 or Ig G2 c compared with BCG and PBS groups. 3, The subunit vaccine LT70 induced higher protective efficacy(5.4±0.38 Log10 CFU in lung) than traditional vaccine BCG(6.01±0.33 Log10 CFU) and PBS control(6.53±0.26 Log10 CFU) at 30 weeks post vaccination(ten weeks post-challenge) against M. tuberculosis infection(p<0.05). After BCG prime, the subunit vaccine LT70(5.62±0.64 Log10 CFU) can strengthen the protective efficacy of BCG(6.01±0.33 Log10 CFU)(p=0.26).Conclusion The subunit vaccine LT70 could induce strong antigen-specific cellular and humoral immune responses; the protective effect of subunit vaccine LT70 is stronger than BCG, which would be a promising subunit vaccine candidate against M. tuberculosis infection.Part Three: Development of the adjuvant DPC in tuberculosis vaccinesNovel tuberculosis subunit vaccines require adjuvant to induce a stronger immune response. Choose a suitable adjuvant not only increases the level of immune response may also change the type of immune response. Vaccines targeting intracellular pathogens such as M. tuberculosis, often required an induction of cellular immune responses, including T helper 1(Th1 cell) type cells and cytotoxic T lymphocytes(CTL). However, adjuvant currently in clinical use only aluminum adjuvant, which promotes the humoral Th2-type immune response, can not induce the Th1 cellmediated immune response. Therefore, new adjuvant which induced cell-mediated immune response is essential to the development of vaccines. In our previous study, the adjuvant DDA combined with toll-like receptor 3 agonist Poly I: C could induce the Th1-type cellular immune response and immune protection. However, adjuvant DDA-Poly I: C(DP for short) was less stable, prone to flocculation, unable to meet the needs of clinical vaccine.Objective Based on the adjuvant DP, research for a new adjuvant to induce the Th1 cellmediated immune response and enhance the stability of tuberculosis subunit vaccine, which could also use in clinic.Methods 1, Based on adjuvant DP, combine with different accessories and evaluate the physical and chemical properties and stability of the adjuvant. So that screening the reagents to enhance the stability of adjuvant DP. 2, We used the emulsion- solvent evaporation method to make the micron-sized cationic liposome adjuvant, the adjuvant DPC in the fusion protein LT70 to construct the subunit vaccine LT70-DPC. 3, The subunit vaccine-induced immune responses and protective efficacy against M. tuberculosis H37 Rv infection in C57BL/6 mice were investigated.Results 1, Construction of the new adjuvant DPC including DDA, Poly I: C and cholesterol. 2, The physical and chemical properties analysis results show that, the particle size reduced from 500 nm to 400 nm, zeta potential increased from 23.48(±2.93) to 41.22(±4.04) with adding of cholesterol. 3, LT70 in the adjuvant of DPC generated higher levels of antigen-specific IFN-γ and antibodies Ig G1 or Ig G2 c compared with BCG and PBS groups, and induced long-term higher protective efficacy against M. tuberculosis infection(5.43±0.37 Log10 CFU) than traditional vaccine BCG(6.01±0.33 Log10 CFU) and PBS control(6.53±0.26 Log10 CFU) at 30 weeks post vaccination. Meanwhile, the pathological lesion area in LT70-DPC vaccine group(7.4±0.04%) was significantly lower than the vaccine LT70-DP group(12.2±0.01%) after M. tuberculosis H37 Rv infection.Conclusion The novel adjuvant DPC increased the stability of adjuvant DP, in favor of future development and production. DPC would be a promising vaccine adjuvant with the ability to stimulate Th1 type cell-mediated immunity, and could be used in TB subunit vaccine.