| Objective:About1/3population around the world is infected with Mycobacterium tuberculosis (M. tuberculosis, MTB). Among them, most are in latent infection. Preventing the latently infected individuals from progressing to active disease could make a major impact on tuberculosis (TB) control worldwide. The purpose of this study was to screen the effective MTB antigens in latent infection, construct TB subunit vaccine and evaluate the therapeutic effect of TB subunit vaccines in rabbit pulmonary latent BCG infection model.Methods:(1) To screen the effective MTB antigens in latent infection:We got258up-regulated MTB antigens from seven vitro models of latent M. tuberculosis infection through literature reviewing, and selected the five genes (Rv2031c, Rv2626c, Rv2007c, Rv1738, Rv1733c) with high-level expression and immunogenicity in MTB latent infection for construction of fusion proteins.(2) Construction of fusion protein FdxA-Hrpl(F6):The DNA sequence coding mature protein of FdxA and Hrpl were generated by the PCR amplification using Mycobacterium tuberculosis H37Ra DNA as template. The plasmid encoding FdxA-Hrpl was generated by inserting the genes Rv2007c(FdxA) and Rv2626c (Hrpl) into the multiple cloning sites of the expression vector pET30a respectively. The recombinant plasmid was transforme into E. coli BL21for expressing fusion protein F6. Fusion protein F6with His-tagged was purified by Nickel ion exchange chromatography method.(3) To set up the rabbit pulmonary latent BCG infection model:Rabbits were infected with BCG by intratracheal administration method. At12weeks after BCG infection, the rabbits were treated with dexamethasone by intramuscular injection at0.5mg/kg daily for five weeks; then rabbits were deeply anesthetized (intravenous injection with urethane,2g/kg) and sacrificed to obtain lung, spleen and thymus; At last, rabbit lungs were used for the determination of bacillary load and the pathological analysis.(4) Evaluation of the therapeutic effect of the fusion protein vaccines:First, we constructed subunit vaccine FdxA-Hrpl(F6) and HspX-Rv1738-Rvl733c(H83) including MTB antigens Rv2031c (HspX),Rv2626c (Hrp1), Rv2007c (FdxA), Rv1738and Rv1733c which highly expressed in dormant bacilli; in addition, another subunit vaccine ESAT6-Ag85B-MPT64190-198-Mtb8.4(EAMM) incuding mainly antigens expressed in proliferation bacteria was constructed previous in our lab and has proven to be effective against Mtb infection. After infected with BCG, rabbits were divided into five groups in a complete randomized design:BCG and normal saline group (BCG group); BCG and adjuvant group (adjuvant group); BCG and F6plus H83group (F6+H83group); BCG and EAMM group (EAMM group); BCG and F6plus H83plus EAMM group (F6+H83+EAMM group). Rabbits were immunized with vaccines by subcutaneous injection on left forearm method at5,7,9weeks after BCG infection, and then evaluate the therapeutic effect on the rabbit pulmonary latent BCG infection model as above described.Results:1. We got258up-regulated genes related to MTB latency by antigen screening, and selected five genes of high-level expression and immunogenicity in MTB latent infection to construct therapeutic fusion proteins. The five genes include Rv2031c (HspX), Rv2626c (Hrpl), Rv2007c (FdxA), Rv1738and Rv1733c.2. The fusion protein F6was expressed soluble in E. coli and could be purified by Nickel ion exchange chromatography method.3. We didn’t detect any pulmonary bacterial on lung tissue at four weeks after BCG infection, but large numbers of pulmonary mycobacteria emerged after the dexamethasone treated.4. The mycbacteria (BCG) colony forming units results:BCG group1.2×105, DP group5.7×104,EAMM group7.9×103, F6+H83group3.2×103, EAMM+F6+H83group1.6×103. The EAMM+F6+H83group showed significant lower CFU counts than EAMM group and F6+H83group, p(EAMM+F6+H83vs EAMM)=0.00003,p(EAMM+F6+H83vs F6+H83)=0.0216.The F6+H83group showed significant lower CFU counts than EAMM group, p(F6+H83vs EAMM)=0.0209. All the three vaccine groups showed significant lower CFU counts than the control groups,p(EAMM vs BCG)=0.0015,p(F6+H83vs BCG=0.0015, p(EAMM+F6+H83vs BCG=0.0001),p(EAMM vs DP)=0.0368, p(F6+H83vs DP)=0.00326, p(EAMM+F6+H83vs DP)=0.0001.Conclusions:1. The rabbit pulmonary latent BCG infection model could be used to evaluate the therapeutic effect of the fusion protein vaccines, and provided a simple, safe, effective research model for TB vaccines, TB drugs and immune modulators.2. The newly constructed fusion protein vaccine F6has some ability to clear the latent BCG.3. The combination of vaccine (F6+H83) based on MTB persister-associated antigens with EAMM based on proliferating bacteria-associated antigens can significantly reduce the rabbit lung BCG colony-forming unit (CFU) number. |
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