| Background and aimsBCG is the only available vaccine for the prevention of TB and the coverage rate is about 90% in the most developing countries worldwild. However, the efficacy of BCG vaccine in preventing adult TB is highly variable and and mechanisms for this have not been confirmed yet. There are 16 RDs identified in the genome of M.tb H37Rv so far. The primary deletion of RD1 between 1908 and 1921, which is absent from all BCG substrains but present in virulent M. tuberculosis and M. bovis, is thought to be the most obvious reason for the attenuation of BCG. Moreover, by the deletion of RD2 region during 1927 and 1931, BCG substrains are divided into early substrains ( BCG Japan, Russia, Moreau, Sweden, and Birkhaug) and late substrains (BCG Tice, Frappier, Phipps, Prague, Danish, Pasteur, Glaxo). Recent studies suggest that RD1 could enhance the protective efficacy of BCG in the forms of recombinant BCG or subunit vaccine. The effect of lack of RD2 on the protective efficacy of BCG substrains against TB remains unknown. CFP10(Rv3874) and ESAT-6(Rv3875), CFP21(Rv1984c) and MPT64(Rv1980c), have been considered important immunodominant antigens encoded by RD1 and RD2 of M. tuberculosis, respectively. In order to elucidate the effect of loss of RD2 on the efficacy of BCG, the immune responses generated against DNA vaccines expressing CFP21-MPT64 and ESAT-6-CFP10 fusion proteins and the immunogenicity of both fusion proteins in tuberculin skin test (TST) positive healthy population were compared in the present study. More importantly, we compared the use of a BCG prime-DNA vaccines boost strategy to induce protection against virulent M. tb challenge in C57BL/6 mice.Methods1. The coding sequences of both EAST-6 and CFP10 proteins were amplified by PCR .The esat-6-cfp10 was generated by a second PCR according to the method of gene splicing with overlap extension. The fusion gene was inserted into procaryotic expression vector pProEXHTb and eucaryotic expression vector pcDNA3.1(-), resulting in the recombinant plasmids pPro610 and pcD610, respectively. Meanwhile, pPro2164 was digested with BamHI and HindIII and then inserted into pcDNA3.1(-), resulting in the recombinant plasmid pcD2164.2. E. coli BL21 (DE3) strains harboring the plasmid pPro610 or pPro2164 were cultured and induced with IPTG to express rEC and rCM fusion protein, respectively. Protein purification was performed using a Ni-NTA purification system. These processes were examined by SDS-PAGE and verified by Western blotting. The purified products of both rEC and rCM proteins were further quantitated by BCA method.3. Plasmids pcD610 and pcD2164 for immunization were transformed into E. coli DH5αand endotoxin-free plasmid DNA was purified using the Qiagen Plasmid Giga kit.4. Heparinized whole blood from 18 household contacts with recent active TB patients was collected. Whole blood IFN-γassay(WBIA) stimulated with the rEC or rCM fusion proteins was performed in order to compare the immunogenicity of rCM with rEC.5. Mice were vaccined with a BCG(China substrain) prime- DNA vaccines heterologous boost strategy. Two weeks after the last DNA vaccination, antigen-specific IgG antibody and IFN-γproduction by splenocytes were detected by ELISA. Expression of iNOS and cytokines in lungs were determined by qRT-PCR.6. Two weeks after the last DNA vaccination, mice were challenged with M.tb H37Rv strain, and the protective immunity was determined by bacterial loads in spleen and lung and lung histopathological examination of infected mice.Results1. The desired constructs were confirmed by DNA sequencing and enzyme digestion.2. The expression of rEC was examined by SDS-PAGE and verified by Western blotting. The purified proteins of rEC and rCM were congfirmed by SDS-PAGE.3. Analysis of the distribution of IFN-γlevels in TST-positive and TST-negative samples showed a significant correlation between TST positivity and IFN-γlevel, regardless of the proteins used (both p<0.05). The mean IFN-γlevels in samples from both TST-positive group and TST-negative group detected by rCM-WBIA were higher than those of rEC-WBIA, but the differences were not found to be statistically significant (both p >0.05).4. As expected, the most significant IgG-specific response to rEC protein was induced in both pcD610 and BCG plus pcD610 group, when rEC protein was used for detection. Both pcD2164 and BCG plus pcD2164-treated mice produced the strongest IgG response to rCM protein. No antigen-specific antibodies were detected in other groups, regardless of the proteins used.5. Splenocytes from mice vaccinated with DNA vaccines and BCG prime plus DNA vaccines booster produced higher level of IFN-γthan BCG (p<0.05), when stimulated with rEC or rCM protein. Moreover, BCG plus pcD2164 vaccination induced the strongest IFN-γresponse to rCM protein.6. qRT-PCR resluts showed that the expression levels of iNOS, IFN-γand TNF-αin BCG prime plus DNA vaccines booster were not less than them in BCG group. Relatively, compared with BCG group, BCG plus DNA vaccines groups had some decrease of the expression of IL-10 or TGF-β.7. After challenged with virulent M. tb H37Rv, decrease in the bacterial loads of lung and spleen with different degree was seen in vaccinated mice. Moreover, BCG plus DNA vaccines groups had lower value than BCG alone or DNA vaccination alone. Moreover, BCG plus pcD2164 group had the lowest value of all.8. As above, slight interstitial pneumonia appeared in BCG, BCG plus vector and BCG plus pcD610 groups. Alveolar tissue from mice vaccination with BCG plus pcD2164 appeared to be intact with very limited lung inflammation and little granuloma formation around alveolar tissues.ConclusionsOur results clearly indicate that pcD610 and pcD2164 DNA vaccine are two promising TB candidates with a BCG prime and a heterologous vaccine boost strategy, and the loss of RD2 has an important influence on the protective efficacy of different BCG substrains. These findings will benefit the optimal choice of BCG substrain for neonatal immunization and rational design of new vaccines for the prevention of TB. |
PDF Full Text Request