Characterization of a novel DNA vaccine against tuberculosis, using two different antigens: the HSP65 gene from Mycobacterium leprae and Ag85A gene from Mycobacterium tuberculosis.
Hori, J.I1; Zarate-Blades, C.R1; Coelho-Castelo, A.A.M1; Huygen, K2 and Silva, C.L1
1Center for Tuberculosis Research, Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, USP; 2 Institute Pasteur Brussels, Belgium
Introduction: Tuberculosis (TB) remains a major health problem affecting millions of people worldwide. The only TB vaccine currently available is BCG, and although it continues to be used in several countries, its efficacy is questionable. Our group has previously demonstrated that DNA vaccine encoding the 65kDa heat shock protein from M. leprae (DNA-HSP65) presented a prophylactic and therapeutic effect in experimental TB. Another promising candidate for TB vaccines is the Ag85A from M. tuberculosis that have been showing strong T-cell responses. Objective: To construct a DNA vaccine encoding these two antigens and evaluate its prophylactic effects in experimental TB. Methods: The gene Ag85A was amplified by PCR and subcloned into expressions plasmids pVAX1 vector and pVAX-HSP65 to permanence in frame with HSP65. The characterizations were done by PCR, nucleotides sequence and restriction analysis. Furthermore macrophages J774 were transfected with the different constructs and the total RNA or cell lisate were obtained to confirm the in vitro fusion protein expression by RT-PCR and Western blot. Balb/c mice were immunized with the constructs by intramuscular route with 4 doses (100ug/douse) at 2 weeks intervals, and the presence of HSP65 and Ag85A antibodies were detected 15 days after the last immunization by ELISA assay. Results/Discussion: The restriction analysis reveled a band with 4,0Kb corresponding to both genes and the RT-PCR analysis detected mRNA expression for Ag85A. Besides that, Western blot analysis confirmed the expression of fusion protein showing a band with approximately 100kDa corresponding to 65kDa from HSP65 plus 32kDa from Ag85A. The animals immunized with pVAX-Ag85A presented an increase of Ag85A specific antibodies with prevalence of IgG2a subtype, indicating Th1 pattern responses. In another way, the pVAX-Ag85/Hsp65 construction didn’t stimulate a significant production of either specific antibody anti-HSP65 or anti-Ag85A. Perhaps this lower antibody production observed could be related to presence of conformational epitopes, only present in native protein but that could be missing or modified in fusion constructs. Conclusions: These preliminary results showed that the multiple DNA vaccine has been transcript and translated correctly. The next step will be start protection trials to experimental TB.
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