PLASMID VECTOR BASED DNA VACCINES AGAINST TUBERCULOSIS

Abstract

Tuberculosis (TB) is a pre-historic disease and it is a significant cause of morbidity and mortality in human and cattle, in many parts of world, particularly in developing nations such as Pakistan, where this disease is uncontrolled and accounts for substantial human and economic losses. The problem has further exacerbated due to emergence of increasingly more resistant strains of M. tb to antibiotics and failure of BCG vaccine. Therefore, new and state of the art strategies like DNA vaccination which is more efficacious and cost effective vaccine to protect against TB must be sought. In this study six genes belonging to M. tb (hspx, cfp10, ag85a, ag85b, ag85c and esat6) were selected and used as a potential candidate for DNA vaccination. Each gene was cloned in pTZR57 cloning vector (Fermentas, USA) with out Kozak sequences upstream the gene and in pcDNA3.1 Topo (Invitrogen, USA) vector with Kozak sequences. Finally the genes were sub-cloned in pND-a mammalian expression vector to get transgene expression under in vitro and in vivo conditions. Almost all of the conscructs without Kozak sequences were unable to produce detectable protein in animal cell lines as checked by Western blots except hspx-pND, which has a natural Kozak in its sequence. The pND having M. tb genes constructs with Kozak sequence gave significantly high expressions. The expression comparison of M. tb genes with Kozak sequences cloned in pcDNA3.1 and pND was also done under in vitro cell line conditions and constructs in pND were found the best. On the basis of in vitro cell line expressions, the endotoxin free pND-M. tb gene constructs preparations were made and subjected to eight weeks old female Balb/c mice’s quadriceps muscles and intradermas @ of 50 μg DNA/leg and 25 μg interdermally at the base of tail. In this way total 125 μg DNA solubilized in normal saline was used for inoculation of an animal. No booster injections were given. The animals were divided into six groups including positive and negative control groups. Eight animals were used for hspx-pND vaccine, eight for cfp10-pND vaccine, two for esat6-pND vaccine and two for equally mixed ag85a, b and c in pND vaccines. Four animals were used as positive control group and injected with Np-pND constructs, similarly four animals were used for negative control (normal saline) group. The animals were bled after nine weeks of post vaccination through tail and finally with cardiac puncture technique. The antibodies were confirmed by Western blot analyses and their level was monitored by indigenously standardized Multiplex Microbead Assay. The best humoral response was shown by hspx-pND vaccinated animals both on Western blots and Multiplex Microbead Assay. Fairly positive response was obtained in animals vaccinated with esat6-pND14 and ag85 a, b and c genes in pND by Western blots and Multiplex Microbead Assay. Whereas undetectable level of antibodies from cfp10-pND vaccinated animals was noted. It is therefore, concluded based on results that all of the M. tb gene constructs in pND gave good expression under in vitro conditions except esat6 gene and under in vivo conditions except cfp10-pND constructs. Therefore, in general the results are promising and need more animal studies before these constructs go to clinical trials.