dc.description.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. |
en_US |