Immune responses generated by M.w & BCG in animal models of tuberculosis: a comparative study

Co-Investigator
Pramod Upadhyay

The present study aims to investigate the protective efficacy of M.w immunisation in live or killed form, against subsequent infection with M.tuberculosis in animal models as well as to analyse the immune responses to M.tuberculosis in animals immunised with M.w to examine novel ways of altering protective immunity against M.tuberculosis. These responses will be compared with those generated in BCG immunised mice. A parallel study, with the aim to identify new antigenic proteins from culture filtrate of M.tuberculosis, relevant for induction of acquired immunity to tuberculosis is being pursued.

M.tuberculosis specific immune responses induced by immunisation with M.w were studied in mice model. Four groups of mice were immunized with live M.w killed M.w, BCG, and normal saline respectively. In each group, subgroups were immunized with different numbers of M.w bacilli (106-109). At each dose three mice were immunized by s.c. injection. Booster dose was given after 3 wks. Four weeks after the booster dose, mice were euthanised and splenocytes were stimulated in-vitro with M.tuberculosis antigens and M.w antigens. It was found that immunisation with live M.w induced higher T-cell response (proliferation & IFN-g release) as compared to killed M.w Mice immunized with 107 live M.w generated optimum immune response to M.tuberculosis antigens and these responses were higher as compared to mice immunized with BCG.

To evaluate the protective immune response of rCFP6 and its Plasmid DNA, five groups of mice were immunized with rCFP6 in saline/rCFP6+Alum/rCFP6+IFA/saline+ IFA or Alum/Plasmid DNA. In each group, subgroups were given different concentrations of protein and DNA. Four weeks after the booster dose, mice were euthanised and splenocytes were restimulated in-vitro with
M.tuberculosis antigens and purified CFP6. It was observed that mice immunized with rCFP6 in saline induced moderate protective response while the response of CFP6 along with adjuvants was significantly high. Highest response was obtained with CFP6 + IFA both in terms of IFN3 release and proliferation. 100¼g Plasmid DNA by i.m. injection gave optimum response but response with DNA was less in magnitude as compared to response with rCFP6.

Hence, in an attempt to improve the response induced by plasmid DNA, we used electroporation method to introduce DNA in mice. Initial results have shown no significant difference in the immune response generated by the two methods of DNA immunisation.

Simultaneously, we are also exploring the possibility of immunisation through aerogenic route. We have immunized the mice with aerosol of M.w and BCG. Immune response induced by this method of immunisation are being studied

Recently we have initiated, study of antitumor activity of conjugates of taxol. The antineoplastic agent taxol has demonstrated significant efficacy as an antitumor chemotherapeutic in human clinical trials. Primarily it has shown activity in ovarian and breast cancer, small-cell and non-small cell lung cancer, head and neck cancers. However, a major difficulty in the clinical trial use has been its insolubility in water. Paclitaxel is typically formulated as concentrated solution in Cremophor EL and dehydrated alcohol (50%v/v) and must be further diluted before administration. The amount of Cremophor EL necessary to deliver the required doses of paclitaxel is quite high and several toxic effects have been attributed to it. Conjugation of chemotherapeutic agents to water-soluble macromolecular carriers is an alternative approach to improve the solubility of drug. In addition, polymer-drug conjugates may provide desirable pharmacokinetics and an improved therapeutic index, because of EPR effect of macromolecules in solid tumors.The chemotherapeutic capacity of taxol is realized through multiple mechanisms. Taxol’s primary antitumor mechanism is by polymerizing microtubules which halts cell-cycle progression. Besides this, taxol has profound cell cycle-independent effects also, which are similar to those induced by bacterial LPS, activating expression of all characterized LPS-inducible genes. LPS mimetic activity of taxol is independent of its microtubule stabilizing action. These findings lead us to explore the possibility of making taxol soluble by conjugating with non-toxic derivatives of LPS, viz. succinylated and phthalylated LPS and at the same time possibly increasing the anti-tumor efficacy of taxol derivative by combining the chemotherapeutic and immunotherapeutic activities. Conjugation of taxol with non-toxic derivative of LPS is being studied.

Publications

Original peer-reviewed articles

1.     Bhaskar S and Upadhyay P (2003) Design and evaluation of an aerosol infection chamber for small animals. Int J Pharm (in press).