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

Co-Investigator
Parmod K Upadhyay

Two different strategies are being pursued to develop vaccine against tuberculosis, the whole bacterial vaccine approach relying on multiple antigens and built-in-adjuvanticity. Other mycobacterial strains which share cross-reactive antigens with M.tuberculosis are being considered as alternatives to M.bovis for vaccine use. One such strain M.w has shown protective effect of killed M.w against subsequent tuberculosis in preliminary studies. Second, the subunit vaccine approach attempting to induce efficacious immunity by unique antigens in defined adjuvants. It has been suggested that proteins secreted from the multiplying and metabolizing bacilli at the early stage of infection are responsible for the recognition of infected macrophages by T cells. Thus they are believed to be involved in inducing protective immunity against tuberculosis.

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.

As lung is the primary target organ for this disease, immunisation potential by aerogenic route is being studied. Inhalation of aerosols provides a non-invasive delivery system that physically targets the lung as the desired site of pharmacological effect. Five groups of mice were immunised with M.w aerosol/BCG aerosol/M.w, s.c inj./BCG s.c inj./ normal saline respectively. In aerosol groups, four subgroups were exposed to aerosols of different concentration of bacilli (109/ml to 106/ml). Three weeks after the booster dose, mice were sacrificed and immune response was evaluated.

Since IFN-g is a crucial component of antimycobacterial immunity, the induction of this cytokine in both lungs and spleens was assessed. Lymphocytes collected from broncho-alveolar lavage and spleenocytes were restimulated in vitro with M.tb Ags. IFN-g secreted in the culture supernatant was quantitated. It was found that IFN-g secretion after immunisation with M.w aerosol was three to four times than that with BCG aerosol or M.w given by s.c injection. A consistently high immune response with M.w aerosol was obtained in three sets of experiments.

In order to study the protective efficacy of M.w immunisation in animal models of tuberculosis, a collaborative proposal with CJIL, Agra has been submitted to funding agency. M.tb infection experiments will be done in BSL-3 facility of CJIL.

To explore the possibility of improving immunogenicity of CFP6, with adjuvants, different groups of mice were immunised with rCFP6 along with MPL/IFA/NTLPS by s.c route. T-cell response induced with MPL or IFA were similar in nature and magnitude.

In an attempt to develop an immunisation strategy that will induce higher protective immune response, heterologus prime-boost strategy is being studied. In this strategy, primary immunisation is done with plasmid DNA of CFP6 and booster is given with purified recombinant CFP6.

Recently we have initiated, study of antitumor activity of conjugates of taxol. Taxol has demonstrated significant efficacy as an antitumor chemotherapeutic in human clinical trials. However, a major difficulty in the clinical trial use has been its insolubility in water. Taxol 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 taxol 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. 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 and at the same time possibly increasing the anti-tumor efficacy of taxol derivative, by combining the chemotherapeutic and immunotherapeutic activities.

LPS derivative was made by alkylation with n-amino butyric acid. In this alkylation reaction, hydroxyl groups of lipid A moiety of LPS react with carboxylic group of butyric acid. In this conjugate, an amino group is made available for further reaction. In another reaction, taxol-succinate derivative was made by reacting hydroxyl group of taxol with succinic anhydride. The final conjugation of taxol-succinate and amino butyric LPS was carried out in aqueous medium by using a water soluble carbodimide reagent. Spectroscopic characterization of various intermediate derivatives and final conjugate is being done.

Publications

Original peer-reviewed articles

1.   Bhaskar S, Banavaliker JN and Hanif M (2003) Large scale validation of latex agglutination test for diagnosis of tuberculosis. FEMS Immunol Med Microbiol 1613:1-5.

2.     *Bhaskar S and Upadhyay P (2003) Design and evaluation of an aerosol infection chamber for small animals. Int J Pharmaceutics 255:43-48 (*in press last year, since published).