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

Co-Investigators
Parmod K 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.

An indigenous aerosol infection chamber has been designed and used to infect the mice with non-pathogenic mycobacteria through aerogenic route. Respirable aerosol of the bacterial suspension has been made by passing compressed air through a nebulizer holding the suspension. Uniform distribution of aerosol inside an air tight chamber has been ensured by fans fitted inside the chamber. This was checked by keeping LJ plates at different corners of the chamber and exposing them to aerosol for few minutes. There was uniform spread of colonies over all the plates. All safety requirements have been ensured in this aerosol infection system. The aerosol coming out of the chamber is made to pass through incinerator followed by disinfection fluid and 0.22mm filter. The device has been checked for sterility of air coming out of it. In order to ensure double safety to the experimenter the device could be placed in a Bio safety hood and controlled remotely. To ease the operation and to make it more reliable, the assembly is controlled by a microprocessor which controls the time of exposure by actuating relays and valves.

The concentration of bacterial suspension to deliver about 100 bacilli per mice during 5 minutes exposure has been standardised. Presently experiments to optimize the dose of live and killed M. w. to generate optimum immune response are being conducted.

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. Towards this end, eight low molecular weight proteins were characterised immunologically. Out of eight proteins studied, three proteins gave significantly higher proliferative responses in PBMCs collected from healthy contacts of TB patients. These proteins seems to be immunologically important. Two proteins were found to be new.

To get large quantity of these proteins for further characterisation and study their protective efficacy in animal models, genes of these proteins have been cloned and expressed in E. coli. One of the proteins (CFP6) has been purified in soluble form in large quantities. Lymphocyte proliferative responses of tuberculosis patients and healthy contacts (hospital staff) to purified recombinant protein (rCFP6) were studied. Blood samples were collected from 10 patients and 10 healthy contacts. Recombinant CFP6 gave significantly higher mean proliferative responses in PBMCs collected from healthy contacts as compared to patients. To study the protective efficacy of CFP6 as DNA subunit vaccine, we made recombinant plasmid carrying gene for CFP6 under the control of eukaryotic promoter (CMV promoter). This plasmid construct has been purified in large amount. Recombinant CFP6 and its plasmid construct will be evaluated for their potential to generate protective immune response in mice.

Publications

1.     Bhaskar S, Banavaliker JN, Bhardwaj K and Upadhyay PK (2002) A novel ultrasound-enhanced latex agglutination test for the detection of antibodies against Mycobacterium tuberculosis in serum, J Immunol Meth (in press).