Achievements

Prof. Anil K. Tyagi:
The work is focused on M. tuberculosis. The emphasis is on gene regulation, mechanism of pathogenesis and development of a new vaccine against tuberculosis by using recombinant BCG and DNA vaccine approach. We have shown that mycobacterial transcriptional elements function poorly in E.coli. RNA polymerases from mycobacteria recognises promoter elements with comparable efficiency. Analysis of these promoters has revealed that their -10 regions are highly similar to those of E.coli promoters in contrast to their -35 regions which tolerate a greater variety of sequences. We have also developed a versatile expression system, which permits stable expression of homologous and heterologous genes in mycobacteria and enables the expression of a gene to be regulated several hundred fold. This expression system has been used for the expression of several immunoprotective antigens of M.tuberculosis, for the developments of recombinant BCG vaccines. Besides, the laboratory has also generated several candidate DNA vaccines. We had identified a gene from M.tuberculosis, the 38 kDa protein product of which (VirS) bears homology with VirF, VirFy, Cfad, Rns and FapR which act as positive regulators of transcription and control the expression of structural genes required for establishment of infection and pathogenesis. Another gene mymA expressing a monooxygenase homologue is located divergent to virS. Deletion analysis of upstream region of mymA showed that its expression is subjected to regulation by transacting factor(s). mymA represents the first gene of an operon which is homologous to an operon present in Aceinetobacter wherein it is used for metabolizing unusual substrates and converting them into TCA cycle intermediates for the production of energy. We have shown that this operon is induced 6-7 fold when M. tuberculosis infects macrophages. Thus, mymA could function for production of energy for mycobacteria apparently striving for survival within macrophages.

During the next five years, the recombinant BCG and DNA vaccine candidates will be evaluated in animal models for their protective efficacy and the promising candidates will be subjected to toxicity studies. Besides, by using the promising candidates, heterologous prime boost approach will also be studied to achieve more promising results. The work on the characterization of promoters will be further extended by using foot-printing approach to elucidate the exact sequences of mycobacterial promoters. The mymA and virS genes will be disrupted and their consequences on virulence will be studied.

Prof. Vijay K. Chaudhary:
Our laboratory has focussed on engineering of antibodies for therapeutic and diagnostic application. Using state-of-the-art techniques of genetic engineering and protein expression, molecules have been produced that have diagnostic value. One example of such research is the development of NEVA-HIV, a diagnostic test for the detection of HIV infection within five minutes in a drop of blood with requiring any sophisticated instrument. The test uses recombinant proteins consisting of a monovalent fragment of an anti-human RBC monoclonal antibody fused to a specific protein antigen derived from HIV. These proteins cross-link RBCs in the presence of anti-HIV antibodies, which are present in the blood of HIV-infected individuals. The cross-linking of RBCs is seen as agglutination (clumping) by naked eyes. This novel technology of HIV detection was ilaunched to the Nation by Hon’ble Vice President of India, on the occasion of the NATIONAL TECHNOLOGY DAY, the 11th May, 2001. The technology has been successfully transferred to Cadila Pharmaceutical Limited, who after fulfilling the regulatory requirements have started marketing of the kit in India with plans for marketing in African countries in the near future.

We have pioneered the system of Phage Display and employed it for a variety of applications. We have developed various phage vectors and optimised display of peptides and proteins as fusion partners with different coat proteins of phage M13. One of our recently developed phage display vector for making gene fragment libraries has been highly appreciated and several groups worldwide have requested the vector for their work. With Genomics coming up in a big way recently, we have used phage display technology for Functional Genomics. For this, we have developed M13 and lambda phage based display vectors and are making cDNA libraries of human cells to study protein-protein interactions. We have also made whole genome libraries of pathogenic organisms like HIV and HEV and have mapped immunodominant regions of these organisms that will be useful in diagnostics and therapeutics. Recently, we have made a whole genome library of Mycobacterium tuberculosis H37Rv. Each and every reading frame encoded by Mycobacterium genome is displayed on phage as peptides. This library, perhaps the first whole genome display of such a large genome (4 Mb) will be a great resource for the entire scientific community working on tuberculosis and can be used to study pathogenesis and interaction of the microbial proteins with host tissues, identification of bacterial proteins interacting with host cellular proteins, delineation of proteins of mycobacterial eliciting immune response in infected patients to identify epitopes crucial for early and sensitive diagnosis of infection, and innumerable other studies of this pathogen. The future work plan of the laboratory will involve: Development of Phage display technology for functional genomics. Identification of specific immunodominant epitopes of Mycobacterium tuberculosis using whole genome phage display library. Identification of immunodominant epitopes of HIV for early detection of infection. Engineering of anti-RBC antibodies to improve affinity and folding. Human antibodies for therapeutics and imaging based diagnostics.

Prof. Prahlad C. Ghosh:
An active area of research concerns with the studies on the targeted delivery of drugs, toxins and genetic materials using liposomes and nanoparticles as carriers for the treatment of cancerous and infectious diseases. A liposome formulation of amphotericin-B is developed which is less toxic and more effective as compared to free drugs in controlling fungal infections (Aspergillosis) at experimental level. After testing the safety and efficacy of this formulation in human patients at various centres this formulation has been transferred to the industry. We have demonstrated for the first time that liposome can be used as an effective means in vivo for delivery of monensin to specific cells and have potential application for cancer and malaria therapy. We are also the first to develop a colorimetric method for estimation of polyethylene glycol in free as well as protein and lipid-bound form. This method can be employed in the assessment of the pharmacokinetics property of therapeutically important pegylated proteins and pegylated sterically stabilized liposomes in clinics. Very recently we have demonstrated for the first time that carboxylic ionophore and lysosomotropic amines significantly enhances the cytotoxicity of liposomal ricin in Chinese hamster ovary cells. This raises an interesting possibility of delivering ricin in intact form into the cytosol of mammalian cells using liposomes as a carrier. This may open up a new way of delivering macromolecules into the cytosol of mammalian cells including tumor cells. Recently we have demonstrated that doxorubicin entrapped in hydrogel chitosan nanoparticles showed effective tumor regression and increased life-span of animals as compared to free drug treated groups. The improved therapeutic efficacy of chitosan nanoparticles was found to be due to higher accumulation of doxorubicin at the tumor site from chitosan nanoparticles due to the enhanced permeability and retention (EPR) effect, reduced immunosuppressive activity of doxorubicin and low uptake in the cardiac tissue. The results obtained are promising and clearly warrant clinical investigation of this novel long circulating nanoparticles for future application in the treatment of tumor.
During the next five years, we plan to study the targeted delivery of toxins, drugs, genes for the treatment of cancerous and infectious diseases using sterically stabilized liposomes and nanoparticles as a carrier.

Prof. Debi P. Sarkar:
The major focus of the laboratory is to develop and use Sendai virus envelopes, F-Virosomes for encapsulating a variety of macromolecules (Drugs and Genes) and carry out basic and applied work. We have prepared two types of reconstituted Sendai virus envelopes, F-virosomes containing only the fusion protein (F) and F,HN-virosomes containing both the F protein and the hemagglutinin-neuraminidase (HN) by solubilization of the intact virus with Triton X-100 followed by its removal using SM2 Bio-Beads. We also prepared reduced F,HN-virosomes (F,HNred-virosomes), by treating the F,HN-virosomes with dithiothreitol and its subsequent removal by dialysis. We demonstrated the fusion of F- and F,HNred-virosomes with HepG2 and HeLa cells respectively, using an assay method based on the relief of self quenching (dequenching) of the lipid probe N-4-Nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine (NBD-PE) incorporated in the Sendai envelopes. On the basis of results obtained, we conclude that the presence of HN in the viral membrane is not necessary for its fusion with target cells. The potential of reconstituted Sendai Viral envelopes containing only the fusion protein (F-Virosomes) was studied for targeted cytosolic delivery of lysozyme to human hepatoblastoma cells (HepG2) in culture. Based on the results obtained, our work establishes for the first time that a heterologous membrane ligand like HA with strong binding affinity with target cells is sufficient to replace the role of HN to a high extent. We have discovered a previously unknown glycoprotein of Mr 45K in the envelope of Sendai virus and characterized its role in F protein-mediated membrane fusion. The Potential of F-virosomes in the targeted cytosolic delivery of genes (DNA) in vitro and in vivo has been recently established (USA patent granted, 1997). Recently in collaboration with Dr. Vijay Kumar, ICGEB, New Delhi, we have demonstrated for the first time the role of Hepatits B Virus X protein in stimulating mitogenic cell signaling cascades under in vivo conditions (whole animal). During the next five years, we plan to focus our research activities in the areas of role of matrix and fusion proteins in budding of Sendai virus from host cells and targeted delivery of DNA vaccines into hepatocytes through engineered sendai viral envelopes (in collaboration with ICGEB, New Delhi).

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