Molecular Immunotechnology

WHO-sponsored Filarial Genome project

Prof. Kaliraj's group was involved in sequencing a total of 17,000 ESTs comprising of 7000 genes of B.malayi. Functional genomic analysis revealed that 30% of the genes were unique for filarial parasite, 27% being structural genes and 20% being enzymes. Genes such as SXP-1, Abundant Larval Transcript 2(ALT), Translationally Controlled Tumor Protein (TCTP), VAH, Thioredoxin, Transglutaminase, Thioredoxin peroxidase, MIF-1, HSP70 and endochitinase from the filarial parasites, Brugia malayi and Wuchereria bancrofti, have been identified as candidates for diagnosis and prophylactic measures. The immunodiagnostic and immunoprophylactic applications using these parasitic gene products have been extensively studied.

Immuno diagnosis of human lymphatic filariasis

As a part of global effort to eliminate lymphatic filariasis, a major public health problem for India and many other countries in the world, highly sensitive and specific diagnostic tests are being developed for close monitoring and evaluation of the control programs.Newer diagnostic tools reported based on antigen detection (Og4C3 and ICT card test kits) are limited by their ability to detect only bancroftian infection and not brugian infections. It is also essential to develop a diagnostic kit to identify prevalence of mixed filarial infection due to W. Bancrofti and B.malayi.

Prof. Kaliraj’s group has identified WbSXP-1 as a promising and a more reliable candidate for the identification of active infections (Microfileamics) in both bancroftian and brugian filariasis. Simple and rapid qualitative Immunodiagnostic test kits for the identification of antigen (monoclonal antibody to WbSXP-1 based) and antibody (recombinant filarial antigen WbSXP-1 based) from individuals with W.bancrofti/ B.malayi/mixed infections by Flow-through Assay (antibody test) and Immuno-chromatographic card test (antigen test) have been developed in collaboration with SPAN Diagnostics, Surat, P.A.R.I.S, Immunologicals, and UTC, Compiegne France. This product is currently under field evaluation for commercialization. Confidentiality and Material Transfer agreements have been executed by Prof. Kaliraj with M/S Span Diagnostics Surat for Manufacturing and Marketing in India and with M/S PANBIO Australia for Global marketing of these kits. This (is being worked out) / (serves) as a successful model for commercialization of a research product involving university, national and international commercial and noncommercial organizations / partners.

In the next phase stage-specific detection kits are being developed using recombinant DNA Technology and monoclonal antibodies for effective control of the disease. Compact Disc based high throughput immunoassays In many immunoassays like ELISA antigen or antibody is adsorbed onto the plastic surface of a microtitre plate. In collaboration with Glasgow university, UK., University of Heidelberg, Germany and Aims Sham's University, Egypt under EC project Prof. Kaliraj and his group have designed a compact disc (CD) platform which can replace the microtitre plate for antigen capture immunoassays as a cost effective and versatile method for mass diagnosis.

Immunoprophylaxis

The need for a lymphatic filariasis vaccine for complete eradication of the disease is imperative. Prof. Kaliraj's work carried out at the Centre in the past 10 years in collaboration with NIH, USA; Fermentile Hospital, Australia and JBTDRC, Wardha has resulted in the identification of novel stage-specific antigen ALT2 and other targets like VAH, transglutaminase, SXP, endochitinase, TCTP, thioredoxin, etc. as potential vaccine candidates. The results of the immunoprophylactic studies in mouse and gerbil models using ALT2 as a protein vaccine as well as a DNA vaccine are promising. Three putative vaccine candidates having partial lengths of ALT2, VAH and Thioredoxin peroxidase genes have been identified by screening the T7 phage display library of L3 stage using putative immune sera. This important work is pursued in collaboration with UIC College of medicine, Rockford USA. Optimization of protective efficacy of DNA vaccine by bimodal and polycistronic based multiple antigens approach is being carried out with the financial assistance from Department of Biotechnology and Indian Council of Medical Research, India.

Immunotechnology

Research Activities

Prof.R.B. Narayanan's immunotechnology group is currently working on delineating the immunological mechanisms involved in the pathogenesis of human lymphatic filariasis, a parasitic disease caused by the nematode parasites Wuchreria bancrofti and Brugia malayi affecting the developing countries including India. This is achieved by studying the interaction of parasite-derived antigens with T cells and macrophages of normal healthy individuals and filarial patients. Efforts are also on to identify antigens for diagnostic use in viral infections affecting dogs, shrimps and crabs. Immunotechnology lab has all the facilities to undertake research in Immunology of Infectious Diseases.

I. Immunopathology of Filariasis
a. T cell and macrophage function in filarial patients.

T cells and macrophages are purified from the blood of patients with bancroftian filariasis. Purified filarial recombinant proteins and crude parasite antigens are allowed to interact with these cells and functional status like lymphoproliferation, cytokine production and other biological mediators release is assessed. In addition studies on the co stimulatory molecules and T cell receptor is being pursued.

b. Wolbachia genes.

Wolbachia is an endosymbiont bacteria that resides in the live filarial worms of either sex or microfilaria. The worm depends on wolbachia its metabolism and survival. We have identified new genes from wolbachia of W.bancrofti and extended these studies to the cattle filarial parasite Setaria digitata. These genes are being cloned, expressed, purified and used for immunological studies in filarial patients.

c. Identification of new genes from W.bancrofti.

Lymphodema is a severe clinical condition in filarial patients with chronic pathology. At the present time there is no reliable test available to distinguish between the lymphodema of filarial or non-filarial origin. Screening cDNA libraries of W.bancrofti adult and microfilaria is being carried out to identify genes that react at the level various antibody isotypes using patients serum in a hope that new genes will be identified that may be of diagnostic importance.

II. Immunodiagnosis and Immunophylaxis of Canine Distemper Virus infection in Dogs.

The need for a lymphatic filariasis vaccine for complete eradication of the disease is imperative. Prof. Kaliraj's work carried out at the Centre in the past 10 years in collaboration with NIH, USA; Fermentile Hospital, Australia and JBTDRC, Wardha has resulted in the identification of novel stage-specific antigen ALT2 and other targets like VAH, transglutaminase, SXP, endochitinase, TCTP, thioredoxin, etc. as potential vaccine candidates. The results of the immunoprophylactic studies in mouse and gerbil models using ALT2 as a protein vaccine as well as a DNA vaccine are promising. Three putative vaccine candidates having partial lengths of ALT2, VAH and Thioredoxin peroxidase genes have been identified by screening the T7 phage display library of L3 stage using putative immune sera. This important work is pursued in collaboration with UIC College of medicine, Rockford USA. Optimization of protective efficacy of DNA vaccine by bimodal and polycistronic based multiple antigens approach is being carried out with the financial assistance from Department of Biotechnology and Indian Council of Medical Research, India.

Canine Distemper Virus (CDV) infection is prevalent in dogs and its prevention is necessary. Efforts are underway to produce recombinant proteins from this virus for diagnostic and prophylactic studies using molecular and immunological approaches.

III. Production of recombinant proteins to White Spot Syndrome Virus in Shrimps.

White Spot Syndrome Virus (WSSV) infects a sizable population of shrimps and crabs. Our lab has recently developed an immunodiagnostic assay to detect WSSV in shrimps and its possible commercialization is being explored. Efforts are on to produce well-characterized recombinant proteins from WSSV for functional studies.

IV. Spermicidal effects of neem oil and its fractions.

Neem oil fractions have various biological functions and therapeutic uses. This is a collaborative project between the Centre for Biotechnology and TTK LIG limited, Chennai to screen neem oil fractions for spermicidal action on human sperms.