PUBLIC SECTOR UNDERTAKINGS
Bharat Immunologicals & Biologicals Corporation Limited, Bulandshahr
Bharat Immunologicals & Biologicals Corporation Limited (BIBCOL) was incorporated in March 1989 as a Public Sector Company at village Chola, Distt. Bulandshahr, Uttar Pradesh, to manufacture Oral Polio Vaccine (OPV) and other immunobiologicals cGMP conditions as specified by WHO and Federal Standards.
The Company has been formulating OPV from bulk since January 1996 and about 1016 million doses have been supplied to the National Immunization Programme so far. This includes OPV supplied through UNICEF. The company has settled all its outstanding loans with financial institutes and banks including loan liability of the Government of India. Now the company has become debt free.
During 2004-05, 120 million doses of OPV has been formulated and supplied to the National Immunization Programme (NIP). During the year 2005-06, the company is expected to formulate about 125 million doses of OPV from bulk and supply the same to NIP.
Indian Vaccines Corporation Limited, Gurgaon
The Indian Vaccines Corporation Limited (IVCOL) was incorporated as a joint venture company in March, 1989 to undertake research and development and manufacture of viral vaccines. Due to change in product mix policy and non-availability of vero cell technology from Pasteur Merieux Serum & Vaccines (PMSV), France the company was on hold since February, 1992.
In pursuance of the Cabinet decision in September, 1998 restructuring process of the company has been undertaken to utilize the assets created under the project. Accordingly, the National Brain Research Centre has been established at part of IVCOL premises. The proposal to transfer some of DBT shares, which were acquired, from PMSV, was offered to Indian Petrochemicals Corporation Limited, one of the promoters of the company. The proposal has been submitted to the competent authority for final decision.
International Centre for Genetic Engineering and Biotechnology
International Centre for Genetic Engineering and Biotechnology is an independent intergovernmental organisation under the UN system. The Department of Biotechnology provides the host country contribution to ICGEB. The primary mandate of the organisation is to carry out basic research in areas related to human health and agriculture useful for developing countries. The Centre also imparts training and education and promotes development of products and transfer technologies to industrial partners.
The Centre continued efforts in developing cost effective technologies and their subsequent transfer to the industry. “Bioprahar” a biopesticidal formulation effective against lepidopteron pests was developed and successfully transferred to an industrial partner in India. A recombinant multi-epitope protein based HCV detection system was developed and transferred to the industry. The Centre filed three new patent applications and extended three patents to PCT. Under the manpower-training programme, three training courses were conducted in the field of malaria, plant molecular biology and bioinformatics.
Human Health
Malaria
Vaccine Research : Adhesion of Plasmodiumfalciparum-infected erythrocytes in brain capillaries is implicated in cerebral malaria and adhesion in placenta leads to complications in pregnancy that put the lives of both mother and child at risk. Efforts are being made to study the molecular interactions between host receptors and parasite ligands that mediate red cell invasion and cytoadherence by malaria parasites and use some of these molecules on the surface of the parasite as vaccine candidates. The malaria vaccine programme is focused on developing the procedure for the production of recombinant vaccine candidate molecules like MSP19 and binding domain of erythrocyte binding antigen of (EBA175)
Lab scale procedures have been developed and transferred to industrial partner for the production of GMP grade material to be used in preclinical and clinical trials. The immunogenic response for a combination vaccine of the above two antigens, in small animals, using different adjuvant, has been concluded. Based on these studies, an appropriate adjuvant for the development of final combination vaccine formulation has been selected. This project has been supported by the Prime Minister’s Initiative on Vaccine, Jai Vigyan Mission, through the Department of Biotechnology, Government of India as well as the European Malaria Vaccine Initiative (EMVI). The corresponding vaccine for Plasmodiumvivax is also being developed in parallel with major financial support from the Malaria Vaccine Initiative (MVI).
In addition, a number of other vaccine candidates such as Apical Membrane Antigen I (AMA I), Merozoite Surface Protein 3 (MSP3) and Merozoite Surface Protein 9 (MSP9) are also being developed by the Malaria Group.
Development and application of RNA interference in malaria parasite : With the completion of Plasmodium falciparum genome, it is now known that there are more than 5000 genes in Plasmodium falciparum genome and the function of more than 60% of these genes is unknown. The malaria group carried out in-depth study to establish RNAi in P. falciparum to study the function of the genes. Using dsRNAs corresponding to cysteine protease genes of P. falciparum (falcipain 1&2), the group demonstrated that falcipains play an important role in the hemoglobin degradation. It also demonstrated that the mechanism of RNAi is conserved in P. falciparum i.e. dsRNA is cleaved to 25nt RNA species (siRNAs) which direct RNA induced silencing complex (RISC) to cleave mRNA.
Malaria drug development and structural peptides
This group is involved in studies towards de novo design of mini proteins, drug discovery, drug design and drug action. Conformationally constrained amino acids like didehydro phenylalanine and dimethylglycine are being used to synthesize peptides exhibiting super-secondary structures, ordered molecular assemblies or potent antibiotic action. X ray diffraction data has been used to examine the high-resolution architecture of these super-secondary structures or ordered molecular assemblies and using this strategy the solid-state crystal structures and solution state circular dischroism structures of two different helical hairpin peptides have been prevented.
This group has invented and successfully put to use a high throughput anti-malarial drug discovery method using a complex of recombinant Pf HRPII and heme to look for visual color change induced by potential blood stage antimalarials. The method of screening molecules for anti-malarial activity has been validated. This format has worked very well in a robotic mode where over 10,000 compounds from chemical combinatorial libraries have been screened in a microtiter format and around ten promising hits identified. A patent application has been filed for the method of screening in the Indian Patent Office.
Virology
The Virology Group is currently exploring select aspects of the biology of the following viruses: the hepatitis B virus (HBV), the hepatitis E virus (HEV), the human immunodeficiency virus type 1 (HIV-1) and the SARS corona virus. Infections due to these viruses contribute significantly to morbidity and mortality across the world, especially in developing countries. The structure-function relationships between viral proteins as well as those between viral and host proteins are being investigated to understand viral pathogenesis. The molecular and genetic tools being used include cloning and expression of viral proteins, sequencing and mutagenesis, protein-protein interaction technologies (yeast two-hybrid and FRET), analysis of signal transduction pathways, confocal microscopy and transgenic mouse models.
Immunology
Main focus of areas of research are biology of B Lymphocytes, and examining host-pathogen interaction in Mycobacterium tuberculosis (Mtb) infections. In study of B lymphocytes, a novel feedback mechanism has been identified that controls both the amplitude and duration of B cell receptor signaling. In addition the mechanisms have been delineated for controlling transcription from themurine CD80 gene. It is demonstrated that nucleosomes located at distal positions from the transcription start site can play critical roles in this process, by exploiting the superstructure of the intervening DNA segment. Studies in the area of Mtb have primarily focused on the effects of a 10 kDa antigen that is expressed from the RD-1 region of the Mtb genome called ycobacterium tuberculosis Secretory Antigen (MTSA-10) also known as CFP-10. The studies revealed that this antigen controls macrophage responsiveness both in terms of pro-inflammatory cytokine and nitric oxide production, and the induction of expression of costimulatory molecules. In addition, it was also shown that MTSA-can induce differentiation and maturation of dendritic cells from bone marrow precursors. Interestingly, DCs thus generated were biased towards generating Th2-type responses, suggesting that antigens secreted by Mtb may be involved in down-regulating host immune responses in order to facilitate the infection process.
Recombinant gene products
The group primarily focused on the identification, design and development of laboratoryscale technologies for the production of recombinant proteins of medical importance and the transfer of these technologies to the pharmaceutical industry. Novel strategies are being developed to make designer proteins with potentially useful therapeutic properties.
The current focus is on dengue research. The group is investigating the possibility of developing subunit vaccine candidates using both prokaryotic andeukaryotic expression systems. In addition, the utility of novel multi-epitope proteins is being explored in the rapid and reliable detection of dengue infections, in order to develop inexpensive diagnostic kits.
Structural and computational biology
In the ‘Post-Genomic’ era of modern molecular biology, structural and functional studies of proteins have necessarily taken the center-stage. The elucidation of cellular mechanisms, which function via a network of interacting proteins require a detailed structure-function thrust. The Structural Biology Group aimed at understanding the structural principles that govern protein-based biomolecularinteractions. Structure-function studies on malaria parasite proteins, viral proteins and protein involved in hypertension are currently on-going. Additionally, bioinformatics-based approaches are being used to model three-dimensional structures of a large number of parasite proteins.
Plant Biotechnology
The group is actively involved in understanding the mechanisms of plant adaptation in response to abiotic stresses and mechanism of DNA replication following virus invasion. The final aim is to develop abiotic stress tolerant and virus resistant plants using transgenic approaches. Towards this end genes are being identified that are regulated under stress and analyzing the mechanisms of their expression including characterization of transcription factors and stress inducible promoters. Functional validation of the genes is being undertaken using a transgenic approach to identify the most potential genes for manipulation in crop plants, like rice. The group has manipulated glyoxalase pathway by over expressing glyoxase I and II genes and also manipulated the expression of helicase genes to confer salinity stress tolerance in plants. For virus resistance, a detailed analysis of proteins involved in the replication of mung bean yellow mosaic virus is being studied using a yeast model system that was developed in the lab. Further, virus induced gene silencing vectors are also being developed.
Insect resistance
Efforts continue of transferring the insecticidal protein coding genes of Bacillus thuringiensis to relevant crop plants for protection against targeted pests. Together with Plant Transformation group the following genes have been transformed into cotton coker 310 plants, cry1Ac, cry1a5, vip and cry2Ab. The transgenic plants have been analyzed for the presence of gene and protection against predation by targeted pest. Plants offering varying degrees of protection have been transferred to commercial partners for breeding into elite cultivars.
Under an agreement with Nirmal Seeds Ltd. constructs bearing cry1Ia5 and vip were transformed into eggplant. The pest protection results with transgenic plants revealed a high degree of protection against eggplant borer (Leucinoides orbanalis). These plants are being grown further in the green house.
Development and evaluation of biopesticidal formulation
A microbe based biopesticidal sprayable formulation has been developed. The efficacy of formulation was examined against specific agriculture, horticulture and forestry pests. Field tests were conducted in agriculture fields at Punjabrao Deshmukh Krishi Vidhalaya, Akola; Vasant Dada Sugar Institute, Pune; and fields of commercial alliance partner Nirmal Biotech Ltd. The formulation was effective in controlling the following pests: Diamond back moth – cabbage and cauliflower, white woolly aphids – sugarcane, mealy bugs- grapes, citrus and mango and white ants-teak plantations . A specific regimen of spraying on each crop was developed and the results obtained after two growing seasons revealed excellent control of targeted pests. Evaluation of mammalian toxicity at two different independent laboratories demonstrated the safety of formulation based on standard tests. The technology of formulation preparation has been transferred to Nirmal Biotech and the product has been launched as BIOPRAHAR. An Indian patent and a PCT has been filed.
Plant transformation
The main focus of the group is to introduce new genes/traits into various crop plants. The target crop plants included cotton, rice, tomato andsunflower. The traits focused included insect and fungal resistance, nutritional quality and to develop new expression systems that allow to regulate and over express foreign proteins useful in agriculture and in industry. Both nuclear and chloroplast genomes have been targeted for the introduction of foreign genes. More recently, a major focus was to develop regulated and tissue specific expression of foreign genes in plants through T7 RNA polymerase directed transcription. Studies were carried out using both monocot and dicot plants. In case of cotton the group has introduced four Bt gene namely, cry1Ia5, cry1A/ c, vip and cry2A/b that are important to provide durable and broad spectrum of insect resistance. The cotton plants transformed with cry1Ia5, cry1A/c, vip are being used in backcrossing with Indian elite cultivars by commercial collaborators (MSSCL, Nimal Seeds). In case of rice the group has introduced cry1Ia5 and vip genes and stable transgenic lines were developed and tested for their ability for insect resistance against yellow stem borer and leaf folder. To improve the nutrition quality of rice and tomato, the iron storage protein gene (ferritin), was over expressed under T7 system for specific expression in endosperm and fruit tissues respectively.
Plant resistance
The group is concentrating on the tagging and genetic and physical mapping, marker-assisted selection of gall midge resistance genes for use in pyramiding into important rice cultivars. The markers are also being used for map-based gene cloning of Gm2 — a gall midge resistance gene. Besides, as a part of integrated pest management, the group has developed molecular markers that can distinguish the different biotypes of this insect without resorting to host-based screening. Many gall midge resistance genes, Gm2, Gm4t, Gm7 and Gm8 have been mapped and tagged in rice that confer resistance against different biotypes of gall midge. Gm2 and Gm7 are mapped on to chromosome 4 and Gm4t and Gm8 are mapped on to chromosome 8 of rice. Marker-assisted selection (MAS) protocol for selection of resistant plants containing Gm2, Gm4t,Gm7, and Gm8 genes in rice has also been developed. This is being routinely used at plant breeding stations in the country. These markers are also being used in pyramiding Gm2, Gm4t, Gm7 and Gm8 genes along with bacterial blight resistance (BLB) genes in elite rice cultivars to provide durable resistance against gall midge. This is being carried out at Directorate of Rice Research (DRR), and Central Rice Research Institute (CRRI) in India.
Publications
The Centre published around 70 papers in national and international peer reviewed journals during the year 2004.
Technology Transfers
During the past year, the following technology transfers were successfully accomplished.
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Multi-epitope protein-based HCV detection
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Tulip Group of Cos., India; 2004
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Biopesticidal formulation
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Patents filed / extended by the center during 2004
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Biopesticidal formulation
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Bacteriophage T7 RNA polymerase – based transcription system for over expression of foreign proteins in plants
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Novel recombinant dengue multi- epitope (r-DME) proteins as diagnostic intermediates (jointly with DRDE)
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Molecular markers for mapping and tagging GM8 gene and their application in marker assisted selection of gallmidge resistant/ susceptible phenotypes
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Extraction of latex of Calotropis procera and a process for the preparation thereof. (jointly with AIIMS)
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Regeneration of plants and medium of use
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Novel selectable marker system for transgenic plants
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Training Activities
Four training programmes were conducted during the year. The programmes focused on Plant Molecular Biology, Malaria and Bioinformatics. About 60 scientists from the member countries including India attended the programmes. The bioinformatics facility in the Centre is a WHO/TDR recognized training centre for bioinformatics for regional scientists working on tropical diseases, (one amongst the 4 other such WHO/TDR recognized centers www.icgeb.res.in/ and http://www.who.int/tdr/publications/tdrnews/news68/bioinformatics.htm).
Regular bioinformatics workshops are also conducted by the Centre.