international centre for genetic engineering and biotechnology (icgeb)

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International Centre for Genetic Engineering and Biotechnology (ICGEB)
209
International Centre for Genetic Engineering
and Biotechnology is an independent inter-
governmental 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.
13.1 Human Health
Malaria
Vaccine Research : Adhesion of Plasmodium
falciparum-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 MSP
19
and binding domain of
erythrocyte binding antigen of (EBA
175
)
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 Plasmodium
vivax 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 (MSP
3
) and
Merozoite Surface Protein 9 (MSP
9
) 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.
INTERNATIONAL CENTRE FOR GENETIC
ENGINEERING AND BIOTECHNOLOGY (ICGEB)
CHAPTER 13
DBT Annual Report 2004 - 2005
210
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 the
murine CD80 gene. It is demonstrated that
nucleosomes located at distal positions from the
Screening antimalarial drugs in HTP microtiter
format
The Virology Group is currently exploring select
aspects of the biology of the following viruses:
HBV, HEV, HIV-1 and SARS
International Centre for Genetic Engineering and Biotechnology (ICGEB)
211
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 Mycobacterium
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 laboratory-
scale 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 and
eukaryotic 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 biomolecular
interactions. 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
13.2 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, cry1a
5
, 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 cry1Ia
5
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.
DBT Annual Report 2004 - 2005
212
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 and
sunflower. 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.
13.3 Publications
The Centre published around 70 papers in
national and international peer reviewed journals
during the year 2004.
13.4 Technology Transfers
During the past year, the following technology
transfers were successfully accomplished.
No.Technology Transferred to
1 Multi-epitope protein-based Tulip Group of Cos.,
HCV detection India; 2004
2 Biopesticidal formulation Nirmal Organo Biotech
International Centre for Genetic Engineering and Biotechnology (ICGEB)
213
13.5 Patents filed / extended by the centre
during 2004
No.Patent Filed / Extended
1 Biopesticidal formulation Extended to PCT
2 Bacteriophage T7 RNA polymerase – based transcription system
for over expression of foreign proteins in plants Extended to PCT
3 Novel recombinant dengue multi- epitope (r-DME) proteins as
diagnostic intermediates (jointly with DRDE) Extended to PCT
4.Molecular markers for mapping and tagging GM8 gene and their
application in marker assisted selection of gallmidge resistant/
susceptible phenotypes Extended to PCT
5.Extraction of latex of Calotropis procera and a process for the
preparation thereof. (jointly with AIIMS) Filed-India
6 Regeneration of plants and medium of use.Filed -India
7 Novel selectable marker system for transgenic plants Filed -India
13.6 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.
Launching of the Bioprahar a micro based sprayable biopesticide by the Hon’ble Minister for Science and Technology and Ocean Development at
ICGEB, New Delhi. Dr. M.K.Bhan, Secretary, Department of Biotechnology, Prof.V.S.Chauhan, Director, ICGEB, New Delhi, Dr.Patil, Dean PDKVV
Akola and Dr.Rajput, Director, Nirmal Seed are also seen in the picture.
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/
~whotdr and http://www.who.int/tdr/publications/
tdrnews/news68/bioinformatics.htm). Regular
bioinformatics workshops are also conducted by the
Centre.