international centre for genetic engineering and biotechnology (icgeb)

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11 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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International Centre for Genetic Engineering and Biotechnology (ICGEB)
207
CHAPTER 13
ICGEB continued its efforts in promoting
biotechnology and genetic engineering research in
the member countries. Two Technologies where
transferred and seven patents were filed. The
manpower-training programme continued at the
Centre with five training courses conducted in the
field of malaria, plant transformation, recombinant
gene products, virology and bioinformatics.
Human Health
Malaria: Malaria continues to be a major public
health problem in the tropical world. The malaria
group is trying to understand the biology of the
malaria parasite and its interactions with the human
host and using this information to develop novel
prophylactic and therapeutic strategies against
malaria.
Red cell invasion by malaria parasites and
cytoadherence by P. falciparum: A family of
erythrocyte binding proteins (EBPs), which includes
the Plasmodium vivax and P. knowlesi duffy binding
proteins (PkDBP and PvDBP) and P. falciparum sialic
acid binding protein, also known as EBA-175,
mediate critical interactions with erythrocyte
receptors to mediate invasion. The studies reveled
that P. knowlesi a gene knock out (PkaKO) parasites
loose the ability to form a junction with human
erythrocytes and can no longer invade human
erythrocytes. The binding domains of EBPs have
been mapped to N-terminal, conserved, cysteine-
rich regions, that are referred to as region II, or
Duffy-binding-like (DBL) domains. The binding
domains of PvDBP and EBA-175, (PvRII and PfF2)
contain ~300 amino acid residues including 12
conserved cysteines. Binding residues to the central
~100 amino acid residues of these functional
domains. Site-directed mutagenesis of residues
within the central region of PvRII has shown that
hydrophobic interactions play a critical role in
interaction of PvDBP with the Duffy antigen.
Adhesion of P. falciparum-infected erythrocytes
to vascular endothelium in diverse host organs is
implicated in pathological outcomes such as cerebral
malaria. Binding to host endothelium is mediated
by parasite proteins belonging to the PfEMP-1
family, which are encoded by var genes and contain
multiple extracellular DBL domains that mediate
binding. The group has demonstrated that three
diverse P. falciparum field isolates bind the same
region of ICAM-1 although there are subtle
differences in the contact residues used for binding.
The binding domains for ICAM-1 from three diverse
ICAM-1 binding P. falciparum isolates have been
mapped to DBLbC2 regions of PfEMP-1 indicating
that binding sites for ICAM-1 may be conserved. A
strategy to block interaction with ICAM-1 and
prevent severe disease may thus be possible.
Mechanisms of immune suppression in
malaria: Understanding how blood stage malaria
antigens regulate the immune responses may aid
in the design of vaccines. Dendritic cell (DC)
function can be modulated by the parasite to
stimulate either effector CD4 T cells for long-lived
immune responses, or regulatory T (Tr) cells that
suppress immune responses. The group has
demonstrated that the 19kD C-terminal fragment
of merozoite surface protein-1 (PfMSP-119) inhibits
the maturation of splenic DCs and induces
extracellullar stress-related kinase-1/2 (ERK-1/2)-
dependent IL-10 expression in mice. Adoptive
transfer of PfMSP-119 pulsed splenic DCs induced
CD4 T cell unresponsiveness by generating IL-10
producing CD4 Tr cells in recipient mice. These
results show that splenic DCs exposed to PfMSP-
119 induces differentiation of Tr cells and represents
a novel immune subversion strategy.
RNA interference (RNAi) in malaria parasites:
Number of proteins involved in RNAi such as Dicer,
Argounate, Tudor-SNc, VIG, Fragile X have been
identified in C. elegans, Drosophila and human.
However, in protozoan, fungi and algae where RNAi
has been demonstrated, RNAi related proteins have
not been identified. The group has identified one
such protein, Tudor-SNc, in Plasmodium sp. In-silico
comparison of P. falciparum Tudor-SNc (PfTudor
INTERNATIONAL CENTRE FOR GENETIC
ENGINEERING AND BIOTECHNOLOGY (ICGEB)
DBT Annual Report 2005-06
208
SNc) with other Tudor-SNc proteins revealed that
PfTudor-SNc protein is quite distinct from
vertebrateTudor-SNc proteins. The group has
expressed and purified two different fragments of
PfTudor-SNc protein and found that PfTudor-SNc has
a Ca+ dependent nuclease activity and binds RNA.
Using specific inhibitor against Tudor-SNc protein,
it was demonstrated that PfTudor-SNc is an essential
enzyme in the parasite life cycle and can thus be
an important target for malaria drug development.
Malaria vaccine development: The lead
candidates for development of recombinant malaria
vaccines include the binding domains, PvRII and
PfF2, of the P. vivax and P. falciparum erythrocyte
binding proteins PvDBP and EBA-175, respectively,
and the C-terminal 19kD cysteine-rich fragment of
MSP-1 (PfMSP-119). Methods have been developed
to produce recombinant PvRII, PfF2 and PfMSP-119
in their correctly folded conformations by expression
in E. coli. Recombinant PvRII formulated in alum
and ASO2A is immunogenic in mice and monkeys.
Methods to produce recombinant PvRII have been
scaled up in collaboration with an industrial partner,
Bharat Biotech International Ltd. (BBIL), Hyderabad.
The safety and Phase I clinical trial studies are being
planned. Immunization with the combination of PfF2
and PfMSP-119 formulated in Montanide ISA 720
produce high titer invasion inhibitory antibodies
against both antigens with no interference in
immune responses or invasion inhibitory activity.
In addition to these lead candidates, efforts are
underway to develop recombinant vaccines based
on PfMSP-3, PfMSP-9 and PvMSP-119.
This study has been supported by the Prime
Ministers initiative on Jai Vigyan Mission on vaccine,
through the Department as well as the European
Malaria Vaccine Initiative (EMVI). The corresponding
vaccine for P. vivax is also being developed in
parallel with major financial support from the
Malaria Vaccine Initiative (MVI).
De novo design of antibiotic peptides: In the
programme of de novo design and chemical
synthesis of potent antibiotic peptides,
conformationally restrictive unnatural amino acid
such as a, b didehydrophenylalanine (DF) has been
used. The Group has delineated the contributions
of aromaticity, helicity and amphipathicity to the
potency of antibiotic effect. The group observed that
dendramers have much higher antibiotic activity
than monomeric peptides. The mechanisms of
action is being examined of these antibiotic peptides
by way of assays like minimum inhibitory
concentration, endotoxin (LPS) binding, inner and
outer membrane permeabilization, translocation
into the cell and proteomic identification of the
intracellular targets. Studies are also being
conducted on DPhe containing small self organising
peptides which can form nanotubes.
Virology: The virology group is 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 coronavirus. The structure-function
relationships between viral proteins as well as those
between viral and host proteins are being
investigated to understand viral pathogenesis, using
the molecular and genetic tools such as cloning and
expression of viral proteins, sequencing and
mutagenesis,protein-protein interaction
technologies, analysis of signal transduction
pathways, confocal microscopy, transgenic mouse
models, microarrays and mass spectrometry. In
another initiative the group is setting up assay
systems to characterize T cell memory and function
in HIV-infected persons and uninfected volunteers
given experimental HIV vaccines.
Immunology : The tuberculosis research program
has a focus on delineating interaction of
Mycobacterium tuberculosis (Mtb) secretory
antigens (MTSA) with the macrophages and
dendritic cells (DCs) using a 10-kDa antigen, as
the model protein. It is demonstrated that MTSA-
10 binds to macrophage surface and induces the
release of the proinflammatory cytokine TNF.
However, priming of macrophages with MTSA-10
also induced macrophage unresponsiveness to
subsequent induction of microbicidal free radical
nitric oxide (NO) by LPS or whole mycobacteria.
Furthermore, retroviral transduction of MTSA-10 in
J774 macrophages markedly reduced the latters
ability for NO synthesis, as well as constitutive
expression of the co-stimulatory molecule B7.1.
MTSA-10-pulsed dendritic cells failed to respond to
secondary antigenic challenge and down-regulated
T cell recall responses to mycobacterial proteins
suggesting that mycobacterial secretory proteins
may contribute to putatively protective as well as
deleterious immune functions.
Using proteomics approach, it was found that
recombinant MTSA-10 could induce J774.1 cells to
International Centre for Genetic Engineering and Biotechnology (ICGEB)
209
undergo major de-phosphorylation of the total
phospho-proteome. Furthermore, MTSA-10
stimulation activated the membrane-associated
phosphatases, which might be the cause for global
de-phosphorylation of macrophage proteins. The
results indicate that MTSA-10 plays an important
role in modulating the overall signaling process
by changing the cellular equilibrium of kinases
and phosphatases. Such molecular mechanisms
might give Mycobacterium an advantage over
macrophages not only to evade their own
destruction but also survive within the infected host
cell.
In another study various Mtb secretory proteins
were evaluated as vaccine candidates. The genes
encoding Mtb proteins viz. MTSA-10, CFP-21, ERP,
and Ag85B were cloned in DNA vaccine vectors.
These constructs induced reasonable levels of
Th1 kind of immune responses in mice. Further
studies on induction of protective responses in
mice have been carried out in collaboration
with PGIMER, Chandigarh and results are being
analysed.
Recombinant gene products laboratory: The
group focused primarily 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. A novel
method developed to generate artificial proteins
using codon-shuffled genes and is being explored
to make therapeutically useful designer proteins.
The current focus is on dengue and tuberculosis,
two major re-emerging infectious diseases of
tremendous global public health significance.
A cost-effective, simple and rapid diagnostic
test for dengue detection has been developed that
combines sensitivity and specificity. Two synthetic
protein antigens, one specific for immunoglobulin
G (IgG) and the other specific for IgM class of
dengue antibodies have been designed and
expressed. Preliminary evaluation of these two
recombinant multiepitope proteins against a
commercially available dengue diagnostic kit
(PanBio Australia), using a panel of dengue patient
sera, has yielded encouraging results. The strategy
of using recombinant multiepitope proteins
completely obviates multiple peptide synthesis and
avoids expensive and time-consuming virus culture
for antigen production.
Structural and computational biology : The
structural biology group aims at understanding the
structural principles that govern protein-based
biomolecular interactions. Structure-function studies
on malaria parasite proteins, are currently on-going.
Additionally, bioinformatic-based approaches are
being used to model three-dimensional structures of
a large number of parasite proteins
Plant Biotechnology
Plant molecular biology : The group is actively
involved in understanding the mechanisms of plant
adaptation in response to abitoic 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 the genes are being
identified that are regulated under stress as also
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 overexpressing glyoxase I and II genes
and DNA- RNA encoding and helicase gene and
vaculor sodium proton antiporter gene 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 : The group has continued its
efforts 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 analysed 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
DBT Annual Report 2005-06
210
orbanalis). These plants are being grown further in
the green house.
Plant transformation: The main focus of research
include use of plants as bioreactors to produce novel
compounds, high level foreign gene expression
through T7 RNA polymerase dependent
transcription, novel and improved methodology for
the transformation of crop plants that are difficult
by conventional methods, genetic engineering of
chloroplasts to increase crop productivity and
nutritional genomics. The target crop plants include
cotton, rice, tomato and sunflower.
Plant resistance : Main areas of interest are
identification and cloning of gall midge resistance
genes and to develop marker-assisted selection for
gall midge resistance genes with a view to pyramid
different gall midge resistance genes for durable
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
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 80 papers in
National and International peer reviewed journal
during the year 2005. Some major publications
are:
1. The Strength of Receptor Signalling is Centrally
Controlled through a Cooperative Loop between
Ca2+ and an Oxidant Signal. Cell. 121: 281-293.
(2005).
2. Structural basis for Duffy recognition by the
malaria parasite Duffy-binding-like domain. Nature.
2005 Dec 21.
3. Receptor binding residues lie in central regions
of Duffy- binding like domains involved in red cell
invasion. Blood. September 2004
4. Transcripation regulation from TATA and inr-less
promotor function. The EMBO journal. 2006
Technology Transfers and Patents
During the past year, the following technology
transfers were successfully accomplished.
No.Technology Transferred to
1 Multiepitope protein- J.Mitra & Company
based HCV detection New Delhi
2 E.coli clone expressing Zephyr
a tetravalent dengue Biomedicals Goa
multiepitope protein
No.Patent Filed
1.Salt tolerent tobacco India
2.Salt tolerent Rice India
3.Salt tolerent Chickpea India
4.Antibiotic potential India
5.Tetravalent dengue..India
antibodies
6.Development of salt India
tolerent plnats
7.Development of MYMIV India
based gene silencing
vectors
Training Activities
The centre conducted four training programmes
during the year with focus on plant transformation,
malaria virology, recombinant gene products and
bioinformatics. About 60 scientists from the member
countries and South East Asian region including
India attended the programmes.