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Biology of Japanese encephalitis virus |
| Principal Investigator : Sudhanshu Vrati
Project Associates
/ Assistants Ph D Students Japanese
encephalitis virus (JEV) is a member of the flaviviridae family of animal
viruses that contains several other medically important viruses such as Dengue
and Yellow fever. JEV is a major cause of human encephalitis and is
responsible for considerable mortality and morbidity in India. Frequent
epidemics of JEV are being reported from various parts of India and the virus
has become endemic in several parts of the country. Different aspects of JEV
biology are being studied in our laboratory, some of which are outlined below. A. Development of naked DNA based vaccine for JEV The
objective of this work is to develop naked DNA based immunization procedures
for vaccination against JEV. To this end, we intend to make recombinant
plasmids carrying various structural and non-structural protein genes of JEV
under the control of a strong eukaryotic promoter. These plasmid constructs
will be evaluated for their potential to generate protective immunity in
experimental animals against JEV. Plasmid
constructs used in our earlier studies are based on a large plasmid pCEP4 of
about 10 kilo bases (KB). During in vitro transfections, it is known that
smaller plasmids have better transfection efficiency. We have now constructed
a pcDNA vector (about 5 KB) based expression plasmid for JEV proteins. This
vector synthesized JEV proteins in vitro as efficiently as the previous
construct. We are now comparing the immunogenicity in mice of the two plasmids. For
carrying out the immunogenicity of the above DNA constructs in rhesus monkeys,
we have established B-cell lines from at least two monkeys so far. This would
enable us to do CTL assays following the DNA immunizations. More
transformations are currently in progress to establish more B-cell lines. Plasmid
constructs have been made that should synthesize the non-structural NS1
protein besides the structural E and the M proteins of JEV. Proteins
synthesized by these plasmids are being characterized by in vitro transfection
of tissue cultured cells. B. Development of a tissue culture derived vaccine against JEV A
mouse brain derived vaccine using the Japanese Nakayama strain of JEV is
currently being produced at the Central Research Institute, Kasauli. However,
the cost of this vaccine precludes its large-scale usage for mass vaccination
as may be required for the effective control of the virus activity in field.
In order to bring down the cost of vaccine production, efforts are being made
to grow the virus to high titers in tissue cultured cells. Mice
immunizations were carried out with tissue culture grown, formaline-inactivated
JEV to test the potential of this virus for vaccine preparation in mice.
Antibodies generated in mice neutralized the Indian (Vellore P20778) and the
Japanese (JaOAr) strains of JEV, albeit with different titers. Immunized mice
showed complete protection against the lethal JEV challenge. C. Interaction of cellular proteins with JEV RNA JEV
genome is a plus-sense single-stranded RNA of ~11 KB. A minus-sense RNA
template is generated during virus replication, which is then copied to
produce lots of plus-sense genomic RNA molecules. Based on the amino acid
sequence homologies with other replicases, a couple of viral proteins have
been speculated to be involved in replication of the JEV genome. However, we
do not know if any of the cellular proteins also are needed for viral
replication. We are, therefore, studying cellular proteins that interact with
JEV genome sequences, which are likely to be involved in viral replication. An
80-kDa protein identified as SV2A was found to interact with JEV RNA. The
protein was synthesized in E.coli and the lysate was used to demonstrate the
JEV NCR RNA interaction with SV2A. Antibodies to SV2A peptide were made and
these are to be used for further confirming the SV2A binding to JEV genome. In
an alternate effort to purify and identify the proteins in the neonatal mouse
brain cell lysate that bind to JEV RNA, we fractionated the proteins contained
in the lysate by the classical protein purification methods. These have
resulted in partial purification of the proteins binding to JEV RNA. Efforts
to purify the proteins further are in progress. D. Use of a self assembling plant virus protein for presenting antigenic peptides from Japanese encephalitis virus Peptide
vaccines have a number of potential advantages in terms of production, safety
and stability. However, peptides are poorly immunogenic on their own and hence
need to be presented with strong adjuvants such as Freund’s adjuvant. Such
adjuvants are not permissible for use in humans and so there is need to
identify newer adjuvants that may be safe and effective. Studies have shown
that presentation of peptides in a highly ordered aggregate form can result in
enhanced immune response. We are, therefore, investigating the use of a
self-polymerizing coat protein from Johnson grass mosaic virus (JGMV) for
presenting antigenic peptides from JEV. Mice
immunized with VLPs presenting a 27-amino acid sequence from the JEV E protein
generated low titers of JEV neutralizing antibodies. Immunized mice showed
about 60% protection against the lethal JEV challenge. We have now prepared
VLPs presenting over 10 different JEV peptides. Mice have been immunized with
these recombinant VLPs and their immunogenicity results are awaited. E. Use of adenovirus recombinant synthesizing Japanese encephalitis virus E protein for immunization against Japanese encephalitis: Human
adenoviruses cause mild, benign infections and in recent years these viruses
are increasingly being used for human gene therapy and recombinant vaccine
development. From the vaccine point of view, the added advantage that
adenoviruses present is that they can be delivered orally. We are developing
adenovirus recombinants that synthesise JEV E protein as potential vaccine
candidate for immunization against Japanese encephalitis. The
cDNA encoding JEV E protein together with the prM protein was placed under the
CMV promoter and this expression cassette was transferred to adenovirus
shuttle vector. The JEV sequence was then inserted into the E1 transcription
unit of human adenovirus 5. Recombinant adenovirus synthesizing JEV proteins
have been produced in our laboratory and grown in bulk for immunization of
mice. Publications Original
peer-reviewed articles 1.
Saini M and Vrati S (2003) A Japanese encephalitis virus peptide present on
Johnson grass mosaic virus-like particles induces virus-neutralizing
antibodies and protects mice against lethal challenge. J Virol 77:3487-3494. 2.
Saini M and Vrati S (2003) High level expression of Johnson grass mosaic virus
coat protein in E.coli and its auto-assembly to form virus-like particles. Protein
Exp Purif 28:86-92. 3.
Rath A, Batra D, Kaur R, Vrati S and Gupta SK (2003) Characterization of
immune response in mice to plasmid DNA encoding dog zona pellucida
glycoprotein-3. Vaccine (in press). 4.
Kaur R and Vrati S (2003) Development of recombinant vaccine against Japanese
encephalitis. J Neurovirol (in press). |