|
Biology
of Japanese encephalitis virus |
| Principal Investigator :
Sudhanshu Vrati Project Associates
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.
Molecular determinants of JEV virulence This
project aims at identifying the genetic determinants of JEV virulence by
studying the genetic and biological phenotypes of naturally occurring isolates
of the virus that show marked change in their mouse virulence compared to the
virulent prototype JaOArS982 strain. An Indian isolate of JEV which shows
significantly altered biological properties in tissue cultured cells and mice
is being studied at the genetic level. The
construct made last year, where the viral genome segment was placed under the
control of the T7 promoter was transcribed in vitro. This did not
succeed and hence alternate constructs are being made with different
nucleotide sequence that forms the transcription start site within the cloned
DNA. Efficiency of RNA transcription from these constructs is to be tested.
Besides, we have now cloned the rest of the genome segments and are in the
process of assembling them to generate the full-length clone. B.
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. In
the studies described above, 100 mg DNA was injected
intra-muscular per mouse. We have examined alternate ways to deliver the DNA
so that we could cut down on the amount of DNA used per animal. We have used
chemical methods to prepare micro and nano particles of DNA. The DNA
microparticles were found to be very efficient; only 1 mg
of plasmid DNA per mouse injected intra-muscular was immunogenic. Studies on
DNA nanoparticles are in progress. In
order to enhance antibody response to a DNA based immunization, a protein
boost strategy has been found to be useful. We have also examined the
potential of DNA priming followed by protein booster in our studies. We have
synthesized JEV E protein in E. coli and used this for immunizations.
We found the DNA-protein boost to be superior to DNA-DNA, protein-protein, or
protein-DNA immunization regimen. Plasmid
constructs used in our studies above are based on a large plasmid pCEP4 of
about 10 kilo bases. During in vitro transfections, it is known that
smaller plasmids have better transfection efficiency. We have now constructed
a new plasmid synthesizing JEV protein and this plasmid has smaller vector
backbone. We will be comparing the immunogenicity in mice of the two plasmids,
one large and another small, synthesizing the same protein. Approval
for carrying out immunogenicity of the above DNA constructs in rhesus monkeys
has been obtained and currently efforts are underway to establish B-cell lines
from individual monkeys to enable us to do CTL assays following the DNA
immunizations. C.
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. We
have been able to upscale the microcarrier culture technique for JEV growth in
vero cells. Now we can grow 0.5 liter of virus in one spinner flask at a titer
of 107 pfu/ml or higher. Several
methods for purifying JEV from the tissue culture supernatant were tested.
Mice immunizations were carried out with tissue culture grown, formaline-inactivated
JEV to test the potential of this virus for vaccine preparation in a small
number of mice which indicated to the potential of the tissue cultured virus
for use in immunization against JEV. D.
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. Full
length clone of the 80-kDa protein has been obtained from the mouse brain RNA
and the cDNA encoding the 80-kDa protein was cloned in an E. coli
expression vector pVex which contains the T7 promoter for the foreign gene
expression. Our extensive efforts to synthesize the 80-kDa protein in this
system have not been successful. The 80-kDa protein cDNA has now been placed
under the control of the T5 promoter in vector pQE30 that should enable us to
synthesize histidine-tagged 80-kDa protein in E. coli. In
an effort to purify and identify the proteins in the neonatal mouse brain cell
lysate that bind to JEV RNA, we have begun to fractionate the proteins
contained in the lysate by ion-exchange chromatography and gel filtration.
These processes have resulted in partial purification of the proteins binding
to JEV RNA. Efforts to purify the proteins further are in progress. E. 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. Fusions
of four peptide sequences chosen from JEV E protein were made to JGMV CP using
the recombinant DNA means. The over-expressed fusion protein made in E.
coli was purified and was shown to form virus-like-particles (VLPs). The
same JEV peptide sequences were chemically synthesized and conjugated to BSA.
Mice were immunized with BSA-conjugated-peptide preparations (using CFA) or
with the VLPs presenting the JEV peptides (without the use of any adjuvant).
It was found that the VLPs induced vigorous anti-peptide and anti-JEV
antibodies and the titers were significantly higher than those obtained with
BSA-conjugated-peptide preparation. This indicated to a very high potential of
JGMV VLPs for peptide presentation. A 27 amino acid sequence from JEV E
protein presented on JGMV VLPs was shown to induce JEV neutralizing
antibodies. Publications Original
peer-reviewed articles 1. Kaur
R, Sachdeva G and Vrati S (2002) Plasmid DNA immunization against Japanese
encephalitis virus: Immunogenicity of membrane-anchored and secretory envelope
protein. J Infect Dis 185:1-12. |