Note: Descriptions are shown in the official language in which they were submitted.
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NEW DENGUE AND VIiEST NILE VIRUSES PROTEINS AND GENES
CODING THE FOREGOING, AND THEIR USE IN VACCINAL, THERAPEUTIC
AND DIAGNOSTIC APPLICATIONS
FIELD OF THE INVENTION
The present invention relates to West-Niie virus (WNV) andlor Dengue virus
derived peptides, and more particularly to polypeptides or polynucleotides
derived
from WNV andlor Dengue virus polypeptides or polynucleotides and their use in
the preparation of compositions and vaccines. More specifically, the present
invention is concerned with compositions, vaccines and methods for providing
an
immune response andlor a protective immunity to animals against a West-Nile
virus or a Dengue virus and methods for the diagnosis of West-Nile virus or
Dengue virus infection.
BACKGROUND OF THE INVENTION
Flaviviridae are arboviruses (arthropod-borne virus) mainly transported by
mosquitoes and blood-sucking ticks. They are small encapsidated viruses and
their genomes consist of infectious single-stranded and linear RNA of positive
polarity. In Man, flaviviruses cause deadly hemorrhagic fever or meningo-
encephalitis. Yellow fever, dengue fever and Japanese encephalitis are the
main
tropical flaviviroses. Other important human flaviviroses are Saint Louis
encephalitis, tick-born European encephalitis and West Nile fever.
West Nile fever is a zoonosis associated with a flavivirus which was first
isolated in Uganda in 1937. Its transmission cycle calls for birds as the main
reservoir and for blood sucking mosquitoes of the Culex genus as vectors.
Migratory viremic birds transport the virus to far-away regions where they
transmit
it anew to ornithophile mosquitoes of the Culex genus. Many species of mammals
are permissive for the West Nile virus. Horses are particularly sensitive to
the
disease but do not participate in the cycle of transmission. West Nile fever
is
endemic in Africa, Asia, Europe and Australia. Phylogenic studies have
revealed
the existence of two strains of viruses : viral line 1 has a worldwide
distribution,
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and viral line 2 is essentially African. Viral line 1 was responsible for
enzooties in
Romania (1996), Russia (1999), Israel (1998-2000) and more recently in North ,
America where the virus had never been detected before 1999. The viral strains
isolated during the recent epidemics in Israel and the United-States are more
than
99,7 % identical. In the IVliddle-East and North America, where the virus has
taken
root, an important bird mortality rate has been observed among infected birds,
notably in Corvidae. In North America, over 4000 subjects were infected with
the
West Nile virus, 250 of which died between the months of August and December
2002. At the present time, zoonosis is observed in all regions of the United
States.
At the moment, there exists no human vaccine or specific therapy against West
Nile fever.
In temperate and sub-tropics( regions, human infections may occur during
the fall season. When a subject is bitten by an infected mosquito, the
incubation
period lasts approximately one week but less than 20 % of people infected with
the West Nile virus ever go on to clinical manifestations. In its benignant
form, the
viral infection manifests itself by an undifferentiated febrile state
associated with
muscular weakness, headaches and abdominal pain. In less than 1 % of infected
subjects, encephalitis or acute aseptic meningitis may occur. Splenomegaly,
hepatitis, pancreatitis and myocarditis are also observed. Flask paralyses
similar
to a poliomyelitis syndrome have recently been reported, but fatal cases of
viral
encephalitis (5% of patients having severe neurological disorders} mainly
concern
fragile subjects and the aged. Inter-human transmission of the virus has also
recently been observed in the United-States in subjects having undergone organ
transplants or having been perfused with contaminated blood products. Intra-
uterine transmission of the virus has been reported in the United-States. The
development of a human vaccine against the West Nile fever is a priority in
view of
the fact that the zoonosis has taken root in Nortih America and is expected to
propagate in the coming months to Central America, South America and the
Caribbean where dengue fever and yellow fever are already rampant.
Therefore, there is a need for West-Nile virus (WNV) andlor Dengue virus
derived peptides, and more particularly to pofypeptides or polynucleotides
derived
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from WNV and/or Dengue virus polypeptides or polynucieotides and their use in
the preparation of compositions and vaccines..
The present invention fulfils these needs and also other needs which will be
apparent to those skilled in the art upon reading the following specification:
SUMMAIRY CAF THE IN1lENTI~N
The present invention relates to West-Nile virus andlor Dengue virus
derived polypeptides.
More specifically, one object of the invention concerns a purified
polypeptide wherein it derives from a West-Nile virus antigen or a Dengue
virus
antigen.
Another object of the invention concerns a purified polyclonal or monoclonal
antibody capable of specifically binding to a polypeptide of the invention.
Another object of the invention concerns a purified polynucleotide sequence
coding for the polypeptide of the invention and its use for detecting the
presence
or absence of a West-Nile virus antigen or a Dengue virus antigen in a
biological
sample.
A further object of the invention concerns a recombinant viral vector which
is a recombinant virus comprising a polynucleotide sequence of the invention.
Another object of the invention is a recombinant measles virus capable of
expressing a polypeptide of the invention or comprising, in its genome, a
polynucleotide of the invention.
Yet, another object of the invention relates to a pharmaceutical composition
comprising:
a) at feast one component selected from the group consisting of:
- a polypeptide of the invention or a functional derivative thereof;
- an antibody as defined above;
- an expression vector as defined above;
- a pofynucleotide of the invention or a fragment thereof,
- a recombinant viral vector of the invention; and
- a recombinant measles virus of the invention;
and
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b) a pharmaceutically acceptable vehicle or carrier.
Another object of the invention concerns the use of the pharmaceutical
composition of the invention, as an anti-West-Nile virus and/or an anti-Dengue
virus agent, or for the preparation of an anti-West-Nile virus andlor an anti-
Dengue
virus vaccine.
Another object of the invention relates to a host cell incorporating an
expression vector as defined above or a recombinant viral vector as defined
above.
Furthermore, another object of the inventin concerns a method of producing
a recombinant virus for the preparation of an anti-West--Nile virus vaccine or
an
anti-Dengue virus vaccine, the method comprising the steps of:
a) providing a host cell as defined above;
b) placing the host cell from step a) in conditions permitting the replication
of a recombinant virus capable of expressing a polypeptide of the
invention; and
c) isolating the recombinant virus produced in step b).
Another object of the invention concerns a West-Nile virus neutralization
assay, comprising the steps of
a) contacting VERO cells with West-Nile virus and an antibody;
b) culturing said VERO cells under conditions which allow for West-Nile
virus replication; and
c) measuring reduction of West-Nile virus replication foci on said VERO
cells.
A further object of the invention is to provide a method for treating andlor
preventing a WNV- or Dengue virus- associated disease or infection in an
animal,
the method comprising the step of administering to the animal an effective
amount
of at least one element selected from the group consisting of
- a polypeptide or a functional derivative thereof as defined above;
- an antibody as defined above;
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- an expression vector as defined above;
- a polynucleotide or a fragment thereof as defined above;
- a recombinant viral vector as defined above; and
- a recombinant measles virus as defined above.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the nucleic acid sequence encoding the secreted
glycoprotein E from WNV and identified as SEQ 1D NO. 1.
Figure 2 shows the amino acid sequence of the secreted glycoprotein E
from WNV and identified as SEQ ID NO 5.
Figure 3 shows the nucleic acid sequence encoding the preM plus E
glycoproteins from WNV and identified as SEQ ID NO. 2.
Figure 4 shows the amino acid sequence of the preM plus E glycoproteins
from WNV and identified as SEQ ID NO 6.
Figure 5 shows the nucleic acid sequence encoding the NS1 protein from
WNV and identified as SEQ ID NO. 3.
Figure 6 shows the amino acid sequence of the NS1 protein from WNV and
identified as SEQ ID NO 7.
Figure 7 shows the nucleic acid sequence encoding the preM-E gene from
Dengue type 1 virus and identified as SEQ ID NO. 4.
Figure 8 shows the amino acid sequence of the preM-E gene from Dengue
type 1 virus and identified as SEQ ID NO 8.
Figure 9 is a schematic map of the pTM-MVSchw recombinant plasmids
according to preferred embodiments of the invention.
Figure 10 shows the expression of sEWNV by MVSchw-sEWNV recombinant
MV in Vero cells. (A) Schematic diagram of MVschw-sEWNV and virus growth. The
IS-98-ST1 cDNA coding for sEW~v was inserted into the Schwarz MV genome
between the BsiW~ and BssHll sites of the ATU at position 2. The MV genes are
indicated: N (nucleoprotein), PVC (phosphoprotein and V, C proteins), M
(matrix),
F (fusion), H (hemagglutinin), L (polymerise). T7 : T7 RNA polymerise
promoter;
hh : hammerhead ribozyme, T7t : T7 RNA polymerise terminator; ~ : hepatitis
delta virus (HDV) ribozyme; ATU : additional transcription unit. (B) Growth
curves
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of MV. Vero cells were infected with MVschw (open box) or MVschw-sEw~v {black
box} at a multiplicity of infection (m.o.i) of 0.01 TCIDSO/cell. At various
times post-
infection, infectious virus particles were titered as described in the
Methods. (C)
Immunofluorescence staining of sEwNV glycoprotein in syncitia of MVschw-sEWNV-
infected Vero cells fixed 30 h post-infection. Cells were permeabilized (A, B)
or not
(C, D) with Triton X-100 and then immunostained using anti-WNV HMAF.
Magnification : x 1000. No positive signal was observed in MVschw-infected
cells.
(D) Radioimmunoprecipitation (RIP) assay showing the release of SEwN~ from
MVschw-sEWN~-infected cells. Vero cells were infected with WNV strain !S-98-
ST1
(m.o.i of 5) for 24 h, MVschw (m.o.i. of 0.1 ), MVschw-sEWNV (m.o.i. of 0.1 )
for 40 h,
or mock-infected (MI}. Radioiabeled supernatants and cell lysates were
immunoprecipitated with specific anti-MV (a-MV) or anti-WNV {a-WNV) polyclonal
antibodies. WNV E glycoprotein (open arrow head) and sEWNV (black arrow head)
are shown.
Figure 11 shows anti-MVSchw-sEWNV antibodies recognizing the WNV E
glycoprotein. Vero cells were infected with WNV strain IS-98-ST1 (WNV) or mock-
infected (No virus). Labeled cell lysates were immunoprecipitated with pooled
immune sera (dilution 1:100} from mice inoculated with WNV, MVSchw, MVSchw-
sEWN" as described in the legend to Fig. 10D. Specific anti-
lymphochoriomeningitis
virus (LCMV) antibodies were used as a negative control. WNV structural
glycoproteins prM and E and non structural proteins NS3, NSS, NS2A and NS2B
are shown. p.c., post-challenge.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to West-Nile virus (WNV) andlor Dengue virus
derived peptides, and more particularly to polypeptides or polynucleotides
derived
from WNV andlor Dengue virus polypeptides or polynucleotides and their use in
the preparation of compositions and vaccines. More specifically, the present
invention is concerned with compositions, vaccines and methods for providing
an
immune response andlor a protective immunity to animals against a West-Nile
virus or a Dengue virus and methods for the diagnosis of West-Nile virus or
Dengue virus infection.
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As used herein, the term "immune response" refers to the T cell response or
the increased serum levels of antibodies to an antigen, or presence of
neutralizing
antibodies to an antigen, such as a WNV or a Dengue virus antigen. The term
"immune response" is to be understood as including a humoral response andlor a
cellular response and/or an inflammatory response.
An "antigen" refers to a molecule, such as a protein or a polypeptide,
containing one or more epitopes that will stimulate a host's immune system to
make a humoral andlor cellular antigen-specific response. The term is also
used
interchangeably with "immunogen".
The term °'protection°' or '°protective immunity" refers
herein to the ability of
the serum antibodies and ceiluiar response induced during immunization to
protect
(partially or totally) against against a West-Niie virus or a Dengue virus.
Thus, an
animal immunized by the compositions or vaccines of the invention will
experience
limited growth and spread of an infectious WNV or Dengue virus.
As used herein, the term "animal" refers to any animal that is susceptible to
be infected by a West-Nile virus or a Dengue virus. Among the animals which
are
known to be potentially infected by these viruses, there are, but not limited
to,
humans, birds and horses.
1. Potynucleotides and polypeptides
In a first embodiment, the present invention concerns a purified polypeptide
characterized in that it derives from a West-Nile virus antigen or a Dengue
virus
antigen or functional derivative thereof. As can be appreciated, a
protein/peptide is
said to "derive" from a protein/peptide or from a fragment thereof when such
proteinlpeptide comprises at least one portion, substantially similar in its
sequence, to the native prateinlpeptide or to a fragment thereof.
The West-Nile virus antigen of the present invention is preferably selected
from the group consisting of secreted envelope glycoprotein (E), heterodimer
glycoproteins (PreM-E) and NS1 protein. More specifically, the secreted
envelope
gfycoprotein (E) comprises the sequence of SEQ BD NO: 5 or a functional
derivative thereof, the heterodimer glycoproteins (PreM-E) comprises the
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sequence of SEQ ID NO: 6 or a functional derivative thereof, and the NS1
protein
comprises the sequence of SEQ ID NO: 7 or a functional derivative thereof.
The Dengue virus antigen of the invention is preferably selected from the
group consisting of secreted envelope gfycoprotein (E), heterodimer
glycoproteins
(PreM-E) and NS1 protein. More specifically, the heterodimer glycoproteins
(PreM-
E) comprises the sequence of SEQ ID NO: 8 or a functional derivative thereof.
Accoding to a preferred embodiment, the polypeptide of the present
invention has an amino acid sequence having at least 80% homology, or even
preferably 85% homology to part or all of SEQ ID NO:1, of SEQ ID N0:2, of SEQ
ID N0:3 or of SEO ID N0:4.
A "functional derivative", as is generally understood and used herein, refers
to a proteinlpeptide sequence that possesses a functional biological activity
that is
substantially similar to the biological activity of the whole protein/peptide
sequence. In other words, it refers to a polypeptide or fragments) thereof
that
substantially retain the same biological functions as the polypeptide of SEQ
ID
Nos: 5 to 8. A functional derivative of a proteinlpeptide may or may not
contain
post-translational modifications such as covalently finked carbohydrate, if
such
modification is not necessary for the performance of a specific function. The
term
"functions! derivative" is intended to the "fragments", "segments",
"variants",
"analogs" or "chemical derivatives" of a proteir~lpeptide. As used herein, a
protein/peptide is said to be a "chemical derivative" of another
proteinlpeptide
when it contains additional chemical moieties not normally parf of the
protein/peptide, said moieties being added by using techniques well known in
the
art. Such moieties may improve the protein/peptide solubility, absorption,
bioavailability, biological half life, and the like. Any undesirable toxicity
and side-
effects of the protein/peptide may be attenuated and even eliminated by using
such moieties.
Yet, more preferably, the polypeptide comprises an amino acid sequence
substantially the same or having 100% identify with SEQ ID N0:1, SEQ 1D N0:2,
SEQ ID N0:3, or SEQ ID N0:4.
One can use a program such as the CLUSTAL program to compare amino
acid sequences. This program compares amino acid sequences and finds the
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optimal alignment by inserting spaces in either sequence as appropriate. It is
possible to calculate amino acid identity or homology for an optimal
alignment. A
program like BLASTx will align the longest stretch of similar sequences and
assign
a value to the fit. It is thus possible to obtain a comparison where several
regions
of similarity are found, each having a differenfi score. Both types of
identity
analysis are contemplated in the present invention.
As used herein, the term "polypeptide(s)" refers to any peptide or protein
comprising two or more amino acids joined to each other by peptide bonds or
modified peptide bonds. °'Polypeptide(s)" refers to both short chains,
commonly
referred to as peptides, oligopeptides and oligomers and to longer chains
generally referred to as proteins. Polypeptides may contain amino acids other
than
the 20 gene-encoded amino acids. '°Polypeptide(s)'° include
those modified either
by natural processes, such as processing and other post-translational
modifications, but also by chemical modification techniques. Such
modifications
are well described in basic texts and in more detailed monographs, as well as
in a
voluminous research literature, and they are well known to those of skill in
the art.
It wilt be appreciated that the same type of modificatiorl may be present in
the
same or varying degree at several sites in a given polypeptide. Also, a given
polypeptide may contain many types of modifications. Modifications can occur
anywhere in a polypeptide, including the peptide backbone, the amino acid side-
chains, and the amino or carboxyl termini. Modifications include, for example,
acetylation, acylation, ADP-ribosyiation, amidation, covalent attachment of
flavin,
covalent attachment of a heme moiety, covalent attachment of a nucleotide or
nucleotide derivative, covalent attachment of a lipid or lipid derivative,
covalent
attachment of phosphotidyfinositol, cross-linking, cyclization, disulfide bond
formation, demethylation, formation of cysteine, formation of pyroglutamate,
formylation, gamma-carboxylation, GPI anchor formation, hydroxylation,
iodination, methylation, myristoylation, oxidation, proteolytic processing,
phosphorylation, prenylation, racemization, glycosylation, lipid attachment,
sulfation, gamma-carboxylation of giutamic acid residues, hydroxylation,
selenoylation, sulfation and transfer-RNA mediated addition of amino acids to
proteins, such as arginylation, and ubiquitination. See, for instance:
PROTEINS--
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STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W.H.
Freeman and Company, New York (1993); Wold, F., Posttransiational Protein
Modifications.: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL
COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic
5 Press, New York {1983); Seifter et ai., Meth. Enzymol. '182:626-646 (1990);
and
Rattan et al., Protein Synthesis: Posttransfational Modifications and Aging,
Ann.
N.Y. Acad. Sci. 663: 48-62(1992). Polypeptides may be branched or cyclic, with
or
without branching. Cyclic, branched and branched circular polypeptides may
result
from post-translational natural processes and may be made by entirely
synthetic
10 methods, as well.
With respect to protein or polypeptide, the term "isolated polypeptide" or
"isolated and purified polypeptide" is sometimes used herein. This term refers
primarily to a protein produced by expression of an isolated polynucleotide
molecule contemplated by invention. Alternatively, this term may refer to a
protein
which has been sufficiently separated from other proteins with which it would
naturally be associated, so as to exist in "substantially pure" form.
The term "substantially pure" refers to a preparation comprising at least
50-60% by weight the compound of interest (e. g., nucleic acid,
oligonucleotide,
protein, etc.). More preferably, the preparation comprises at least 75% by
weight,
and most preferably 90-99% by weight, the compound of interest.
Purity is measured by methods appropriate for the compound of interest
(e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis,
HPLC analysis, and the like).
In a second embodiment, the present invention concerns a purified
polynucleotide encoding a polypeptide of the invention. Therefore, the
polynucleotide of the invention has a nucleic acid sequence which is at least
65%
identical, more particularly 80% identical and even more particularly 95%
identical
to part or all of any one of SEQ ID NO 5 to 8 or functional fragments thereof.
A "functional fragment", as is generally understood and used herein, refers
to a nucleic acid sequence that encodes for a functional biological activity
that is
substantially similar to the biological activity of the whole nucleic acid
sequence. In
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other words, it refers to a nucleic acid or fragments) thereof that
substantially
retains the capacity of encoding for a polypeptide of the invention.
The term "fragment" as used herein refer to a polynucleotide sequence
(e.g., cDNA) which is an isolated portion of the subject nucleic acid
constructed
artificially (e.g., by chemical synthesis) or by cleaving a natural product
into
multiple pieces, using restriction endonucleases or mechanical shearing, or a
portion of a nucleic acid synthesized by PCR, DNA polymerase or any other
polymerizing technique well known in the art, or expressed in a host cell by
recombinant nucleic acid technology well known to one of skill in the art.
With reference to polynucleotides of the invention, the term "isolated
polynucleotide" is sometimes used. This term, when applied to DNA, refers to a
DNA molecule that is separated from sequences with which it is immediately
contiguous (in the 5' and 3' directions) in the naturally occurring genome of
the
organism from which it was derived. For example, the "isolated polynucleotide"
may comprise a DNA molecule inserted into a vector, such as a piasmid or virus
vector, or integrated into the genomic DNA of a procaryote or eucaryote. An
"isolated polynucleotide molecule°' may also comprise a cDNA molecule.
Amino acid or nucleotide sequence "identity" and "similarity" are determined
from an optimal global alignment between the two sequences being compared. An
optimal global alignment is achieved using, for example, the Needleman-Wunsch
algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453). "Identity"
means that an amino acid or nucleotide at a particular position in a first
polypeptide or polynucleotide is identical to a corresponding amino acid or
nucleotide in a second polypeptide or polynucleotide that is in an optimal
global
alignment with the first polypeptide or polynucleotide. In contrast to
identity,
"similarity" encompasses amino acids that are conservative substitutions. A
"conservative" substitution is any substitution that has a positive score in
the
blosum62 substitution matrix (Henfikoff and Hentikoff, 1992, Proc. Natl. Acad.
Sci.
USA 89: 10915-10919). By the statement "sequence A is n% similar to sequence
B" is meant that n% of the positions of an optimal global alignment between
sequences A and B consists of identical residues or nucleotides and
conservative
substitutions. By the statement "sequence A is n% identical to sequence B" is
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meant that n% of the positions of an optimal global alignment between
sequences
A and B consists of identical residues or nucleotides.
As used herein, the term '°polynucleotide(s)" generally refers to
any
polyribonucleotide or poly-deoxyribonucleotide, which may be unmodified RNA or
DNA or modified RNA or DNA. This definition includes, vvithout limitation,
single
and double-stranded DNA, DNA that is a mixture of single- and double-stranded
regions or single-, double- and triple-stranded regions, cDNA, single- and
double-
stranded RNA, and RNA that is mixture of single- and double-stranded regions,
hybrid molecules comprising DNA and RNA that may be single-stranded or, more
typically, double-stranded, or triple-stranded regions, or a mixture of single-
and
double-stranded regions. In addition, "polynucleotide" as used herein refers
to
triple-stranded regions comprising RNA or DNA or both RNA and DNA. The
strands in such regions may be from the same molecule or from different
molecules. The regions may include all of one or more of the molecules, but
more
typically involve only a region of some of the molecules. One of the molecules
of a
triple-helical region often is an oligonucleotide. As used herein, the term
"polynucieotide(s)" also includes DNAs or RNAs as described above that contain
one or more modified bases. Thus, DNAs or RNAs with backbones modified for
stability or for other reasons are "polynucleotide(s)'° as that term is
intended
herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or
modified bases, such as tritylated bases, to name just two examples, are
polynucleotides as the term is used herein. It will be appreciated that a
great
variety of modifications have been made to DNA and RNA that serve many useful
purposes known to those of skill in the art. "Polynucleotide(s)" embraces
short
polynucleotides or fragments comprising at least 6 nucleotides often referred
to as
oligonucleotide(s). The term '°pofynucleotide(s)" as it is employed
herein thus
embraces such chemically, enzymatically or metabolically modified forms of
polynucleotides, as weft as the chemical forms of DNA and RNA characteristic
of
viruses and cells, including, for example, simple and complex cells which
exhibits
the same biological function as the polypeptide encoded by any one of SEQ ID
NOS.1 to 4. The term "polynucleotide(s)" also embraces short nucleotides or
CA 02456873 2004-02-26
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fragments, often referred to as "oligonucleotides", that due to mutagenesis
are not
100% identical but nevertheless code for the same amino acid sequence.
2. Vectors and Cells
In a third embodiment, the invention is also directed to a host, such as a
genetically modified cell, comprising any of the polynucleotide sequence
according
to the invention and more preferably, a host capable of expressing the
polypeptide
encoded by this pofynucleotide. Even more preferably, the present invention is
concerned with a host cell that incorporates an expression vector or a
recombinant
viral vector as defined herein below.
The host cell may be any type of cell (a transiently-transfected mammalian
cell line, an isolated primary cell, or insect cell, yeast (Saccharomyces
cerevisiae,
Ktuyveromyces lactis, Pichia pastoris), plant cell, rnicroorganism, or a
bacterium
(such as E. coli). The following biological deposit relating to MEF/3T3.Tet-
Off/prME.WN # h2 cell line comprising an expression vector encoding for pseudo-
particles of WNV strain IS-98-ST1 composed of prME complexed glycoproteins
was registered at the Collection Nationale des Cultures de Microorganismes
(CNCM) under accession numbers l-3018 on May 2, 2003.
In a fourth embodiment, the invention is further directed to cloning or
expression vector comprising a polynucleotide sequence as defined above.
As used herein, the term "vector" refers to a polynucleotide construct
designed for transduction/transfection of one or more cell types. Vectors may
be,
for example, "cloning vectors" which are designed for isolation, propagation
and
replication of inserted nucleotides, "expression vectors" which are designed
for
expression of a nucleotide sequence in a host cell, or a '°viral
vector" which is
designed to result in the production of a recombinant virus or virus-like
particle, or
"shuttle vectors", which comprise the attributes of more than one type of
vector.
A number of vectors suitable for stable transfection of cells and bacteria are
available to the public (e.g. plasmids, adenoviruses, baculoviruses, yeast
baculoviruses, plant viruses, adeno-associated viruses, retroviruses, Herpes
Simplex Viruses, Alphaviruses, Lentiviruses}, as are methods for constructing
CA 02456873 2004-02-26
14
such cell lines. It will be understood that the present invention encompasses
any
type of vector comprising any of the polynucleotide molecule of the invention.
According to a preferred embodiment, the vector is a recombinant viral
vector which is a recombinant virus comprising a polynucleotide sequence as
defined above. Preferably the recombinant virus is a five attenuated virus or
a
defective virus, such as a recombinant virus selected from the group
consisting of
measles virus, hepatitis B virus, human papillomavirus, picornaviridae and
lentivirus. More preferably, the recombinant virus is a recombinant measles
virus,
for instance the Schwarz measles virus strain, which is capable of expressing
a
polypeptide as defined above or comprises, in its genome, a poiynucleotide as
defined above.
3. Antibodies
In a fifth embodiment, the invention features purified antibodies that
specifically bind to the isolated or purified polypeptide as defined above or
fragments thereof. The antibodies of the invention may be prepared by a
variety of
methods using the polypeptides described above. For example, the West-Nile or
Dengue virus antigen, or antigenic fragments thereof, may be administered to
an
animal in order to induce the production of polyclonal antibodies.
Alternatively,
antibodies used as described herein may be monoclonal antibodies, which are
prepared using hybridoma technology (see, e.g., Hammerling ef al., In
Monoclonal
Antibodies and T-Cefl Hybridomas, Elsevier, NY, 191 ).
As mentioned above, the present invention is preferably directed to
antibodies that specifically bind to a West-Nile antigen or a Dengue virus
antigen,
or fragments thereof. In particular, the invention features "neutralizing"
antibodies.
By "neutralizing" antibodies is meant antibodies that interfere with any of
the
biological activities of any of the WNV antigen or Dengue virus antigen. Any
standard assay known to one skilled in the art may be used to assess
potentially
neutralizing antibodies. ~nce produced, monoclonal and polyclonal antibodies
are
preferably tested for specific WNV or Dengue virus proteins recognition by
Western blot, immunoprecipitation analysis or any other suitable method.
CA 02456873 2004-02-26
Antibodies that recognize WNV or Dengue virus proteins expressing cells
and antibodies that specifically recognize WNV or Dengue virus proteins (or
functional fragments thereof), such as those described herein, are considered
useful to the invention. Such an antibody may be used in any standard
5 immunodetection method for the detection, quantification, and purification
of WNV
or Dengue virus proteins. The antibody may be a monoclonal or a polyclonal
antibody and may be modified for diagnostic purposes. The antibodies of the
invention may, for example, be used in an immunoassay to monitor WNV or
Dengue virus proteins expression levels, to determine the amount of WNV or
10 Dengue virus profieins or fragment thereof in a biological sample and
evaluate the
presence or not of a WNV or Dengue virus. fn addition, the antibodies may be
coupled to compounds for diagnostic and/or therapeutic uses such as gold
particles, alkaline phosphatase, peroxidase for imaging and therapy. The
antibodies may also be labeled (e.g. immunof(uorescence) for easier detection.
15 With respect to antibodies of the invention, the term °'specifically
binds to"
refers to antibodies that bind with a relatively high affinity to one or more
epitopes
of a protein of interest, but which do not substantiaily recognize and bind
molecules other than the ones) of interest. As used herein, the term
"relatively
high affinity" means a binding affinity between the antibody and the protein
of
interest of at least 106 M-', and preferably of at least about 10' M-' and
even more
preferably 10$ M-~ to 10'° M-'. Determination of such affinity is
preferably
conducted under standard competitive binding immunoassay conditions which is
common knowledge to one skilled in the art. As used herein, '°antibody"
and
"antibodies" include all of the possibilities mentioned hereinafter:
antibodies or
fragments thereof obtained by purification, proteolytic treatment or by
genetic
engineering, artificial constructs comprising antibodies or fragments thereof
and
artificial constructs designed to mimic the binding of antibodies or fragments
thereof. Such antibodies are discussed in Colcher et al. (Q J Nucl Med 1998;
42:
225-241 ). They include complete antibodies, F(ab')2 fragments, Fab fragments,
Fv
fragments, scFv fragments, other fragments, CDR peptides and mimetics. These
can easily be obtained and prepared by those skilled in the art. For example,
enzyme digestion can be used to obtain F(ab')z and Fab fragments by subjecting
CA 02456873 2004-02-26
16
an IgG molecule to pepsin or papain cleavage respectively. Recombinant
antibodies are also covered by the present invention,
Alternatively, the antibody of the invention may be an antibody derivative.
Such an antibody may comprise an antigen-binding region linked or not to a non-
immunogiobulin region. The antigen binding region is an antibody light chain
variable domain or heavy chain variable domain. Typically, the antibody
comprises
both light and heavy chain variable domains, that can be inserted in
constructs
such as single chain Fv (scFv) fragments, disulfide=stabilized Fv (dsFv)
fragments,
muftimeric scFv fragments, diabodies, minibodies or other related forms
(Colcher
et al. Q J Nucl Med 1998; 42: 225-241 ). Such a derivatized antibody may
sometimes be preferable since it is devoid of the Fc portion of the natural
antibody
that can bind to several efifectors of the immune system and elicit an immune
response when administered to a human or an animal. Indeed, derivatized
antibody normally do not lead to immuno-complex disease and complement
activation (type ill hypersensitivity reaction).
Alternatively, a non-immunoglobulin region is fused to the antigen-binding
region of the antibody of the invention. The non-immunoglobuiin region is
typically
a non-immunoglobuiin moiety and may be an enzyme, a region derived from a
protein having known binding specificity, a region derived from a protein
toxin or
indeed from any protein expressed by a gene, or a chemical entity showing
inhibitory or blocking activity(ies) against WNV or Dengue virus proteins. The
two
regions of that modified antibody may be connected via a cieavable or a
permanent linker sequence.
Preferably, the antibody of the invention is a human or animal
immunoglobulin such as IgG1, IgG2, IgG3, IgG4, IgM, IgA, igE or 1gD carrying
rat
or mouse variable regions (chimeric) or CDRs (humanized or "animalized").
Furthermore, the antibody of the invention may also be conjugated to any
suitable
carrier known to one skilled in the art in order to provide, for instance, a
specific
delivery and prolonged retention of the antibody, either in a targeted local
area or
for a systemic application.
The term "humanized antibody" refers to an antibody derived from a non-
human antibody, typically rnurine, that retains or substantially retains the
antigen-
CA 02456873 2004-02-26
17
binding properties of the parent antibody but which is less immunogenic in
humans. This may be achieved by various methods including (a) grafting only
the
non-human CDRs onto human framework and constant regions with or without
retention of critical framework residues, or (b) transplanting the entire non-
human
variable domains, but "cloaking" them with a human-tike section by replacement
of
surface residues. Such methods are well known to one skilled in the art.
As mentioned above, the antibody of the invention is immunologically
specific to the polypeptide of the present invention and irnmunological
derivatives
thereof. As used herein, the term "immunological derivative" refers to a
polypeptide that possesses an immunological activity that is substantially
similar to
the immunological activity of the whole polypeptide, and such immunological
activity refers to the capacity of stimulating the production of antibodies
immunologically specific to the WNV or Dengue virus proteins or derivative
thereof. The term "immunological derivative" therefore encompass "fragments",
"segments", "variants", or "analogs" of a polypeptide.
4. Compositions and vaccines
The polypeptides of the present invention, the polynucleotides coding the
same, the polyclonal or monoclonal antibadies, the recombinant measles virus
produced according to the invention, may be used in many ways for the
diagnosis,
the treatment or the prevention of WNV- or Dengue virus- associated diseases
or
infection.
In a sixth embodiment, the present invention relates to a composition for
eliciting an immune response or a protective immunity against a WNV or a
Dengue
virus. According to a related aspect, the present invention relates to a
vaccine for
preventing andlor treating a WNV- or Dengue virus- associated disease or
infection. As used herein, the term "treating" refers to a process by which
the
symptoms of a WNV- or Dengue virus- associated disease or infection are
alleviated or completely eliminated. As used herein, the term "preventing"
refers to
a process by which a WNV- or Dengue virus- associated disease or infection is
obstructed or delayed. The composition or the vaccine of the invention
comprises
a polynucleotide, a polypeptide, an expression vector, a recombinant viral
vector,
CA 02456873 2004-02-26
18
a recombinant measles virus and/or an antibody as defined above and an
acceptable carrier.
As used herein, the expression "an acceptable carrier" means a vehicle for
containing the poiynucleotide, the polypeptide, the expression vector, the
recombinant viral vector, the recombinant measles virus and/or the antibody of
the
invention that can be injected into an animal host without adverse effects.
Suitable
carriers known in the art include, but are not limited to, gold particles,
sterile water,
saline, glucose, dextrose, or buffered solutions. Carriers may include
auxiliary
agents including, but not limited to, diluents, stabilizers (i. e.-, sugars
and amino
acids), preservatives, wetting agents, emulsifying agents, pH buffering
agents,
viscosity enhancing additives, colors and the like.
Further agents can be added to the composition and vaccine of the
invention. For instance, the composition of the invention may also comprise
agents
such as drugs, immunostimulants (such as a-interferon, (3-interferon, y-
interferon,
granulocyte macrophage colony stimulator factor (GM-CSF), macrophage colony
stimulator factor (M-CSF), interleukin 2 (IL2), infierleukin 12 (IL12), and
CpG
oligonucleotides), antioxidants, surfactants, flavoring agents, volatile oils,
buffering
agents, dispersants, propellants, and preservatives. For preparing such
compositions, methods well known in the art may be used.
The amount of polynucleotide, polypeptide, expression vector, recombinant
viral vector, recombinant measles virus and/or antibody present in the
compositions or in the vaccines of the present invention is preferably a
therapeutically effective amount. A therapeutically effective amount of the
polynucleotide, the polypeptide, the expression vector, the recombinant viral
vector, the recombinant measles virus and/or the antibody of the invention is
that
amaunt necessary to allow the same to perform their immunological role without
causing, overly negative effects in the host to which the composition is
administered. The exact amount of polynucleotide, polypeptide, expression
vector,
recombinant viral vector, recombinant measles virus and/or antibody to be used
and the compositionfvaccine to be administered will vary according to factors
such
as the type of condition being treated, the mode of administration, as well as
the
other ingredients in the composition.
CA 02456873 2004-02-26
19
5. Methods of use
In a seventh embodiment, the present invention relates to methods for
treating andlor preventing a WNV- or Dengue virus- associated disease or
infection in an animal are provided. The method comprises the step of
administering to the animal an effective amount of at least one element
selected
from the group consisting of
- a polypeptide of the invention or a functional derivative thereof;
- an antibody as defined above;
- an expression vector as defined above;
- a polynucleotide of the invention or a fragment thereof,
- a recombinant viral vector of the invention; and
- a recombinant measles virus of the invention.
The vaccine, antibody and composition of the invention may be given to an
animal through various routes of administration. For instance, the composition
may
be administered in the form of sterile injectable preparations, such as
sterile
injectable aqueous or oleaginous suspensions. These suspensions may be
formulated according to techniques known in the art using suitable dispersing
or
wetting agents and suspending agents. The sterile injectable preparations may
also be sterile injectable solutions or suspensions in non-toxic parenterally-
acceptable diluents or solvents. They may be given parenterally, for example
intravenously, intramuscularly or sub-cutaneously by injection; by infusion or
per
os. The vaccine and the composition of the invention may also be formulated as
creams, ointments, lotions, gels, drops, suppositories, sprays, liquids or
powders.
for topical administration. They may also be administered into the airways of
a
subject by way of a pressurized aerosol dispenser, a nasal sprayer, a
nebulizer, a
metered dose inhaler, a dry powder inhaler, or a capsule. Suitable dosages
will
vary, depending upon factors such as the amount of each of the components in
the composition, the desired effect (short or long term), the route of
administration,
the age and the weight of the animal to be treated. Any other methods well
known
CA 02456873 2004-02-26
in the art may be used for administering the vaccine, antibody and the
composition
of the invention.
The present invention is also directed to a method of producing a
recombinant virus for the preparation of an anti-West-Niie virus vaccine or an
anti-
5 Dengue virus vaccine, the method comprising the steps of:
a) providing a host cell as defined above;
b) placing the host cell from step a) in conditions permitting the replication
of a recombinant virus capable of expressing a pofypeptide according to
the invention; and
10 c) isolating the recombinant virus produced in step b).
In a further embodiment, a West-Nile virus neutralisation assay is provided.
Accordingly, the assay comprises the steps of:
a) contacting VERO cells with West-Niie virus and an antibody;
15 b) culturing said VERO cells under conditions which allow for West-Nile
virus replication; and
c) measuring reduction of West-Nile virus replication foci on said VERO
cells.
20 EXAMPLES
The present invention. will be more readily understood by referring to the
following examples. These examples are illustrative of the wide range of
applicability of the present invention and are not intended to limit its
scope.
Modifications and variations can be made therein without departing from the
spirit
and scope of the invention. Although any methods and materials similar or
equivalent to those described herein can be used in the practice for testing
of the
present invention, the preferred methods and materials are described.
CA 02456873 2004-02-26
21
EXAMPLE 1: CONSTRUCTION OF MEASLES VIRUSES (MV) EXPRESSING
WNV AND DEN1 ANTIGENS
In order to test their capacity as vaccine candidates against WNV infection,
recombinant Schwarz measles viruses (MV) expressing these WNV and DEN-1
antigens were constructed. The different genes were introduced .in an
additional
transcription unit in the Schwarz MV cDNA that the inventors previously cloned
(pTM-MVSchw) {European Patent Application N° 02291551.6 filed on June
20,
2002). After rescue of the different recombinant Schwarz measles viruses
expressing the WNV and DEN-1 genes, their capacity to protect mice from a
lethal
WNV intraperitoneal challenge, and monkeys from Dengue virus infection will be
tested.
MV vector
Mass vaccination with live attenuated vaccines has. reduced the incidence
of measles and its complications dramatically since it was introduced in the
60's.
By now, the vaccine has been given to billions of people and is safe and
efficacious. It induces a very efficient, life-long CD4, CD8 and humoral
immunity
after a single injection of 104 TCID50. Moreover, it is easy to produce,
cheap, and
the means to deliver it worldwide already exist. The safety of this vaccine is
due to
several factors: i) The stability of the MV genome which explains that
reversion to
pathogenicity has never been observed. ii)The impossibility for the MV genome
to
integrate in host chromosomes since viral replication is exclusively
cytoplasmic. iii)
The production of the vaccine on safe primary chick embryo fibroblastic cells.
Thus; live attenuated MV could provide a safe and efficient pediatric
vaccination
vector.
MV belongs to the genus Morbillivirus in the family Paramyxoviridae. The
Edmonston MV was isolated in 1954 (32), serially passaged on primary human
kidney and amnion cells, then adapted to chick embryo fibroblasts (CEF) to
produce Edmonston A and B seeds (see (7, 8) for review). Edmonston B was
licensed in 1963 as the first MV vaccine. Further passages of Edmonston A and
B
on CEF produced the more attenuated Schwarz and Moraten viruses (33) whose
sequences have recently been shown to be identical (34, 35). Being
"reactogenic,"
CA 02456873 2004-02-26
22
Edmonston B vaccine was abandoned in 1975 and replaced by the
SchwarzlMoraten vaccine. This is now the most commonly used measles vaccine
(7, 8).
In a previous work, the inventors constructed an infectious cDNA from a
batch of commercial Schwarz vaccine, a widely used MV vaccine (European
Patent Application N° 02291551.6 filed on June 20, 2002). The
extremities of the
cDNA were engineered in order to maximize virus yield during rescue. A
previously described helper cell-based rescue system was adapted by co-
cultivating transfected cells on primary chick embryo fibroblasts, the cells
used to
produce the Schwarz vaccine. After two passages the sequence of the rescued
virus was identical to that of the cDNA and of the published Schwarz sequence.
Two additional transcription units (ATU) were introduced in the cDNA for
cloning
foreign genetic material. The immunogenicity of rescued virus was studied in
mice
transgenic for the CD46 MV receptor and in macaques. Antibody titers in
animals
inoculated with low doses of the rescued virus were identical to those
obtained
with commercial Schwarz MV vaccine. In contrast, the immunogenicity of a
previously described Edmonston strain-derived MV clone was much lower. This
new molecular clone allows producing MV vaccine without having to rely on seed
stocks. The ATUs, allow producing recombinant vaccines based on an approved,
efficient and worldwide used vaccine strain.
EXAMPLE 2: CONSTRUCTi~N OF SCHWAR~ MV-WNV RECOMBINANT
PLASMIDS.
1 ) Secreted glycoprotein E from WNV
The WNV env genie encoding the secreted form of the protein was
generated by RT-PCR amplification of viral RNA purified from viral particles
(WNV
IS-98-ST1 strain). The specific sequence was amplified using PfuTurbo DNA
polymerase (Stratagene) and specific primers that contain unique sites for
subsequent cloning in pTM-MVSchw vector : MV-WNEnv5 5'-
TATCGTACGATGAGAGTTGTGTTTGTCGTGCTA-3' (SEQ ID NO: 9) (BsiWl site
underlined) and MV-WNEnv3 5'-ATAGCGCGCTTAGACAGCCTTCCCAACTGA-3'
(SEQ ID NO: 10} (BssHll site underlined). A start and a stop codon were added
at
CA 02456873 2004-02-26
23
both ends of the gene. The whole sequence generated is 1380 nucleotides long
(see Figure 1 ), including the start and the stop codons and respects the
"rule of
six", stipulating that the nucleotides number of MV genome must be divisible
by 6
(28, 29). The Env protein thus generated contains its signal peptide in N-term
(18
aa) and no transmembrane region. Thus, It represents amino acids 275-732 in
WNV polyprotein and has the sequence shown in Figure 2.
2) preM plus E glycoproteins from WNV
The WNV gene encoding the preM plus E glycoproteins was generated by
PCR amplification of plasmid pVL prM-E.55.1 (clone CNCM I-2732 deposited on
Octobre 15, 2001 ). This expression plasmid encodes the pre-M and E proteins
of
WNV (IS-98-ST1 strain). The sequence was amplified using PfuTurbo DNA
polymerise {Stratagene) and specific primers that contain unique sites for
subsequent cloning in pTM-MVSchw vector : MV-WNpreMES 5'
TATCGTACGATGCAAAAGAAAAGAGGAGGAAAG-3' (SEQ ID NO: 11) (BsiWl
site underlined) and MV-WNpreME3 5'-
ATAGCGCGCTTAAGCGTGCACGTTCACGGAG-3' (SEQ ID NO: 12) (BssHll site
underlined). A start and a stop codon were added at both ends of the gene. The
whole sequence generated is 2076 nucleatides long (see Figure 3), including
the
start and the stop codons and respecfis the MV "rule of six". fn this
construct, the
C-terminus part of the C protein serves as a prM translocation signal. Both
preM
and E viral glycoproteins are transmembrane glycoproteins type I. It is
presumed
that WNV env preME expressing MV will produce and release multimeric forms of
preM-E heterodimers exhibiting high immunogenic potential. The construct
represents amino acids 302-789 in WNV polyprotein and has the sequence shown
in Figure 4.
3) NS1 protein from WNV
The WNV NS1 gene was generated by RT-PCR amplification of viral RNA
purified from viral particles (WNV IS-98-ST1 strain). The specific sequence
was
amplified using PfuTurbo DNA poiymerase (Stratagene) and specific primers : MV
WNNS15 5'- TATCGTACt~ATGAGGTCCATAGCTCTCACG-3' {SEQ ID NO: 13)
CA 02456873 2004-02-26
24
{BsiWi site underlined) arid MV-WNNS13 5'-
ATAGCGCGCTCATTAGGTCTTTTCATCATGTCTC-3' (SECT ID NO: 14) (BssHll
site underlined). A start codon was added at the 5' end and two stop codons at
the
3' end of the sequence. The whole sequence is 1110 nucleotides long (see
Figure
5), including the start and the two stop codons, thus respecting the "rule of
six".
The NS1 protein generated contains its signal peptide sequence in N-term (23
aa).
it represents amino acids 769-1136 in WNV polyprotein and has the sequence
shown in Figure 6.
4) preM-E protein from Dengue type 1 virus
The Dengue virus gene encoding the preM plus E glycoproteins was
generated by PCR amplification of pfasmid pVL pINDJ[prM+E] (clone 2)
(COURAGEOT, M.-P., et al. 2000, A-glucosidase inhibitors reduce dengue virus
production by affecting the initial steps of virion morphogenesis in the
endoplasmic
reticulum. Journal of Virology 74: 564-572). This plasmid encodes the pre-M
and E
glycoproteins of DEN-1 virus (strain FGA/89). The sequence was amplified using
PfuTurbo DNA polymerise (Stratagene) and specific primers that contain unique
sites for subsequent cloning in pTM-MVSchw vector : MV-DEN1preME5 5'-
TATCGTACGATGAACAGGAGGAAAAGATCCGTG-3' (SEQ lD NO: 15) (BsiWl
site underlined) and MV-DEN1 preME3 5'-
ATAGCGCGCTTAAACCATGAGTCCTAGGTACAG-~3' {SEQ ID NO: 16) (BssHll
site underlined). A start and a stop codon were added at both ends of the
gene.
The whole sequence generated is 2040 nucleotides long {see Figure 7),
including
the start and the stop codons and respects the MV "rule of six". In this
construct,
the C-terminus part of the C protein serves as a preM translocation signal.
Both
preM and E viral glycoproteins are transmembrane glycoproteins type I. It is
presumed that DEN-1 env expressing MV will produce and realease multimeric
forms of preM-E heterodimers exhibiting high immunogenic potential. The
construct represents amino acids 95-773 in DEN-1 palyprotein and has the
sequence shown in Figure 3.
The same immunogens can be prepared by the same way from DEN-2,
DEN-3 and DEN-4 serotypes.
CA 02456873 2004-02-26
5} insertion into MV Schwarz vector
The different WNV and DEN-1 nucleotidic sequences were cloned in
pCR2.1-TOPO plasmid (Invitrogen) and sequenced to check that no mutations
5 were introduced. After BsiWIIBssHll digestion of the pCR2.1-TOPO plasmids,
the
DNA fragments were cloned in the pTM-MVSchw vector in ATU position 2 giving
plasmids : pTM-MVSchw-EnvWNV, pTM-MVSchw-preMEwnv, pTM-MVSchw-
NS1WNV and pTM-MVSchw-preMEDEN-1 according to Figure 9.
10 EXAMPLE 3: RECOVERY OF REC~MBINANT MVSCHW-ENVWNV, MVSCHW-
PREMEWNV AND MVSCHW-NS1WNV VIRUSES.
To recover recombinant Schwarz viruses from the plasmids, we used the
helper-cell-based rescue system described by Radecke et al. (11 ) and modified
by
Parks et al. (30). Human helper cells stably expressing T7 RNA polymerase and
15 measles N and P proteins {293-3-46 cells, a kind gift from MA Billeter)
were
transfected using the calcium phosphate procedure with pTM-MVSchw-EnvWNV,
pTM-MVSchw-preMEwnv or pTM-MVSchw-NS1WNV plasmids {5 pg) and a
plasmid expressing the MV polymerise L gene (pEMC-La, 20 ng, a kind gift from
MA Billeter). After overnight incubation at 37° C, the transfection
medium was
20 replaced by fresh medium and a heat shock was applied (43° C for two
hours)
(30). After two days of incubation at 37° C, transfected cells were
transferred on a
CEF cells layer and incubated at 32° C in order to avoid any adaptation
of the
Schwarz vaccine that was originally selected on CEF cells and is currently
grown
on these cells for safety considerations. infectious virus was easily
recovered
25 between 3 and 7 days following cocultivation. Syncytia appeared
occasionally in
CEF, but not systematically. The recombinant viruses were also rescued by the
same technique after cocultivation of transfected 293-3-46 helper cells at
37° C
with primate Vero cells (african green monkey kidney). In this case, syncytia
appeared systematically in all transfections after 2 days of coculture. In
order to
increase the yield of rescue and because these recombinant viruses will be
used
in mice experiments, Vero cells were used as target cells in place of the
usual
chick embryo fibroblasts (CEF) (European Patent Application N°
02297551.6 files
CA 02456873 2004-02-26
2~
on June 20, 2002). Recombinant viruses were passaged two times on Vero cells.
The inventors have previously shown that two passages of the Schwarz virus on
Vero cells did not change its immunogenic capacities in macaques (European
Patent Application N° 02291551.6 files on June 20, 2002).
The recombinant viruses were prepared as described above and the
expression of the transgene in infected cells was checked by
immunofluorescence. To detect WNV Envelope glycoproteins expression, immune
sera from mice resistant to WNV infection were used (international Patent
Application WO 021081741 ).To detect NSI protein expression, the inventors
used
anti-NS1 Monoclonal antiobodies (international Patent Application N° WO
00175665).
EXAMPLE 4: VACCINATION AGAINST WEST-NtL,E VIRUS
West Nile disease has recently emerged as an important mosquito-borne
flavivirus infection with numerous fatal cases of human encephalitis, thus
urging to
develop a safe and efficient vaccine. Measles virus (MV) vaccine, a live-
attenuated
RNA virus, is one of the safest and most effective human vaccine developed so
far. The Schwarz vaccine strain of MV can be used as a vector to immunize
against heterologous viral, thereby offering a novel and attractive
vaccination
strategy against West Nile virus (WNV). We evaluated the efficacy of a Schwarz
measles vaccine-derived vector expressing the secreted form of the WNV
envelope E glycoprotein in a mouse model. Vaccination induced high titers of
specific anti-WNV neutralizing antibodies and protection from a lethal WNV
challenge. Passive administration with antisera from immunized mice also
provided protection, even after challenge with high doses of WNV. Example 4 is
the first report that a live-attenuated recombinant measles virus provides
efficient
protective immunity against an heteroiogous viral disease. The induction of
protective immunity shows that Pive attenuated-MV expressing the secreted form
of
the E giycoprotein is an effective vaccine against West Nile disease.
CA 02456873 2004-02-26
2T
MATERIALS AND METHODS
Cells and virus. Vero-NK (African green monkey kidney) cells were
maintained in DMEM Glutamax (Invitrogen} supplemented with 5% heat-
inactivated fetal bovine serum (FBS). Flelper 293-3-46 cells used for viral
rescue
(11 ) (a kind gift from M. Billeter, Zurich University) were grown in DMEM/10%
FBS
and supplemented with 1.2 mg of G 418 per ml. WNV strain IS-98-ST1 {GenBank
accession number AF 481864} was propagated in mosquito Aeries
pseudoscutellaris AP61 cell monoiayers (13}. Purification on sucrose
gradients,
and virus titration on AP61 cells by focus immurzodetection assay (FIA) were
performed as previously described (13, 27).
Mouse antisera to uVNV. Anti-WNV hyperimmune mouse ascitic fluid
(HMAF) was obtained by repeated immunization of adult mice with WNV strain IS-
98-ST1 followed by the inoculation of sarcoma 180. Mouse polyclonal anti-WNV
antibodies were obtained by immunization of adult BALB/c-MBT congenic mice
with 103 FFU of IS-98-ST1 as described previously (13). The WNV-immune serum
was collected one month after priming.
Construction of pTM-MVSchw-sEWNV plasrr~id. The plasmid pTM-
MVSchw that contains an infectious MV cDNA corresponding to the anti-genome
of the widely used SchwarzlMoraten MV vaccine strain has been reported
elsewhere (10). Additional transcription units were introduced into the viral
genome to turn it into a vector expressing foreign proteins. To construct pTM-
MVSchw-sE~,NV, genomic RNA of WNV was extracted from highly purified IS-98-
ST1 virions and reverse transcribed using Titan One-Step RT-PCR kit (Roche
Molecular Biochemicals} according to the manufacturer's instructions. An RT-
PCR
fragment encoding the internal E translocatian signal (prM-151 to prM-166)
followed by the ectodomain and the stem region of the E protein (E-1 to E-441
)
was generated using the 5' primer MV-WNEnvS 5'-
TATCGTACGATGAGAGTTGTGTTTGTCGTGCTA-3' (SEQ ID NO: 9) containing
a BsiVlll restriction site (underlined) and the 3' primer MV-WNEnv3 5'-
ATAGCGCGCTTAGACAGCCTTCCCAACTGA-3' (SEQ lD NO: 10) containing a
BssHll restriction site (underlined). A start and a stop codon were added at
both
ends of the gene. The sequence respects the «rule of six», stipulating that
the
CA 02456873 2004-02-26
28
nucleotides number of MV genome must be multiple of 6 (28, 29). The PCR
product was directly inserted into pCR2.1-TOPO plasmid (TOPO TA cloning kit,
Invitrogen) according to the manufacturer's instructions ~to give TOPO-sEWNV.
A
1.4-kb fragment containing truncated E protein with translocation signal
sequence
was excised from TOPO-sEWNV using BsiWl and BssHll and then inserted into
BsiWllBssNll-digested pTM-MVSchw-ATU2 which contains the additional
transcription unit (ATU) between the P and M genes of Schwarz MV genome (10,
11 ). The resulting plasmid was designated pTM-MVSchw-sEWNV (named pTM
MVSchw-EnvWVN in the previous Examples). All constructs were verified by
automated sequencing.
Rescue of recombinant MVSchw-sEWNV virus from the coned cDNA.
Rescue of recombinant Schwarz MV from the plasmid pTM-MVSchw-sEWNV was
performed using the helper-cell-based rescue system described by Radecke et
al.
(11 ) and modified by Parks et al. (30). Briefly, human helper cells stably
expressing T7 RNA polymerise and measles N and P proteins (293-3-46 cells, a
kind gift from MA Billeter, Zurich University) were transfected with 5 pg pTM-
MVSchw-sEWNV and 0.02 pg pEMC-La expressing the MV polymerise L gene (a
kind gift from MA Billeter) using the calcium phosphate procedure. After
overnight
incubation at 37° C, a heat shock was applied for 2 h at 43°C.
After two days of
incubation at 37° C, transfected cells were transferred onto a Vero
cell monolayer.
Vero cells were used as target cells in place of the usual chick embryo
fibroblasts
(CEF) in order to increase the yield of rescued virus. The inventors have
previously shown that two passages of the Schwarz virus on Vero cells did not
change its immunogenicity in primates (10). Syncytia that appeared after 2-3
days
of coculture were transferred to 35 mm wells of Vero cells, then expanded in
75-
and then 150-cm~ flasks in DMEM/5% FBS. When syncytia reached 80-90%
confluence (usually 36-48 h post-infection), the cells were scraped in a small
volume of OptiMEM (Invitf-ogen) and frozen and thawed once. After low-speed
centrifugation to pellet cellular debris, the supernatant, which contained
virus, was
stored at -80°C. The titers of MVSchw-sEWNV was determined by an
endpoint limit
dilution assay on Vero cells. The 50% tissue culture infectious doses (TCIDSO)
were calculated using the Karber method.
CA 02456873 2004-02-26
29
Radioimmunoprecipitation assay. Vero cells were starved for 1 h with
DMEM without methionine and cysteine (ICN Biornedicals} and labeled 3 h with
250 pCi/ml Tran35S-label (iCN Biomedicals). Cells were lysed with RIPA buffer
(20
mM TrisCl, pH 8.0, 150 mM NaCI, 10 mM EDTA, 0,1%SDS, 0,5% deoxycholate,
1 % Triton X-100) supplemented with a cocktail of protease inhibitors. R1P
assay
was performed as previously described (31 ). Samples were analyzed by SDS-15%
PAGE under reducing conditions.
Mice experiments. CD46-IFNAR mice were produced as previously
described (10). Adult BALB/c mice were purchased from Janvier Laboratories (Le
Genest St Isle, France). Mice were housed under specific pathogen-free
conditions et the Pasteur Institute. Five to 5-week-old CD45-iFNAR mice were
i.p.
inoculated with 104 or 106 TCID6o of MV. Acute WNV challenge was performed by
i.p. inoculation of neurovirulent WNV strain IS-98-ST1 (i.p.LDSO - 10) in
Dulbecco's modified phosphate saline buffer (DPBS) supplemented with 0.2%
bovine serum albumin (BSA) pH 7.5 (Sigma Chemical Co.). The animals were
monitored daily for signs of morbidity and mortality. All experiments are
approved
and conducted in accordance with the guidelines of the Office Laboratory
Animal
Care at Pasteur Institute.
Anti-WN vaccination test with antigenic boost. Adult CD46+~- IFN-
al~iRr- mice were vaccinated over a four week period with the MV-WN sE virus
at
a dose of 104 DCIP50 (which is a dose recommended far humans) and an
antigenic boost was provided by purified WNV pseudo-particles that were
secreted by MEFl3T3.Tet-Off/WN prME # h2 cells.
Humoral response. To evaluate the specific antibody response in serum,
mice were bled via the periorbital route at different time after inoculation.
Detection
of anti-MV antibodies was performed by ELISA (Trinity Biotech, USA) as
previously described (10). An anti-mouse antibody-HRP conjugate (Amersham)
was used as the secondary antibody. The endpoint titer was calculated as the
reciprocal of the last dilution giving a positive optical density value. The
presence
of anti-WNV antibodies was assessed by ELISA as previously described (13).
Briefly, microtitration plaques were coated with 105 FFU of highly purified
WNV
strain IS-98-ST1 and then incubated with mouse sera dilutions. A test serum
was
CA 02456873 2004-02-26
considered positive if its optical density was twice the optical density of
sera from
immunized control mice.
Neutralization assay. anti-WNV neutralizing antibodies were detected by a
FRNT test. Sera from each mouse group were pooled and heat-inactivated at
5 56°C for 30 min, Vero cells were seeded into 12-well plate (1.5 x 105
cellslwell) for
24 h. Mouse serum samples were serially diluted in MEM Glutamax/2% FBS.
Dilutions (0.1 ml) were incubated at 37°C for 2 h and under gentle
agitation with an
equal volume of WNV strain IS-98-STlcontaining 100 FFU. Remaining infectivity
was then assayed on Vero cell monolayer overlaid with MEM Glutamaxl2%FBS
10 containing 0.8% (Wlv) carboxy methyl cellulose (BDH). After 2 days of
incubation at
37°C with 5% C02, FIA was performed with anti-WNV HMAF as previously
described (27). The highest serum dilution tested that reduced the number of
FFU
by at feast 90% (FRNT9o) was considered the end-point titer.
Passive transfer of immune sera. Pooled immune sera were transferred
15 into 6-week-old female BALBIc mice intraperitoneally. Mice received
injection of
0.1 ml of serial dilutions of pooled serum samples in DPBSI0.2%BSA one day
before WNV inoculation. The challenged mice were observed for more than 3
weeks.
DISCUSSION OF THE RESULTS
20 Since its introduction into the United States in 1999, West Nile virus
(WNV)
infection has been recognized as one of the most serious mosquito-borne
disease
in the Western Hemisphere, causing severe neurological disease
(meningoencephalitis and poliomyelitis-like syndrome) in humans. (3). Within
the
last 4 years, WNV had spread through North America, Central America and the
25 Caribbean (1, 2). It is presumed that it will reach South America in the
coming
years. Since 2002, the US outbreaks were characterized by an apparent increase
in human disease severity with 13,000 cases and 500 deaths. Although mosquito-
borne transmission of WNV predominates, WNV is also transmitted by blood
transfusion, organ donations and transplacentaly to the fetus (3). Prevention
of
30 West Nile encephalitis is a new public health priority and it is imperative
that a
vaccine be developed (3, 4, 5). No vaccine has been approved for human use so
far.
CA 02456873 2004-02-26
31
Because WNV can be transmitted across species, there is an urgent need
to develop preventive strategies for humans. A rational approach should be to
confer a long-term immunity in large groups of individuals, and to boost this
immunity in case of WNV outbreaks. Measles virus {MV) vaccine can now be used
as a vector to immunize against heterologous viral diseases, thereby offering
a
novel and attractive vaccination strategy against WNV. We have recently tested
this vector against HIV infection (6). MV vaccine, a live-attenuated RNA
virus, is
one of the safest and most effective human vaccine developed so far. It
induces a
very efficient, life-Tong immunity after a single or two injections (~, 8).
The MV
genome is very stable and reversion of vaccine strains to pathogenicity has
never
been observed. The Schwarz MV strain is used in two widely used measles
vaccines, Attenuavax (Merck and Co. Inc., West Point, USA) and Rouvax (Aventis
Pasteur, Mercy I'Etoile, France), and in the combined measles, mumps, and
rubella vaccine (MMR) (9). We have recently generated an infectious cDNA for
this strain {10) and introduced additional transcription units (ATU) into it
for cloning
foreign genes, based on the work of Radecke et al. (11 ). The vaccine rescued
from the molecular clone was as immunogenic as tree parental vaccine in
primates
and mice susceptible to MV infection. Thus, this approved and widely used MV
vaccine can be used as a vector to immunize individuals simultaneously against
measles and other infectious diseases.
WNV is a single-stranded RNA virus of the Flaviviridae family, genus
flavivirus, within the Japanese encephalitis antigenic complex (2, 3). The
virion is
composed of three structure( proteins, designated C (core protein), M
(membrane
protein) and E (envelope protein). Protein E, which is exposed on the surface
of
the virion, is responsible for virus attachment and virus-specific membrane
fusion.
Because the E glycoprotein can potentially serve as a major protective
immunogen for a WNV vaccine (12), the inventors introduced the WNV cDNA
encoding the carboxyl-terminally truncated E glycoprotein lacking the
transmembrane-anchoring region (residues E-1 to E-441, designated sEWNv
hereinafter) of IS-98-ST1 strain {13) into the infectious cDNA for the Schwarz
MV
vaccine (10) (Fig. 10A). WNV strain IS-98-ST1 has the same neuropathologic
properties than the new variant designated Isr98IN'~99 that has been
responsible
CA 02456873 2004-02-26
32
for the recent WNV outbreaks in North America and Middle East (13). The WNV
sequence was introduced in an ATU located between the phosphoprotein (P) and
matrix (M) genes in the MV genome. The recombinant MVSchw-sEwrw virus was
produced after transfection of the corresponding plasmid into human helper
cells
allowing the rescue of negative-stranded RNA paramyxoviruses (11 ), then
propagation in Vero cell cultures. The growth of MVschw-sEwNV in Vero cells
was
only slightly delayed as compared to that of standard Schwarz MV (MVscnw)
{Fig.
10B}. After 60 h of infection, the yield of MVscnw-sEwNV was comparable to
that of
MVschw. The expression of sEWNV in MVschw-sEwNV-infected Vero cells was
demonstrated by immunofluorescence and radioimmunoprecipitation (RIP) assays
{Fig. 10C, D). At 40 h post-infection, the cell surface of MVscnw-sEwNV-
induced
syncitia was clearly visualized by anti-WNV immune serum, indicating that
sEWNv
is transported along the compartments of the secretory pathway (Fig. 10C). RIP
analysis revealed that anti-WNV antibodies recognized sEw~,~, that migrated
faster
than authentic E glycoprotein (Fig. 10D). lnfierestingly, sEWNV was detected
in the
supernatants of MVscnw-sEwNV-infected Vero cells at 40 h post-infection (Fig.
10D,
panel Supernatants/ MVscnw-sEWNV, lane a-WNV). Thus, MVscnw-sEWNv
expresses a recombinant E glycoprotein which is secreted efficiently.
Immunoblots
confirmed that sEwNV accumulated in the culture medium of MVscnw-sEwNV
infected Vero cells (data not shown).
Genetically modified mice expressing the human CD46 MV receptor and
lacking the interFeron a/~i receptor (6, 14) (CD46+~- IFN-a/~i R-~-,
abbreviated CD46-
IFNAR) that are susceptible to MV (14) were used to assess the immune response
induced by MVschw-sEwNV. These mice deficient in IFN-a/~i response raise
cellular
and humoral immune responses similar to those of competent mice (6, 10, 15,
16).
Two groups of six CD46-1FNAR mice were inoculated intraperitoneally {i.p.)
with
either 104 or 106 tissue culture infective doses (TCID5o) of MVschw-sEwNV.
Each
group was boosted using the same dose 1 month after the first immunization. As
a
control, CD46-IFNAR mice were immunized with 10& TClDSO of "empty" MVscnw.
One month after the first immunization, specific anti-MV antibodies were
detected
in immune sera from mice inoculated with either MVschw or MVschw-sE~,~v {Table
1 ). Mice that received either dose of MVschw-sEwNV displayed specific anti-
WNV
CA 02456873 2004-02-26
33
antibodies at a dilution of 1:3,000. One month after boosting, the titers of
anti-WNV
antibodies had reached 1:30,000 to 1:200,000 (Table 1 ) and were highly
reactive
with the WNV E glycoprotein (Fig. 11). No anti-WNV antibodies were detected in
the sera of any control mice (Table 1 and Fig. 11 ). These results show that
one
injection of MVschw-sEwNV induces anti-WNV antibodies, and that boosting one
month after priming increases their titers 10 to 60 times.
Anti-WNV neutralizing activity was measured in MVscnw-sEWNV-immune
sera using a focus reduction test (FRNTso) (Table 1 ). As a positive control,
the
WNV-immune serum from immunized BALBIc-MBT congenic mice (13) gave a
FRNTso titer of 50. The immune sera from CD46-IFNAR mice inoculated with
"empty" MVscnw had not detectable neutralizing activity. Immunized CD46-IFNAR
mice which received 104 or 106 TCIDso of MVschw-sEWNV raised neutralizing
antibodies with similar FRNTso titers, and boosting increased their titers
from 10 to
200-300. These data show that mice twice inoculated with the recombinant live-
attenuated MV encoding the secreted form of the IS-98-ST1 E glycoprotein had
high levels of anti-WNV antibody with neutralizing activity, regardless of the
injected dose.
Because antibody-mediated immunity may be critical to protect against
WNV infection (17, 18), the inventors examined if the passive transfer of sera
from
MVSchw-sEw~,v-immunized mice can protect adult BALB/c mice from WNV
infection (Table 2). Groups of six 6-week-ofd BALBIc mice received i.p.
various
amounts of pooled immune sera from MVschw-sEW~v-immunized CD46-IFNAR
mice collected one month after priming or boosting. One day later, the mice
were
challenged with 10 times the i.p. 50% lethal dose (LD5o) of WNV strain IS-98-
ST1
(13, 19). As a positive control, BALBIc mice that received as little as 2 pl
of the
WNV-immune serum were protected from the challenge (Table 2). In contrast, all
mice that received 2 girl of the non-immune mouse serum or serum from "empty"
MVschw-immunized mice died within 11-12 days. Protective passive immunity was
observed in all BALB/c mice following transfer of 2 pl of pooled sera from
CD46-
IFNAR mice immunized once with 106 TCiD5o of MVSchw-sEWNV. As little as 1 pl
of this antisera induced 66% protection. Passive transfer of sera collected
one
month after a single immunization with 104 TCIDSO induced a survival rate of
50 %.
CA 02456873 2004-02-26
34
Remarkably, the administration of 1 pl of MVscnw-sEWNV-immune sera collected 1
month after boosting induced 100% protection. These results indicate that a
single
injection of 106 TCIDSO or two injections of 104 TCIDSa of MVsct,w-sEwNV
elicited
protective humoral response. Because the amount of flavivirus inoculated
during
mosquito feeding is probably in the order of 102 to 104 infectious virus
particles (1 ),
we assessed the capacity of MVschw-sEWN-immune sera to protect against a
range of 102 to 105 focus forming units (FFU) of WNV strain IS-98-ST1. Groups
of
six BALBIc mice were passively immunized with 2 lal of pooled immune sera
collected from CD46-IFNAR mice twice inoculated with 104 TCIDso of MVsct,w-
sEwNV (Table 2). Survival rates of 85-100% were observed in mice that received
the MVschw-sEWNV-immune serum, regardless the lethal doses of IS-98-ST1 (10 to
10,000 i.p. LDSO). These data are consistent with the finding that humoral
response plays a critical role in protection against WNV infection.
Mice which are completely unresponsive to IFN-a/~3 are highly susceptible
to encephalitic flaviviruses (19, 20). Indeed, the inventors previously showed
that
WNV infection of CD46-IFNAR mice was lethal within 3 days instead of 11 days
in
competent mice (19). To assess whether the immunity induced by MVSchw-sEWNv
could protect these compromised animals from WNV infection, three CD46-IFNAR
mice from the group that had received two injections of MVschw-sE~,NV (106
TCID~o), were i.p. inoculated with 100 FFU of IS-98-ST1 one month after the
boost. Mice inoculated with "empty" MVscnw were used as controls. The mice
that
had received MVscnw-sEWNV survived the WNV challenge while control mice died
within 3 days. MVschw-sE~rNV-immunized mice were bled 3 weeks after challenge.
The FRNTso antibody response (titer ~ 100) was comparable to the pre-challenge
response. Notably, post-challenge immune sera did not react with WNV
nonstructural proteins such as NS3 and NS5 as shown by RIP assay (Fig. 11,
panel MVschw-sEw~,v, lane 106 TCID5o, day 20, p.c.), suggesting that no viral
replication occurred after challenge with WNV. These data show that immunizing
with MVschw-sEWNV prevented WNV infection in highly susceptible animals.
The present Example shows for the first time that a live-afitenuated measles
vector derived from the Schwarz MV vaccine can induce a protective immunity
against an heterologous lethal pathogen. These data constitute also the proof
of
CA 02456873 2004-02-26
concept that a live-attenuated Schwarz measles vaccine engineered to express
the secreted form of the WNV E glycoprotein can be used as a vaccine to
prevent
West Nile disease in humans. The MV vaccine vector offers several advantages
over other existing viral vectors. The Schwarz MV vaccine has been used on
5 billions of people since the sixties and shown to be safe and efficacious.
It is easily
produced on a large scale in most countries and can be distributed at Dow
cost.
The MV genome is very stable and reversion to pathogenicity has never been
observed (8). Moreover, MV replicates exclusively in the cytoplasm, ruling out
the
possibility of integration in host DNA. The MV vector has been shown to
express a
10 variety of genes, or combinations of genes, of large size over more than
twelve
passages (6, 16, 21, 22, 23, 24). This stability is likely due to the fact
that there is
little constraint on genome size for pleomorphic viruses with a helical
nucleocapsid. Unlike chimeric viral vectors, the recombinant MV vector is an
authentic MV expressing an additional gene. This greatly reduces the risk of
15 changing the tropism and the pathogenicity of the original vaccine. It
reduces also
the risk of recombination.
The recombinant MV-WNV vaccine according to a preferred embodiment of
the present invention is a promising live-attenuated vector to mass immunize
children and adolescents against both measles and West Nile diseases. Although
20 the existence of an anti-MV immunity in nearly the entire adult human
population
appears to restrict its use to infants, an already worthy goal, recent studies
demonstrated that revaccinating already immunized children results in a boost
of
anti-MV antibodies (25, 26). These and other studies (Ann Arvin) demonstrated
that the presence of passive MV pre-immunity (maternal antibodies) does not
25 circumvent the replication of attenuated MV after a second injection. This
opens
the possibility of using the live-attenuated MV-derived vector to immunize
adults.
Indeed, the inventors reported that a MV-HIV recombinant virus induced anti-
HIV
neutralizing antibodies in mice and macaques even in the presence of pre-
existing
anti-MV immunity (6). Because of cross-species 'transmission, it is feared
that
30 WNV becomes a recurrent zoonosis with repeated seasonal outbreaks in
humans.
The inventors propose that MVSchw-sEWNV could be used to induce long-term
CA 02456873 2004-02-26
36
memory immunity in large groups of children and adults, and to boost this
immunity in case of West Nile disease outbreak.
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22. P. Spielhofer, et af., J. Viral. 72, 2150-2159 (1998).
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23. M. Singly M. Billeter, J. Gen. Virol. 80, 101-6 (1999).
24. Z. Wang, et al., Vaccine 19, 2329-2336 (2001 ).
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CA 02456873 2004-02-26
38
TABLE 1. Antibody response of CD46-iFNAR mice to intraperitoneal
inoculation of MVschw-sEwNv
Immunizing virus MV-specific WN-specific WN-specific
Ab titer 4 Ab titer ~ FRNT9o ~
WNV~ (103 FFU) NT 10,000 50
MVschw 2 (106 TCIDSO) 30,000 < 10 < 10
MVschw-sEwNV 2 (104 TCID~o)15,000 3,000 10
MVSchw-sEwNV 2 (106 TCIDoo)25,000 3,000 10
2 x MVschw-sE,N~v 3 (104 90,000 30,000 200
TCIDSO)
2 x MVschw-sEwNV 3 (106 140,000 200,000 300
TCID~o)
BALB/c-MBT congenic mice were i.p. inoculated with WNV strain IS-98-ST1.
2Virus was given i.p. to CD46-IFNAR mice.
3 Virus was given i.p. twice at 1 month of interval.
4Determined by EL1SA on pooled heat-inactivated sera .
5The highest serum dilution that reduced the number of FFU of WNV by at least
90%.
NT: not tested
CA 02456873 2004-02-26
39
TABLE 2. Protective ability of the MVschw-sEwnv-immune serum
Material used Volume of WNV2 Protection M.D.O.D3
for sera
immunization transferred' (FFU} (no. surviving/(day S.D.}
(Ol) no, tested)
Controls 10 100 0/6 11.5 +_ 1.5
DPBS
W NV4 10 ' - 100 616 -
2 100 5/6 20
MVSchw 5 2 100 0/6 12.0 1.5
MVSchw-sEWNVfi
106 TCID50 (day 2 100 6/6 -
30)
1 100 4/6 11.0 t 1.5
904 TCiD50 (day 10 100 3/6 10.5 2.0
30)
104 TGID50 (day 1 100 616 -
60}
2 100 5/6 11
2 1,000 6/6
2 10,000 516 10
2 100,000 5/6 11
' BALB/c mice received 0.1 ml of DPBS containing the indicated amount of
pooled sera.
2 Mice were challenged with WNV strain IS-98-ST1 one day after passive
transfer.
3 Mean day of death ~ standard deviation.
4 Immune sera from resistant BALB/c-MBT congenic mice (13) inoculated with 103
FFU of
IS-98-ST1 WNV.
5 Immune sera from CD46-IFNAR mice collected 30 days after inoculation of
MVSchw
(106 TCIDSO).
6 Immune sera from CD46-IFNAR mice were collected 30 days after 1 injection or
60 days
after 2 injections of MVSchw-sEWNV.
