Note: Descriptions are shown in the official language in which they were submitted.
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Title: VaccineSagainst West Nile Virus.
FIELD OF THE INVENTION
The invention relates to the field of medicine. It
particularly relates to vaccines against flaviviruses,
more specifically to West Nile Virus, and to methods of
producing same.
BACKGROUND OF THE INVENTION
The Flaviviridae family contains three genera: the
flaviviruses, the pestiviruses and the Hepatitis C
viruses. Flaviviruses are small spherical enveloped
viruses with virions composed of three structural
proteins designated C, M and E, and a single (+)RNA
genome of approximately 11,000 nucleotides (Chambers et
al. 1990; Brinton 2002). The flavivirus genus comprises
more than 60 highly related viruses including several
human pathogens of global and local epidemiological
importance, with most of them being transmitted by
arthropod vectors. With a combined toll of hundreds of
millions of infections around the world annually, yellow
fever virus, Japanese encephalitis virus, St. Louis
encephalitis virus, Murray Valley encephalitis virus,
tick-borne encephalitis virus, dengue virus, and West
Nile Virus continue to be in the focus of epidemiological
surveillance worldwide. While the availability of an
efficient vaccine and control of mosquito vectors have
resulted in significant improvement of the
epidemiological situation in yellow fever, other existing
as well as emerging flavivirus-associated diseases, for
which vaccines are not yet available, continue to
challenge experimental virology. West Nile Virus was
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first identified in 1937 in the West Nile district in
Uganda (Smithburn et al. 1940) and has now been
recognized as the most widespread of the flaviviruses,
with a geographic distribution in Africa, Asia, Europe,
Australia and North America (Campbell et al. 2002).
Recent outbreaks have been reported in Russia, Israel,
Romania, and the United States (Hubalek and Halouzka
1999; Anderson et al. 1999; Jia et al. 1999; Lanciotti et
al. 1999), with over 3000 individuals tested positive and
nearly 300 people killed. The virus was found to have
caused infections in persons in over 40 different states
in the US. Symptoms vary from fever, headache, skin rash,
swollen lymph glands, neck stiffness, stupor,
disorientation, tremors, convulsions, muscle weakness,
pancreatitis, myocarditis, paralysis to coma,, while in
15% of the cases the disease progresses to a more severe
state (e.g., West Nile encephalitis), which can lead to
death. Besides infecting humans, West Nile Viruses are
also known to infect horses and several bird species and
cause severe illness and death. The outbreak in New York
in 1999 started with massive death among crows and
several lethal cases in horses.
Several approaches were followed in the art to
counteract the infection and resulting illnesses brought
about by flaviviruses. One proposed approach was to treat
individuals with chemical compounds, such as ribavirin
and nucleoside analogues, and biologicals such as
interferon alpha-2b and/or helioxanthin (WO 00/10991; WO
02/15664; US patent no. 6,306,899). Others have focused
on the development of vaccines containing: chimeric
flaviviruses, (manipulated) yellow fever viruses for
cross-vaccination, sub-viral particles, replication
defective flaviviruses, (naked) nucleic acid, recombinant
sub-units (envelope proteins), or poxviruses containing
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flavivirus antigens (Arroyo et al. 2001; Wang et al.
2001;~Chang et al. 2001; WO 00/12128; WO 01/03729; WO
02/72036; WO 99/26653; WO 99/63095; WO 01/60315; WO
02/68637; EP 0869184 A; WO 00/14245; WO 02/74963 EP
0691404 A WO 98/37911; WO 01/39802; WO 01/60847; EP
0102228 A; EP 0872553 A; US patent no. 6,184,024,
5,514,375, 5,744,140, 5,744,141 6,416,763, 6,432,411,
5,494671, 6,258,788). One veterinary vaccine containing
inactivated West Nile Viruses was approved in August
2001, solely for the use in horses. In Israel, an
approach was taken to produce a West Nile Virus strain
isolated from geese (Goose Israel 1998) in mouse brains,
to inactivate it by formaldehyde and to apply.the vaccine
in geese flocks (Malkinson et al. 2001). This veterinary
vaccine (for use in geese flocks) was approved by the,
Israelian authorities in July/August 2001. Numerous
disadvantages exist with the treatments and vaccines
mentioned above, related to dosages, (in-) effectiveness,
required titers in production, and side effects (Monath
et al. 2001). Disadvantages in the production of vaccines
on systems such as mouse brains are clearly related to
safety, animal welfare, adverse side effects, allergic
properties, titers and scalability. No human vaccines
have been produced to date. To elicit a proper immune
response against the wild type virus it would be clearly
desirable to have a vaccine comprising a virus that
contains most if not all of its antigenic proteinaceous
molecules in its wild type configuration, but that does
not replicate and that is able to elicit a significant
immune response, resulting in a proper protection against
subsequent infections. Such vaccines should preferably
contain whole-inactivated viruses. However, safe and
large-scale production methods to obtain such whole-
inactivated viruses are not available in the art for
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vaccines directed against West Nile Virus. A cell-based
system based on the use of animal cells such as Vero
cells has disadvantages since Vero cells are normally
grown on microcarriers and therefore highly suited for
large scale production, and the culture is by definition
not free from animal-derived components. It is an object
of the present invention to provide novel methods for
producing West Nile Virus, preferably on large scale, for
the production of novel vaccines based on West Nile Virus
particles.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the Reverse Transcriptase PCR (RT-PCR)
products using samples of cells as deposited under ECACC
no. 96022940 infected with West Nile Virus.
Figure 2 shows the immune fluorescent staining of cells
as deposited under ECACC no. 96022940 infected with West
Nile Virus using a human serum as a negative control and
a serum derived from a monkey that was infected with
yellow fever virus.
Figure 3 is a phylogenetic tree of a large set of West
Nile Viruses based on sequences of the envelope protein.
In the upper part, the lineage I strains are given
(generally associated with severe disease) and in the
lower part, the lineage II strains are given (generally
associated with mild disease).
Figure 4 is a diagram showing the titer of the virus
preparation produced on cells as deposited under ECACC
no. 96022940 and in Mouse Brain. The titration was
determined using VERO cells and given in CCID50/ml.
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Figure 5 is a diagram showing the titer of the virus
preparation produced on cells as deposited under ECACC
no. 96022940 and in Mouse Brain. The titration was
determined using suckling mice and given in MLD50/0.1 ml.
Figure 6 are diagrams showing the time to disease or
death of the geese challenged with the West Nile Virus
strain Goose Israel 1998, vaccinated with a volume of
0.25 ml (A), 0.5 ml (B) or 1.0 ml (C) as compared to the
control geese that did not receive vaccination (negative
control); given here as line `4'. The vaccine comprising
the inactivated West Nile Virus produced on mouse brain
was taken as a positive control and given as a dotted
line `1'. The PER.C6-based vaccine including the adjuvant
is represented by line 12', and the PER.C6-based vaccine
without adjuvant is represented by line 13'.
Figure 7 is a diagram showing the time to disease or
death of all geese combined from the three volumes of
figure 6. Line 11' (dotted) represents the vaccine with
the inactivated West Nile Virus produced on mouse brain.
Line 12' represents the PER.C6-based vaccine including
the adjuvant and `3' is the negative control. The group
receiving the PER.C6-based vaccine without adjuvant is
not given here.
Figure 8 shows the TCID50 titers obtained with several
West Nile Virus strains grown on cells as deposited under
ECACC no. 96022940, using different dilutions of virus:
(A) New York 1999 (USA99b); (B) Kunjin 1991 (Aus9l); (C)
Madagascar 1978 (Mad78); (D) Kunjin 1960 (Aus60); (E)
Cyprus 1968 (Cyp68); (F) Goose Israel 1998 (Isr98).
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Figure 9 is a graphical representation of part of the
data shown in Table XVII for the Mouse Brain-MO and
PER.C6-WN-MO vaccines. Animals that died before challenge
(2 in both groups, leaving 58 animals in both groups for
challenge) were not included in this figure.
Figure 10 is a graphical representation of part of the
data shown in Table XVII for the different PER.C6 based
vaccines with or without aluminium-based adjuvants.
Animals that died in these groups before challenge (1 for
PER.C6-WN-AlOH leaving 59 animals; 4 in PER.C6-WN-AlPh,
leaving 56 animals; 3 in PER.C6-WN, leaving 57 animals)
were not included in this figure.
SUMMARY OF THE INVENTION
The present invention relates to methods for
producing a West Nile Virus comprising the steps of:
either infecting a cell, or a culture of cells, with
a West Nile Virus, or
providing a cell or a cell culture, with nucleic
acid coding for said West Nile Virus; and
culturing the cell, or the culture of cells,
obtained in either one of the previous steps in a
suitable medium under conditions that cause (or allow)
said virus to replicate in the cell, or that cause (or
allow) expression of said nucleic acid coding for said
West Nile Virus, or the culture of cells, thereby causing
the West Nile Virus to be produced, wherein the cell, or
the culture of cells, is characterized in that it
expresses at least an E1A protein of an adenovirus.
Products obtainable by the methods of the invention
are also part of the invention and may be used for
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vaccines in veterinary and human applications. The
invention thus also provides methods for producing,
inactivating and disrupting West Nile Viruses to be used
in veterinary applications.
The invention further relates to West Nile Viruses
or a West Nile viral proteins for use in a vaccine
obtainable by a method or a use according to the
invention, said West Nile Virus or said West Nile viral
protein being free of any non-human mammalian
proteinaceous material. Such West Nile Viruses and/or
West Nile viral proteins are suitable for the production
of (human and/or veterinary) vaccines against flavivirus
infections, such as infections by West Nile Virus, but
also to viruses highly related to West Nile Virus. The
invention also relates to such vaccines. Moreover, the
invention relates to human cells having a sequence
encoding at least one El gene product of an adenovirus in
its genome and having a nucleic acid encoding a West Nile
Virus or at least one West Nile viral protein.
The invention also relates to the production of a
lineage II strain of West Nile Virus that can be used in
a vaccine which can subsequently be applied for
protection against a lineage I strain infection, via a
mechanism referred to as cross-protection.
DETAILED DESCRIPTION
It is an object of the present invention to overcome
at least some of the problems outlined above. The present
invention relates to new vaccines against West Nile Virus
and to methods of producing same. It is appreciated in
the art that there is a great need for a potent vaccine
against West Nile Virus that should be sufficiently
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protective and that could be produced to a large scale.
The invention relates to the production of a whole-
inactivated (or killed) virus, based on West Nile Virus.
More specifically, it relates to a whole-inactivated West
Nile Virus that is being produced using cells that grow
in suspension and under serum-free conditions (animal
component free) to enable one to produce large-scale
batches. It further relates to the down-stream processes
for inactivating and purifying the produced viruses and
to the compositions for prophylactic and therapeutic
treatment.
In the art, West Nile Viruses have been grown and
passaged on a limited number of cells: Baby Hamster
Kidney (BHK) cells, fibroblasts, monkey kidney cells
(Vero), murine macrophages, C6/36 cells, and HeLa cells
(Dunster et al. 1990; Kurane et al. 1992; Wengler et al.
1990). Moreover, West Nile Virus has also been produced
on infant mouse brains. None of these systems is highly
suitable for producing West Nile Viruses in a combination
of large-scale production, in suspension and under animal
component-free and/or serum-free conditions. Especially
these procedures are not suited for the production of
vaccines that are to be used in humans. Vero cells grow
on micro carriers, not in suspension, while HeLa cells
are aggressive tumour cells and BHK is generally not
found to be safe for the production of therapeutics. The
other systems do not provide a platform for large
production scale since cells need to grow indefinitely
and should not be derived from a tumour. Also, vaccines
produced on mouse brains are not desired due to safety,
animal welfare, possible adverse side effects, allergic
reactions and scalability. Importantly, an embryonic
human retinoblast cell line obtained by transformation
and immortalisation through the early region-1 (El) from
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Adenovirus serotype 5 (cells as deposited under ECACC no.
96022940), a cell-based platform technology marketed by
Crucell Holland B.V. under the trade name PER.C601, has
been described to support the growth of influenza virus,
measles virus and Herpes simplex virus type 1 and 2 (WO
01/38362, Although the use of cells as deposited under
ECACC no. 96022940 for the production of flaviviruses has
been suggested in WO 01/38362, no specific mention is
made about West Nile Virus. The present invention
discloses that El-transformed human cells are able to
sustain the growth of West Nile Virus, thereby providing
a highly useful tool in the production of large batches
of West Nile Virus that are to be used for subsequent
purification and inactivation, and for use in vaccines.
The present invention relates to methods for
producing a West Nile virus comprising the steps of
infecting a cell, or a culture of cells, with a West Nile
Virus; and culturing the cell, or the culture of cells,
obtained in the previous step in a suitable medium under
conditions that cause said virus to replicate in the
cell, or the culture of cells, thereby causing the West
Nile Virus to be produced, wherein the cell, or the
culture of cells, is characterized in that it expresses
at least an E1A protein of an adenovirus. In another
embodiment of the invention, the invention relates to a
method for producing a West Nile Virus comprising the
steps of: providing a cell, or a culture of cells, with
nucleic acid coding for said West Nile Virus; and
culturing the cell, or the culture of cells, obtained in
the previous step in a suitable medium under conditions
that cause expression of said nucleic acid coding for
said West Nile Virus, thereby causing the West Nile Virus
to be produced, wherein the cell, or the culture of
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cells, is characterized in that it expresses at least an
E1A protein of an adenovirus. The El region of adenovirus
comprises several sub-regions, e.g., E1A and E1B. Many
studies have been performed in the past that revealed
that the different proteins expressed from the E1 region
play different roles in transformation of cells and
subsequent immortalisation to obtain cell lines. The
invention therefore also relates to a method according to
the invention, further characterized in that the cell, or
the culture of cells, expresses at least one protein of
the E1B region of an adenovirus. For large-scale
production it is required to have a stable cell line that
grows indefinitely. In a preferred embodiment the
invention provides methods according to the invention
wherein the cell, or the culture of cell, comprises a
(functional) E1 region of an adenovirus, and wherein the
El region is (stably) integrated in the chromosomal
genome of said cell.
Different types of cells can be used to apply the
methods of the present invention, however, the cell of
choice should be a cell that is stable, safe and non-
tumorigenic. Therefore the invention also relates to
cells, or cultures of cells, that are derived from a non-
tumorous human cell, and/or derived from a retinoblast,
and/or that is derived from an embryonic cell. More
preferably, said cell is derived from an embryonic
retinoblast. Highly preferred are methods according to
the invention in which the cell is a cell as deposited
under ECACC no. 96022940, or a derivative thereof. Other
cells that may be applied are cells derived from a kidney
cell or an amniocyte.
The nucleic acid that is administered to the cells
in the methods of the present invention is preferably
RNA, while said RNA is preferably delivered to the cell,
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or to the culture of cells, by means of a West Nile
Virus.
The West Nile Virus to be produced is preferably
lineage II strain West Nile B956, lineage II strain
Madagascar 1978, lineage II strain Cyprus 1968, lineage I
strain Kunjin 1960, lineage I strain Kunjin 1991, lineage
I strain Goose Israel 1998 or lineage I strain New York
1999. In another preferred embodiment said West Nile
Virus strain to be produced is a lineage II strain
selected from the group consisting of: Kenya, Uganda,
Senegal 1990, Uganda 1937, Uganda 1959, Central African
Republic 1972a, Central African Republic 1972b, Central
African Republic 1983, Madagascar 1986 and Madagascar
1988. Such West Nile Virus strains may therefore also be
used to deliver the nucleic acid to said cells, or said
culture of cells.
In another aspect of the invention, the invention
relates to methods according to the invention, wherein
said West Nile Virus is providing the nucleic acid to the
cells in a multiplicity of infection ranging from 5 to
5x10-7 plaque-forming units per cell. This range is
sufficient to obtain high titers of replicating West Nile
Viruses being produced, as can be seen in the examples
herein.
The invention also relates to methods according to
the invention, further comprising the steps of optionally
harvesting the produced West Nile Virus; and inactivating
the produced West Nile Virus; or to methods according to
the invention, further comprising the steps of, in either
order, inactivating the produced West Nile Virus; and
harvesting the produced West Nile Virus.
Inactivation of the produced West Nile Viruses is
achieved through methods known to persons skilled in the
art. Examples of inactivation are the use of UV-light, or
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the use of beta-propiolactone. A preferred inactivation
takes place through the use of formaldehyde (formalin-
induced inactivation).
In yet another embodiment, the invention provides
methods for obtaining West Nile Viruses that can be used
in sub-unit vaccines; therefore the invention also
relates to a method according to the invention wherein
the West Nile Viruses are produced, further comprising
the steps of disrupting the produced West Nile Virus; and
purifying one or more antigenic components of said West
Nile Virus, disrupted in the previous step. Such
antigenic components are generally the capsid (or
envelope) proteins of the West Nile Virus particle,
although it cannot be excluded that other (antigenic)
proteins, peptides, or entities from the virus can
obtained after using the methods of the present
invention. Such products are also encompassed within the
claims of the present invention.
The invention also relates to the use of a human
cell having a sequence encoding at least the E1A protein
of an adenovirus in its genome, which cell does not
produce structural adenoviral proteins for the production
of a West Nile Virus. Preferably, such cell also
comprises at least one E1B sequence coding for an E1B
protein, and more preferably, said cell comprises stably
integrated into its genome, an El region from an
adenovirus. A highly preferred adenovirus serotype that
is used for providing the El region is adenovirus
serotype 5.
A preferred use according to the invention is a use
of a human cell, wherein said human cell is derived from
an embryonic retinoblast. Highly preferred is the use of
a cell as deposited under ECACC no. 96022940, or a
derivative thereof.
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The invention also relates to the products obtained
by the methods of the present invention and to certain
applications therewith, such as the use in vaccines.
Therefore it also relates to a West Nile Virus obtainable
by a method according to the invention or by a use
according to the invention, for use in a vaccine, said
West Nile Virus being free of any non-human mammalian
proteinaceous material, while it also relates to vaccines
comprising a West Nile Virus, or a West Nile Virus
protein according to the invention, a pharmaceutically
acceptable carrier and optionally, an adjuvant. Possible
adjuvants that may be applied are mineral oil (generally
accepted for veterinary use) or alum-based adjuvants,
which may be applied for human use. Pharmaceutically
acceptable carriers are widely used and are well known in
the art.
To obtain cross-protection, which is a mechanism
through which a vaccine based on a relatively harmless
virus is used to raise protection against a virus that
would normally give rise to a relatively harmful disease,
it is preferred to use a vaccine comprising a whole-
inactivated lineage II West Nile Virus, a
pharmaceutically acceptable carrier and optionally, an
adjuvant. Such vaccines are provided by the present
invention.
The invention furthermore relates to a human cell
having a sequence encoding at least an E1A gene product
of an adenovirus (and preferably also a sequence coding
for an E1B gene product, and more preferably an El
region) in its genome and having a nucleic acid coding
for a West Nile Virus. Highly preferred is a human cell
according to the invention, wherein said human cell is a
derivative of a cell as deposited under ECACC no.
96022940, or a derivative thereof.
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The invention also provides a method of vaccinating
an animal or human subject against West Nile Virus
infection, comprising administering a vaccine according
to the invention to said animal or human subject.
The present invention relates to a method for
producing a West Nile Virus and/or a West Nile viral
protein for use as a vaccine, comprising: a) providing a
cell having at least a sequence encoding at least one
gene product of the El region of an adenovirus, with a
nucleic acid encoding said West Nile Virus and/or said
West Nile viral protein; b) culturing the cell obtained
in the previous step in a suitable medium; and c)
allowing for expression of said West Nile Virus and/or
said West Nile viral protein in said medium and/or said
cell. Preferably, said method also comprises the step of
purifying the produced West Nile Virus and/or West Nile
viral protein from the tissue culture supernatant and/or
the cells. Purification steps that can or may be used for
obtaining a purified West Nile Virus according to the
present invention include (sterile) filtration,
chromatography (e.g., using heparin sulphate),
diafiltration and/or (an)ion exchange chromatography.
Also preferred are methods of the invention, comprising
the step of inactivating the obtained West Nile Virus.
Inactivation is performed by using one or more of the
inactivation methods available, such as polysorbate
inactivation by for instance using Tween 20, Tween 40,
Tween 60 and/or Tween 80; and/or by long wavelength
ultraviolet radiation, and/or by furocoumarin, and/or by
ascorbic acid and/or a salt thereof. Preferably, the West
Nile Virus obtained by a method or a use according to the
invention is (whole-) inactivated by formalin and/or by
beta-propiolactone treatment. The viral RNA may be
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inactivated by nucleic acid disrupting agents such as
RNase.
The invention also relates to methods for the
production of West Nile viral proteins, and to the West
Nile viral proteins obtained by the methods of the
invention. The West Nile viral proteins can be obtained
by methods comprising a step of disrupting a West Nile
Virus obtained by a method of the present invention,
resulting in a subunit of the West Nile Virus. Such a
subunit, generally comprising at least one antigenic
component of the West Nile Virus, such as the envelope
protein(s) and/or fragments thereof can then be used to
produce a vaccine composition. The subunit of the West
Nile Virus can also be obtained by methods according to
the invention, wherein a nucleic acid encoding said
subunit is provided to a cell having at least a sequence
encoding at least one gene product of the El region of an
adenovirus, by means other than a West Nile Virus.
Therefore, said nucleic acid can be RNA, cDNA and DNA.
Preferably, said nucleic acid is RNA and also preferably,
the nucleic acid delivery vehicle is a West Nile Virus.
In another preferred embodiment, said cells used for
the production of West Nile Virus and/or West Nile viral
proteins are cultured in suspension and/or in serum-free
conditions. More preferably, said cells are cultured in
mammalian-component free medium. Therefore, the invention
also relates to methods for producing a West Nile Virus
and/or a West Nile viral protein for use as a vaccine,
comprising: a) providing a cell with a nucleic acid
encoding said West Nile Virus and/or said West Nile viral
protein; b) culturing the cell obtained in the previous
step in a suitable medium; and c) allowing for expression
of said West Nile Virus and/or said West Nile viral
protein in said medium and/or said cell, wherein said
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cell is cultured in suspension (non-adherent).
Preferably, a suitable medium for the methods of the
present invention is a medium lacking mammalian-derived
components or a serum-free medium, and optionally factors
that are recombinantly produced. The growing and
culturing of the cells for the methods of the present
invention may be performed by using different large-scale
set-ups, such as fed-batch, perfusion culture and wave
bags.
Providing the nucleic acid may occur during
different stages in the cell-culture process, and by
several different methods such as transfection,
electroporation, infection through viral-based delivery
(by carriers such as adenoviruses, alphaviruses and
poxviruses) or by complexes such as liposomes, or other
nucleic-acid delivery vehicles known in the art.
Purifying the produced West Nile Viruses and/or West Nile
viral proteins according to the methods of the present
invention may be performed by several methods known in
the art, such as single- or multistep (anion and/or
cation) exchange chromatography.
In a preferred embodiment a method is provided,
wherein the cell that is provided with the nucleic acid
encoding a West Nile Virus or a West Nile viral protein,
is derived from a non-tumorous human cell. More
preferably, such a cell is derived from a primary human
embryonic retinoblast. Even more preferred are methods
according to the invention, wherein the sequence encoding
at least a gene product of the El region is present in
the chromosomal genome of said cell. Highly preferred is
a method, wherein the cell provided with the nucleic acid
encoding a West Nile Virus is a cell derived from cells
such as those that are deposited under ECACC no.
96022940. As a `derivative' thereof can be understood any
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such cell that contains (in addition to the El region of
adenovirus serotype 5) another heterologous nucleic acid,
which may or may not be incorporated in the genome of the
cell. Examples of such derivatives are cells that contain
in addition a temperature sensitive E2A gene (PER.tsE2A;
as described in WO 01/38362) or other adenovirus genes
(such as described in WO 02/40665). As `derivatives' can
also be understood descendants of cells as deposited
under ECACC no. 96022940 that have been sub-cultured for
a prolonged period either under selective pressure and/or
mutagenising agents, or otherwise, since the deposit at
the ECACC. It is to be understood that the invention also
encompasses other cell lines that have been transformed
with at least the E1A region of an adenovirus. Other cell
lines that could be used for the present invention
comprise, but are not limited to: 293 cells, 293-E4orf6
cells, 911 cells, PER.E1B55K cells, PER.tsE2A cells,
HT1080 cells, amniocytes transformed with adenovirus El
and A549 cells transformed with adenovirus El.
In another aspect of the invention, the invention
provides methods for producing West Nile Viruses and/or
West Nile proteins according to the invention, wherein
the nucleic acid that is provided to the cell is RNA.
Preferably, said nucleic acid is provided by a West Nile
Virus, which contains (in its wild type form) a single
(+)RNA strand.
Many strains of West Nile Viruses have been
described in the art (Lanciotti et al. 2002). In one
embodiment of the present invention, the West Nile Virus
that provides the nucleic acid to the cell and/or that is
the West Nile to be produced is strain West Nile B956
(lineage II). Lineage II West Nile strains are normally
not related to human illnesses, while lineage I strains
are or can be (Lanciotti et al. 2002). The strains that
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may be produced with methods according to the invention
are given in Table I. More West Nile Virus strains, not
given in Table I, may also be produced with the methods
of the present invention. Preferably, New York 1999 (also
referred to as NY99 or USA99b, see below; several
isolates were reported from the New York area, ranging
from human, equine and avian sources), Israel 1998 (Goose
Israel 1998, sometimes referred to as Isr98 or IS-98-ST1,
see WO 02/081511), NY2000 3282, NY2000 3356, NY 1999
equine, Conn 1999, MD 2000, NJ 2000, Kunjin 1960 or 1991
(Aus6O or Aus9l respectively, also referred to as strains
MRM 16 or K 6453 respectively, see below), Madagascar
1978, Cyprus 1968 and Israel 1999 H are used for the
methods of the present invention. In one preferred
embodiment, West Nile Virus strain NY 1999 hum (a strain
isolated form a human brain in 1999 in the New York area)
or 385-99 (a strain isolated from the organs of a Snowy
Owl, Nyctea scandiaca, of The Bronx Zoo in the New York
outbreak) are produced using methods of the present
invention. It is to be understood that it is also
feasible to grow chimeric flaviviruses known in the art
(WO 98/37911; WO 01/39802; WO 01/60847; EP 0102228 A; EP
0872553 A; US patent no. 6,184,024) by methods of the
present invention, thereby circumventing possible
problems of low titers, high costs and/or safety issues.
In a specific embodiment, the present invention
provides methods according to the invention, wherein a
West Nile Virus is providing the nucleic acid to the
cells in a multiplicity of infection ranging from 5 to
5x10-7 plaque forming units per cell. As shown in the
examples, the inventors of the present invention were
able to show that it is feasible to obtain titers of 109
pfu/ml after three days following an multiplicity of
infection that were as low as 0.005 pfu/cell, using
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19
methods of the present invention, while titers obtained
with higher moi's were most likely even higher.
In one embodiment, the invention relates to the use
of a human cell having a sequence encoding at least one
El protein of an adenovirus in its genome which cell does
not produce structural adenoviral proteins for the
production of a West Nile Virus or at least one West Nile
viral protein. Preferably, said human cell is derived
from a primary retinoblast, and even more preferred are
uses according to the invention, wherein said human cell
is a cell as deposited under ECACC no. 96022940, or a
derivative thereof.
In another embodiment, the present invention relates
to a West Nile Virus or a West Nile viral protein
obtainable by a method according to the invention, or by
a use according to the invention, for use in a vaccine,
said West Nile Virus or said West Nile viral protein
being free of any non-human mammalian proteinaceous
material. A vaccine may be produced with a West Nile
Virus and/or West Nile Virus protein according to the
invention. Such a vaccine is preferably a composition
comprising a West Nile Virus and/or a West Nile viral
protein obtained, and a suitable (pharmaceutically
acceptable) carrier such as regularly used in the art of
preparing vaccine compositions for use in humans and in
veterinary applications. Optionally, said vaccine also
comprises an adjuvant. Preferably the human vaccine
comprises an adjuvant reagent that is acceptable for use
in humans, such as `Alum' or aluminium hydroxide, which
is an adjuvant known to persons skilled in the art.
Another adjuvant that may be applied is aluminium
phosphate. For veterinary use, it is also preferred to
use an adjuvant, for example Mineral Oil. Mineral Oil is
an adjuvant that is widely applied in the veterinary
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vaccine industry, for instance in the West Nile Virus
vaccine produced on mouse brains that has been approved
in Israel for vaccination of geese. The vaccine of the
present invention is applied for prophylactic,
therapeutic and/or diagnostic use. The vaccine according
to the invention is also applied for cross-vaccination
for viruses that are highly similar to West Nile Virus,
within the Flaviviridae family. For safety reasons, it is
preferable to vaccinate animals and human subjects
against West Nile Virus by using a vaccine comprising a
whole-inactivated Lineage II strain (associated with mild
disease), thereby protecting such individuals against a
lineage I strain infection (associated with severe
disease) via a mechanism known as cross-protection. It is
thus a highly preferred embodiment of the present
invention to produce a vaccine based on a Lineage II
strain that gives (cross-) protection against a Lineage I
West Nile Virus in animals as well as in humans. The B956
strain (Lineage II) is just one example of such a strain,
but those of skill in the art would be able to identify
other strains related to mild disease causing strains and
belonging to the Lineage II strains (for instance those
given in Table I), that provide cross-protection against
severe-disease causing strains upon vaccination with a
vaccine comprising the mild-disease causing strain. Other
preferred lineage II strains are Madagascar 1978 and
Cyprus 1968.
In another embodiment, the invention relates to a
human cell having a sequence encoding at least one El
gene product of an adenovirus in its genome and having a
nucleic acid encoding a West Nile Virus. Preferably,
wherein said human cell having a sequence encoding at
least one El gene product of an adenovirus in its genome
and having a nucleic acid encoding a West Nile Virus, is
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21
the cell line as deposited under ECACC no. 96022940, or a
derivative thereof.
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EXAMPLES
Example 1. Infection of cells with strain West Nile B956.
Cells (as deposited under no. 96022940 at the
European Collection of Animal Cell Cultures at the Centre
for Applied Microbiology and Research) and useful in
technologies as marketed as a platform by Crucell Holland
B.V. under the trade name PER.C6124, were banked and
cultured as described (WO 01/38362). A series of cells
were cultured in T80 culture flasks with 107 cells per
flask, and several dilutions of West Nile Virus were
incubated with these cells. The strain that was used was
West Nile B956 (lineage II) (Yamshchikov et al. 2001).
The virus was plaque purified on Vero cells using
techniques known to persons skilled in the art of growing
viruses and the virus was further produced on BHK cells.
A plaque assay was performed on Vero cells to determine
the viral titer of the starting material in the BHK
supernatant. This plaque assay was performed according to
the general methods applied in the art. This titer
appeared to be 5x108 plaque forming units per millilitre
(pfu/ml). A mock-transfection was performed using
dilution buffer (DMEM/5% FBS non-Heat Inactivated [nHI]).
Post-infection, samples were taken each day for `real
time' reverse transcriptase TaqMan PCR analysis
(Lanciotti et al. 2000) to determine the titer of the
virus produced, for Reverse Transcriptase (RT) PCR and
Immune Fluorescence. RT-PCR and TaqMan PCR was performed
using West Nile Virus specific primers (see below).
The input virus was diluted in DMEM/5o FBS nHI and
incubated for 1 h with the PER.C6111 cells in 8 serial
dilutions (10-1 to 10-8, with a multiplicity of infection
(moi) of 5 to 5x10-7 pfu/cell). After incubation of the
virus with the cells, the culture medium was discarded
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23
and replaced with fresh DMEM/5% FBS nHI (not-Heat
Inactivated) . Cells were cultured at 37 C/10% C02 at all
times. Cytopathological effect (CPE) was scored visually
after 24 h, 48 h and 72 h post-infection. Results are
shown in Table II. No CPE was detected at 24 h post-
infection. However, clear CPE was visible at the highest
moi's after 48 h, while in most cases significant CPE was
detected at 72 h post-infection. These results show
clearly that El-transformed human cells, such as PER.C6'
cells are able to sustain the production of West Nile
Viruses.
Example 2. RT-PCR on adenovirus E1-transformed human
embryonic retina cells infected with West Nile Virus.
Reverse Transcriptase Polymerase Chain Reaction (RT-
PCR) was performed on the mock-transfected cells as
deposited under ECACC no. 96022940, and on the 24 h and
48 h post-infection samples of such cells infected with
moi's of 5x101 and 5x10-4 pfu/cell. RNA samples were
diluted 1:10, 1:100 and 1:1000. RT-PCR was performed by
TM
using the Qiagen One-Step RT-PCR Kit (Qiagen) and general
methods known to persons skilled in the art of molecular
biology. The primers used were forward primer WNV 1: 5'-
CCA CCG GA(A/T) GTT GAG TAG ACG-3' (SEQ ID NO:1) and
reverse primer WNV 2: 5'-TTT G(T/G)T CAC CCA GTC CTC CT-
3' (SEQ ID NO:2). The negative control contained solely
water, while the positive control contained input virus
from the BHK supernatant. The PCR program that was used
was as follows: 30 min 50 C, 15 min 95 C, followed by 35
cycles of 30 sec 94 C, 30 sec 50 C and 1 min 72 C and a
final step of once 10 min 72 C. Obtained amplified
nucleic acid was loaded on a gel and visualised. Results
show that West Nile viral RNA is present in all samples
of cells that were infected with West Nile Virus, while
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24
no positive signal could be detected in the mock-
infections (Figure 1). The amount increases from 24 h to
48 h post-infection in the lower moi sample.
Example 3. Immune Fluorescence staining of cells as
deposited under ECACC no. 96022940 infected with West
Nile Virus.
Samples were obtained 48 h post-infection from the
cells as deposited under ECACC no. 96022940 that were
mock-infected and that were infected with an moi of 5xl03
(see Table II) with West Nile Virus B956. These samples
were used for immune fluorescence using, as a negative
control, normal human serum and, as a positive antiserum,
a Monkey derived antiserum directed against the envelope
of another flavivirus, namely Yellow Fever virus. Yellow
Fever virus is highly similar to West Nile Virus, and the
antiserum generally recognizes also the envelope of the
West Nile Virus. The serum was obtained after injection
of Yellow Virus in a monkey and recovering of the serum.
Fixating with aceton and staining procedures were
performed according to general methods known in the art,
and by using a standard fluorescence microscope. Although
the human serum gave a relatively high background, a
positive signal could be determined on the cells stained
with the monkey antiserum as can be seen in figure 2.
These results indicate that West Nile Virus is able to
infect human adenovirus El-transformed cells, such as
PER.C61'A' cells.
Example 4. Real-time TagMan PCR for the detection and
quantification of West Nile Virus RNA in infected PER.C67"
cells.
To determine the titers of the West Nile Virus that
were produced by the cells as deposited under ECACC no.
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96022940, a real-time reverse transcriptase TaqMan PCR
was performed using on all samples, except for the 72 h
post-infection samples of the three highest moils as
shown in Table II.
RNA was extracted using the spin protocol of the
TM
QIAamp Viral RNA Mini Kit (Qiagen) following the
description provided by the manufacturer. 200 }11 cell
culture supernatant was mixed with 60 ul elution volume.
Amplification and real-time detection was performed using
the MasterMix1 without UNG with the RNase Inhibitor Mix
(Applied Biosystems) and the QuantiTect Probe RT-PCR Kit
(Qiagen) using the forward primer WNV 1 and reverse
primer WNV 2, and with the VIC labelled probe: 5'-VIC-TGC
TGC CTG CG(A/G) CTC AAC CC-TAMRA-3' (SEQ ID NO:3). All
protocols were performed using the instructions provided
by the manufacturers and generally following the methods
as described by Hadfield et al. (2001) and Lanciotti et
al. (2000). Concentrations used were: Forward primer 300
nM, Reverse primer 900 nM and VIC-labelled probe 100 rim.
Results are shown in Table III. Clearly, PER.C6
cells are able to sustain growth of at least a titer of
1x109 pfu/ml using an input moi of 5x10-3 pfu/cell, 72 h
post-infection, while probably even higher titers were
obtained in lower dilutions. This indicates that PER.C6 '
is a very useful tool for the production of West Nile
Viruses. Since it has been shown that PER.C62" cells can
grow to very high densities, in serum-free medium in
suspension, and in large incubators (>1000 litre), it is
now possible to obtain very large batches of West Nile
Virus that can be used for inactivation and subsequent
use in vaccines against West Nile viral infections, and
most likely also against other flavivirus infections due
to cross-vaccination. The obtained West Nile Viruses can
also be used to disrupt and thus to make split-vaccines
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26
or to purify separate subunits from the disrupted virus,
such as the envelope protein(s) for the use in so-called
sub-unit vaccines. Methods for disrupting enveloped
viruses are known to the skilled person.
Example 5. Production of West Nile Virus strain Goose
Israel 1998 on human adenovirus El-transformed cells for
an animal-challenge model in geese.
Cells as deposited under ECACC no. 96022940 were
banked and cultured as described (WO 01/38362). West Nile
Virus strain Goose Israel 1998 (Malkinson et al. 1998 and
2001; Lanciotti et al. 1999), was produced by inoculating
sub-confluent (80%) monolayers of such cells, grown in
T175 flasks in DMEM plus 10% Fetal Bovine Serum, with the
virus at an Multiplicity of Infection (MOI) of 0.001
vp/cell. The virus-containing culture supernatant was
harvested at day 6 post-infection and cleared by
centrifugation. The resulting virus preparation was
titrated on Vero cells, using methods known to persons
skilled in the art, and seeded in 96-well plates
following standard procedures (Bin et al. 2001; Malkinson
et al. 2001). The titer was determined to be 1010.66 or
4.57x1010 CCID50/ml (Cell Culture Infectious Dose 50 per
ml) for the virus produced on cells as deposited under
ECACC no. 96022940 and 1010.83 or 6.76x1010 CCID50/ml for
the virus produced on mouse brain. The results of the
titration on Vero cells are given in Figure 4.
The virus preparation was also titrated in suckling
mice by intra-cranial (i.c., NB: i.c. may also indicate
intra-cerebrally; all i.c. injections were meant to be
directly in the brain) injection of 0.03 ml of 10-fold
serial dilutions of the culture supernatant. Mice were
observed for mortality over a 9-day period. The mortality
titer for the PER.C6 produced viruses was found to be
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109.76 MLD50/0.1 ml (mouse lethal dose 50 per 0.1 ml) , while
the titer of the virus preparation from mouse brain was
found to be 109'63 MLD50/0.1 ml, as shown in Figure 5.
The virus preparation was inactivated by adding 1 ml
formaldehyde (stock solution of 4%) to 100 ml virus-
containing PER.C671' supernatant and by stirring this
solution at 4 C for 4 weeks. Inactivation was checked by
titration of the inactivated material in suckling mice as
follows: 0.03 ml of inactivated antigen was injected i.c.
in suckling mice, that were subsequently observed for 14
days for mortality. No mortality was observed in any of
the animals. Thus, it is shown here that El-transformed
cells can sustain the growth of the Goose Israel 1998
West Nile Virus strain to very high titers and that the
produced West Nile viruses can be inactivated
sufficiently by the treatment of formaldehyde.
Example 6. Animal vaccination/challenge study using a
PER.C61"-produced West Nile vaccine in comparison with a
Mouse brain-produced vaccine.
Two-weeks old geese were vaccinated subcutaneous on
day 0 and boosted with the same volume on day 14
according to the scheme as depicted in Table IV. General
methods were as described by Malkinson et al. (2001),
while also the Mouse brain vaccine (serving as a positive
control) was produced as described by Malkinson et al.
(2001). Mineral oil, which is a common additive in
veterinary vaccines and used as an adjuvant, was blended
with the preparation and used to form a suitable
vaccine/adjuvant mixture. Some groups receiving the
inactivated virus produced on PER.C6 received no mineral
oil, while the positive control group did, as shown in
Table IV. Although Mineral Oil is a well-recognized
adjuvant in the art of veterinary vaccines, it is
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unlikely to be used in humans. Therefore, it is also an
aspect of the invention to prepare vaccines comprising
adjuvant reagents that are acceptable for use in humans.
An example of such an adjuvant is `Alum', or aluminium
hydroxide, also known to persons skilled in the art. It
is to be understood that all adjuvant reagents that are
acceptable for use in humans can or may be used with the
inactivated West Nile Viruses according to the present
invention.
All animals were challenged with West Nile Virus
strain Goose Israel 1998 at day 21 after the boost
injection (day 35 from start of vaccination). Animals
were observed daily for signs of disease and mortality
for a period of 3 weeks after challenge. Blood samples
were taken from all animals before each vaccination and
boost and before challenge and from animals surviving the
challenge after 3 weeks.
The results of these experiments are shown in Table
V and VI, and clearly indicate that the PER.C6 produced
West Nile Viruses (in an inactivated form and in the
combination with an adjuvant) are as potent as a vaccine
as the vaccine produced on Mouse Brain. The survival rate
in the animal groups receiving PER.C6 based vaccine plus
mineral oil ranges from 88% to 92.3% (with all three
groups averaged to 89.7%, which equal 70 animals out of
78 being disease-free), while the Mouse Brain based
vaccine plus mineral oil ranges from 89.5% to 95% (with
all three groups averaged to 93%, which equals 53 animals
out of 57 being disease-free). Whether the difference
between the rates found with the PER.C6 versus the Mouse
Brain based vaccine is significant cannot be determined,
since the number of animals in both groups is too low.
The percentage of survival in the negative control group
was 0%, which is a significant difference compared to the
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29
groups that received a vaccine; all animals in the
negative control group died within 6 days upon challenge.
The results are also depicted graphically in figure 6 for
the three separate volumes used (Figure 6A: 0.25 ml,
Figure 6B: 0.5 ml and Figure 6C: 1.0 ml). The group
receiving the Mouse Brain-based vaccine is indicated with
a dotted line 1, the group receiving the PER.C6-based
vaccine + adjuvant is indicated with line 2, the group
receiving the PER.C6-based vaccine minus adjuvant is
indicated with line 3, and the negative control group is
indicated with line 4. Figure 7 shows the results
summarized in Table VI for the three volume groups taken
together and also clearly indicates that the PER.C6 based
vaccine (line 2) is as potent as the Mouse Brain based
vaccine (dotted line 1) as compared to the negative
control (line 3) in this goose model.
Example 7. Cross-protection using different Lineage I and
II West Nile Virus strains in an animal study.
The West Nile Virus strains depicted in Table VII
are used in a cross-protection study to prevent disease.
This is to investigate the possibility of obtaining
cross-protection against a Lineage I strain (associated
with severe disease) by using a vaccine based on a
Lineage II strain (associated with mild disease), when
the vaccine is made of inactivated West Nile Viruses
produced in El transformed cells such as PER.C6. Some
strains exhibit very low neuroinvasion ability when
administered intra-peritoneal (i.p.), while others do
better. For instance, it is known from the art that the
NY 99 (Lineage I) strain has a LD50 (Lethal Dose 50) of
only 0.5 plaque forming units (pfu) when administered
i.p., while other strains such as Kunjin 1960 (Lineage I)
and Cyprus 1968 (Lineage II) have a LD50 dose of more than
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10,000 pfu when administered i.p. If the viruses are
administered intra-cranial no big difference in
neurovirulence is found. These aspects of these strains
are known in the art.
Because of safety reasons it is preferable to use a
Lineage II strain for the production of a vaccine that
will give protection against disease caused by Lineage I
strains. For this, the strains depicted in Table VII were
all grown on PER.C6 and are subsequently inactivated as
described above. The inactivated viruses are mixed with
the appropriate adjuvant (most likely mineral oil for
geese) and used in a vaccination/boost/challenge study as
outlined above. Different groups of animals are used for
the different vaccines, while the challenge is performed
with the Goose Israel 1998 and the New York 1999 (NY 99)
strains. If a Lineage II strain-based vaccine gives
sufficient protection against a Lineage I virus
challenge, and if the passage history of the preferred
strain is acceptable (for safety reasons), such strain is
preferably the basis for a vaccine that is to be used in
humans.
Infection of cells as deposited under ECACC no.
96022940 with the different west Nile Virus strains was
generally performed as follows. Cells were trypsinized,
numbers were determined and cells were seeded in 25 cm2
flasks using 5x106 cells per 5 ml DMEM, 5% FBS medium per
flask. Cells were incubated for 24 h at 37 C under 10%
C02. Before infection the medium was replaced with fresh
DMEM +5% FBS. The general assumption is that the cell
number is doubled since seeding, after which the
multiplicity of infection (MLD50 or TCID50/cell) could be
determined. Then the viruses were added and incubated
with the cells at 37 C 10% C02 for several days. Then,
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31
when full cpe had occurred, cells and medium were
harvested and supernatant was clarified by
centrifugation, 10 min at 2000 rpm. Subsequently,
produced virus titers after using different virus
dilutions were determined. Results regarding cpe are
depicted in Tables VIII to XIII, while the TCID50 titers
obtained using the different virus dilutions are given in
Figures 8A to F. The highest titers in TCID50/ml for each
strain were (with the specific input moi between
brackets):
USA99b: 1xl09 (5x10-7)
Isr98: 5.6x109 (8.9x10-6)
Mad78: 1x107 (1.6x10-4)
Cyp68: 5.6x108 (5x10-4 )
Aus60: 3.2x107 (1.6x10-2)
Aus9l: 1x108 (5x10-3) .
These results show that all tested West Nile Virus
strains grow on PER.C6 to significant titers using
relatively low moi's, albeit with different efficiencies.
The best harvest days seem to be day 5 or day 6 but this
may depend on the culture set-up, the density of the
cells, media, scale, and other culture conditions.
Example 8. Protection study using whole-inactivated
vaccines with West Nile Virus produced on PER.C6 in
combination with different adjuvant compounds.
A double blind vehicle controlled study to assess
the effect of aluminium hydroxide and aluminium phosphate
adjuvants on the efficacy of a PER.C6 based West Nile
vaccine in geese was performed. In a previous proof of
concept experiment, full protection of geese by
vaccination with a PER.C6 based experimental WN vaccine
was found using mineral oil as adjuvant (see Example 6).
However, mineral oil cannot be used in vaccines for human
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applications. This implicates the requirement of testing
alternative adjuvants that were previously approved for
human use.
Aluminum-based adjuvants have been used for decades
to increase the immune response of vaccines for human and
animal use. Two types of aluminum-base adjuvants,
Rehydragel aluminum hydroxide and Rehydraphos aluminum
phosphate were tested for the immune potentiating effect
in PER.C6-based West Nile Virus vaccines. The endpoint
was a percentage of disease free survival in the test
groups with adjuvant that was twice the percentage of
that obtained in the group without adjuvant. Apart from
investigating the effect of aluminum-based adjuvant, the
study aimed at confirming the results of the previous
study, using larger group sizes of geese and a double
blind set-up.
The bulk material of the West Nile Virus produced on
mouse brain in this study was produced as described
(Malkinson et al. 2001) and titrated on VERO cells. The
titer of this control vaccine was 1011.25 TCID50/ml and
referred to as Mouse Brain-MV.
The PER.C6-based West Nile Virus bulk was produced
by inoculating sub-confluent (80%) monolayers of PER.C6
cells, grown in T175 flasks in DMEM with FBS, with strain
Goose Israel 1998 (Mouse brain derived) at a multiplicity
of infection (moi) of 10-3. The virus-containing culture
supernatant was harvested at day 6 post-infection and
clarified by centrifugation at 1000 RPM for 20 min. The
resulting virus preparation was titrated on VERO cells
seeded in 96-well plates. The titer turned out to be
1011'66 TCID50/ml. The virus preparation was subsequently
titrated in suckling mice by intra-cranial injection of
30 }1l 10-fold serial dilutions of the culture
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33
supernatant. Mice were observed for mortality over a 9-
day period. The titer was 109''6 MLD50/ml and referred to
as PER.C6-WN.
A batch of supernatant from PER.C6 cells was
produced under similar conditions as for PER.C6 cells
infected with West Nile Viruses. However, for this, cells
were not infected (`Sham'-infected). Supernatant was
harvested and clarified by centrifugation. This
preparation was used as a negative control and referred
to as PER.C6-SH.
The West Nile Virus bulk preparations from PER.C6
and the supernatant of Sham-infected PER.C6 cells were
inactivated by adding 1 ml formaldehyde (stock solution
of 4%) to 100 ml virus-containing PER.C6 supernatant and
by stirring at 4 C for 4 weeks. Inactivation of the Mouse
Brain-WN was performed using methods known in the art.
Inactivation was controlled by titration of the
inactivated material in suckling mice. To this end, 30 pl
of 10-fold serial dilutions (10 '0 - 1010'0) were injected
intra-cranial in suckling mice. The animals were
subsequently observed for mortality over 9 days. The
resulting titers were sufficiently low; no live harmful
virus was detectable using this animal test assay.
TM
PER.C6-WN was formulated with Rehydragel LV
containing 2% A1203 (Reheis) as follows: 98.75 ml of DMEM,
5% FBS was added to 35 ml of clarified inactivated virus
bulk (pH 7.2-7.4) and mixed in a clean sterile 250 ml
TM
bottle. Subsequently, 6.25 ml of Rehydragel LV was added.
The solution was manually swirled and then set on an
orbital shaker set at 90 rpm for 1 h 25 min at 20 C. The
formulated vaccine, referred to as PER.C6-WN-AlOH was
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then aliquotted aseptically at 23 ml/vial (6 vials) and
stored at 4 C.
TM
PER.C6-WN was formulated with Rehydraphos containing
2% solid aluminium phosphate (Reheis) as follows: 98.75
ml of DMEM, 5% FBS was added to 35 ml of clarified
inactivated virus bulk (pH 7.2-7.4) and mixed in a clean
sterile 250 ml bottle. Subsequently, 6.25 ml of
Rehydraphos was added. The solution was manually swirled
and then set on an orbital shaker set at 90 rpm for 1 h
25 min at 20 C. The formulated vaccine, referred to as
PER.C6-WN-A1Ph was then aliquotted aseptically at 23
ml/vial (6 vials) and stored at 4 C.
PER.C6-WN was formulated as follows to obtain a
vaccine without adjuvant: 105 ml medium was added to 35
ml of clarified inactivated virus bulk (pH 7.2-7.4) in a
clean sterile 250 ml bottle. The solution was manually
swirled and then set on an orbital shaker set at 90 rpm
for 1 h 25 min at 20 C. The vaccine was then aliquotted
aseptically at 23 ml/vial (6 vials) and stored at 4 C.
TM
PER.C6-SH was formulated with Rehydragel as follows:
33.85 ml medium was added to 12 ml of clarified
inactivated virus bulk (pH 7.2-7.4) in a clean sterile
TM
100 ml bottle. Subsequently, 2.15 ml of Rehydragel LV was
added. The solution was manually swirled and then set on
an orbital shaker set at 90 rpm for 1 h 25 min at 20 C.
The vaccine referred to as PER.C6-SH-A1OH, was then
aliquotted aseptically at 24 ml/vial (2 vials) and stored
at 4 C.
TM
PER.C6-SH was formulated with Rehydraphos as
follows: 33.85 ml medium was added to 12 ml of clarified
inactivated virus bulk (pH 7.2-7.4) in a clean sterile
TM
100 ml bottle. Subsequently, 2.15 ml of Rehydraphos was
added. The solution was manually swirled and then set on
an orbital shaker set at 90 rpm for 1 h 25 min at 20 C.
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The vaccine referred to as PER.C6-SH-A1Ph, was then
aliquotted aseptically at 24 ml/vial (2 vials) and stored
at 4 C.
PER.C6-SH was formulated as follows to obtain a
control vaccine without adjuvant: 36 ml medium was added
to 12 ml of clarified inactivated virus bulk (pH 7.2-7.4)
in a clean sterile 100 ml bottle. The solution was
manually swirled and then set on an orbital shaker set at
90 rpm for 1 h 25 min at 20 C. The vaccine was then
aliquotted aseptically at 24 ml/vial (2 vials) and stored
at 4 C.
Mouse Brain-WN, PER.C6-WN and PER.C6-SH vaccines
were formulated with Mineral Oil using methods known to
persons skilled in the art'of adjuvating vaccines. These
preparations were referred to as Mouse Brain-WN-MO,
PER.C6-WN-MO and PER.C6-SH-MO respectively.
The study enrolled 400 geese (Anser anser, females
and males, 3 weeks old). All animals were housed in rooms
grouped according to their vaccination separate from
other animals (Group size: n=20). All the rooms were
fitted with mosquito proof netting. All animals included
in this study were monitored daily for behavior and
general health. At day 0 prior to the administration of
the primary injection and at days 14 and 35, prior to the
second injection and challenge with West Nile Virus,
respectively, blood samples were collected from all
animals from the jugular vein. Sera, processed from blood
samples were used in anti-WN-E protein ELISA assays and
in plaque reduction neutralization (PRNT) assays.
The geese were divided and treated as shown in Table
XIV. Injections were performed on days 0 and 14. One ml
per injection was administered subcutaneously in the neck
(s.c.). On day 35 (3 weeks after the second vaccination)
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all the animals were challenged intra-cerebrally (i.c.).
The West Nile challenge virus (Goose Israel 1998) was
passaged three times in VERO cells and titrated in VERO
cells. A stock of virus was aliquotted and stored at -
70 C. The virus was inoculated intra-cranially into the
geese at a dose of 102.0 TCID50/0.1 ml. After challenge
animals were scored for morbidity and mortality twice
daily. Among the 400 geese included in the experiment, 14
geese died during the study. Two animals died in the
Mouse Brain-MO group (leaving 58 animals for challenge),
while also in the PER.C6-WN-MO group two animals died
before challenge. All the animals that died before
challenge were included in the statistical analysis,
since the risk of vaccination has to be taken into
account (intention-to-treat analysis). The results are
shown in Tables XV to XVIII and figures 9 and 10.
Clearly, the data indicates that a West Nile Virus
vaccine produced on PER.C6 raises a proper protection
against a West Nile Virus challenge, which confirms the
earlier findings with smaller groups of geese. It seems
as if the mineral oil adjuvant works best in these
veterinary vaccines using geese. The difference between
the data obtained with the Mouse Brain vaccine plus
mineral oil (Mouse brain-MO) and PER.C6 based vaccine
plus mineral oil (PER.C6-WN-MO) is not significant, which
indicates that (in this large group of animals) both
vaccines are highly suited for use as a veterinary
vaccine. The data also shows that the difference between
Mouse Brain-MO and PER.C6-WN-AlOH is significant, which
means that in geese a significantly lower number of
animals are protected against disease and death when the
aluminum hydroxide adjuvant is applied, as compared to
the use of mineral oil. The same holds true for the
aluminum phosphate adjuvant in comparison to mineral oil.
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However, since the number of surviving animals in the
groups that received a PER.C6 based West Nile Virus
vaccine without an adjuvant is lower than in the groups
with an adjuvant, this data is indicative that an
adjuvant adds to the protection capacity of the vaccine.
Although these results suggest that mineral oil is
the best-suited adjuvant, this cannot be extrapolated to
the human situation. First, mineral oil is not suited for
human use, so it will be hard to test whether these
results will be reflected in the human situation, and
second, humans may be more responsive towards aluminum-
based adjuvants than animals such as geese. Studies
towards the most suitable adjuvant for human use in
combination with the PER.C6 based West Nile Virus vaccine
are to be performed. We predict that a vaccine
preparation with Alum (aluminium hydroxide or aluminium
phosphate) as adjuvant will be useful to protect humans
against West Nile Virus infections.
Example 9. Production of West Nile Viruses on suspension-
growing PER.C6 cells.
For high volume culture of cells in vitro, it is
preferred to grow the cells in dense cultures to
subsequently reach the highest titers of viruses
produced. For high-density culture it is preferred that
cells grow in suspension instead of in attached settings.
PER.C6 is a cell line that has proven to grow well in
both settings. An experiment was performed to see whether
suspension growing PER.C6 cells could sustain the growth
of West Nile Viruses and to compare these results to the
production in attached settings. Example 7 already showed
that different West Nile Viruses could infect PER.C6
cells and that these cells (in attached setting) were
able to sustain growth to significant titers. For the
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suspension growth, two different available suspension
culture media were used: VPRO (JRH) and APM (Gibco).
The experiment was performed as follows: 6-wells
plates containing 1x106 cells per ml were inoculated with
different input virus moi's. Samples were taken on day 2,
3, 4, 5 and 6 post infection and TCID50/ml titers were
determined. The results are depicted in figure 11A (VPRO
medium) and figure 11B (APM medium).
The results indicate that suspension-growing PER.C6
cells are well capable of sustaining growth of West Nile
Virus to. high titers. Thus, this indicates that PER.C6 is
a proper choice for growing West Nile Viruses for
obtaining large batches and thereby obtaining large
amounts of vaccines for both human as well as veterinary
use in a safe, clean and easy-to-handle high-throughput
way.
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Table I. Identified lineage I and II strains of West Nile
Viruses (from Lanciotti et al. 2002).
Lineage I strains Lineage II strains
Egypt 1951 Kenya
France 1965 Uganda
South Africa Senegal1990
Israel 1952 Uganda 1937
Romania 1996 M C. Afr. Rep 1972a
Kenya 1998 C. Afr. Rep 1972b
Senegal 1993 C. Air. Rep 1983
Morocco 1996 Uganda 1959
Italy 1998 Madagascar 1988
Volgograd 1999 Madagascar 1986
New York 1999 Madagascar 1978
Goose Israel 1998 Cyprus 1968
NY2000 3282
NY2000 3356
NY 1999 equine
NY 1999 hum
Conn 1999
Romania 1996
Romania 1996 H
MD 2000
NJ 2000
Israel 1999 H
C. Afr. Rep 1989
Senegal 1979
Algeria 1968
C. Air. Rep 1967
Ivory Coast 1981
Kunjin (strains 1960, 1973, 1984b, 1991, 1984a, 1966 and 1994)
India (strains 1955a, 1980, 1958,1955b)
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Table II. CPE score (in %) on PER.C6 cells infected with
serial dilutions of West Nile Virus B956 (moils range
from 5 to 5x10-7 pfu/cell). CPE was scored 24 h, 48 h and
72 h post-infection. Input virus titer was 5x106 pfu/ml as
determined in plaque assays using Vero cells, using
general methods applied in the art. CPE was not scored on
the samples depicted as `nd', these were discarded since
full CPE was already detected at 48 h post-infection.
CPE
Dilution moi Input virus 24 h post- 48 h post- 72 h post-
v/v (pfu/cell) fu's/flask infection infection infection
mock 0 0 0 0 0
10' 5 5x10 0 100 nd
10" 5x10 5x10 0 100 nd
10" 5x10" 5x10 0 50 100
10 5x10" 5x10 0 5 100
10- 5x10 5x10 0 0 80
10 5x10 5x10 0 0 50
107 5x10 50 0 0 0
10 5x10" 5 0 0 0
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Table III. West Nile Virus titers as determined by real-
time RT-TaqMan PCR. Titers are given in pfu/ml (right
columns depicted as 24 h, 48 h and 72 h post-infection).
Dilution moi Input virus 24 h post- 48 h post- 72 h post-
fu/cell fu/flask infection infection infection
10'1 5 5x107 3,6x107 5,9x107 nd
10"2 5x10-1 5x106 6,5x106 2,7x107 nd
10"3 5x10-2 5x105 1,9x106 6,1x107 nd
10-4 5x10-3 5x104 2,5x105 1,9x10 1,0x109
,10"5 5x104 5x103 2,2x104 5,7x106 4,3x108
10-6 5x10"5 5x102 5,6x103 7,1x105 1,5x10$
10"7 5x10"6 50 3,4x103 7,9x104 9,8x107
10$ 5x10-7 5 5,5x103 4,2x103 1,2x106
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Table IV. Vaccination scheme using PER.C60" based West
Nile vaccine and a Mouse brain produced West Nile vaccine
in two-week old geese.
PART A
Group Material Volume Vaccination Adjuvant
(n=)
1. 20 WN vaccine PER.C6" 0.25 ml Day 0 None
Day 14
2 22 WN vaccine PER.C6" 0.5 ml Day 0 None
Day 14
3 23 WN vaccine PER.C6" 1 ml Day 0 None
Day 14
PART B
1. 25 WN vaccine PER.C6'" 0.25 ml Day 0 Mineral oil
Day 14
2. 25 WN vaccine PER.C6" 0.5 ml Day 0 Mineral oil
Day 14
3 28 WN vaccine PER.C6" 1.0 ml Day 0 Mineral oil
Day 14
PART C
1. 19 Mouse brain vaccine 0.25 ml Day 0 Mineral oil
Day 14
2. 20 Mouse brain vaccine 0.5 ml Day 0 Mineral oil
Day 14
3 18 Mouse brain vaccine 1.0 ml Day 0 Mineral oil
Day 14
4. 13 No Vaccine
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Table V. Results of the experiment showing the percentage
of geese surviving (and being disease free) upon
vaccination with whole inactivated West Nile Viruses
(WNV) produced in tissue culture using PER.C6''t' cells or
using mouse brains (MB) and challenged with West Nile
Virus strain Goose Israel 1998. Certain West Nile Virus
vaccines held Mineral Oil as an adjuvant, as indicated.
The volume of the vaccine as described in the description
is also indicated. N = the number of animals per group.
The control group of 13 animals did not receive any
vaccine and all died upon challenge.
Vaccine Volume Adjuvant N Disease/ Disease free
Death /survival
WNV PER.C6 1.0 ml None 23 15 34.8%
WNV PER.C6 0.5 ml None 22 10 54.6%
WNV PER.C6 0.25 ml None 20 11 45.0%
WNV PER.C6 1.0 ml Mineral Oil 28 2 92.9%
WNV PER.C6 0.5 ml Mineral Oil 25 3 88.0%
WNV PER.C6 0.25 ml Mineral Oil 25 3 88.0%
WNV MB 1.0 ml Mineral Oil 18 1 94.4%
WNV MB 0.5 ml Mineral Oil 20 1 95.0%
WNV MB 0.25 ml Mineral Oil 19 2 89.5%
Control --- --- 13 13 0%
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Table VI. The results of Table V taken together for the
geese groups that received the Mouse Brain-derived
vaccine and the PER.C601-derived vaccine (all with Mineral
Oil as adjuvant) as compared to the control group that
did not receive vaccination.
Vaccine Adjuvant N Disease/ Disease free
Death /survival
WNV PER.C6 Mineral Oil 78 8 89.7%
WNV MB Mineral Oil 57 4 93.0%
Control --- 13 13 0%
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Table VII. Selection of Lineage I and II West Nile Virus
strains used in a cross-protection assay in geese.
Viruses of the different strains were produced in PER.C6.
They are purified, inactivated, mixed with adjuvant and
used for a protection against a challenge with live Goose
Israel 1998 and New York 1999 West Nile Viruses. The
right two columns give the inoculation LD50 in pfu for
each of the strains as it is known from the art with
respect to neuroinvasion upon intra-peritoneal (i.p.)
administration or with respect to neurovirulence upon
intra-cranial administration.
Lineage Strain i.p. i.c.
I Goose Israel 1998 (Isr98) n.d. 0.1
I New York 1999 (USA99b) 0.5 0.1
I Kunjin 1960 (Aus60) >10,000 n.d.
I Kunjin 1991 (Aus9l) >10,000 3.2
II Cyprus 1968 (Cyp68) >10,000 0.5
II Madagascar 1978 (Mad78) >10,000 n.d.
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Table VIII. cpe scores after infection of PER.C6 cells
with West Nile Virus New York 1999 (NY99 isolated from a
Snowy Owl, The Bronx Zoo, New York)
USA99b
dilutions day 2 da 3 day 4 day 5 day 6
10-3 + <1% + 75% full cpe na na
10-4 + 25% +(75%) full cpe na
10-5 - +1% +1% +10% +75%
10-6 + <1% + <1% +(25-50%)
10-7 1- 1- + <1% + <1% +(5%)
10-8
1- 1-
Table IX. cpe scores after infection of PER.C6 cells with
West Nile Virus Aus9l (strain Kunjin 1991 [K 6453],
isolated in Australia from Culex annulirostris mosquitoes
in 1991)
US91
dilutions day2 da 3 day 4 day 5 day 6
10-3 + 1 % +(1-5%) +(10%) +(1-5%)
10-4 +<1% +1% +1%
10-5
10-6
10-7
10-8
Table X. cpe scores after infection of PER.C6 cells with
West Nile Virus Mad78 (strain Madagascar 1998)
MAD78
dilutions day 2 day 3 day 4 day5 day 6
10-3 + 1% + ON
10-4 +(<l%)
10-5
10-6
10-7
10-8
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Table XI. cpe scores after infection of PER.C6 cells with
West Nile Virus Aus6O (strain Kunjin 1960 [MRM 16),
isolated in Australia from Culex annulirostris mosquitoes
in 1960)
US60
dilutions day 2 day 3 day 4 day 5 day 6
10-3 +(25%) + 50-75% + (75%)
10-4 +(1-5%) +(1-5%) +(10%)
10-5 + <1% + 1%
10-6 + <1
10-7
10-8
Table XII. cpe scores after infection of PER.C6 cells
with West Nile Virus Cyp68 (strain Cyprus 1968)
CYP68
dilutions day 2 day 3 day 4 day5 day 6
10-3 + (1%) + (1%) +(10%) +(25%)
10-4 +1% +1%
10-5
10-6
10-7
10-8
Table XIII. cpe scores after infection of PER.C6 cells
with West Nile Virus Isr98 (strain Goose Israel 1998)
1SR98
dilutions day 2 day 3 day 4 day 5 day 6
10-3 + (<I%) +(50%) full c e na na
10-4 +(25%) +(75%) full cpe na
10-5 + (1%) +(1-5%) + (50-75%) full cpe
10-6 + <1% + <1% + (25%) + (50%)
10-7 + <1
11 1- 1- 1-
110-8
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Table XIV. Vaccination scheme of geese using West Nile
Virus vaccines comprising different adjuvant compounds.
Group Material Volume Injections Adjuvant
size
3 x 20 Mouse Brain WN-MO 1 ml Day 0 Mineral oil
Day 14
3 x 20 PERC6-WN-MO 1 ml Day 0 Mineral oil
Day 14
3 x 20 PERC6-WN-AIOH 1 ml Day 0 Aluminum hydroxide
Day 14
Rehydra el
3 x 20 PEPC6-WN-AIPh 1 ml Day 0 Aluminum phosphate
Day 14
Reh dra hos
3 x 20 PEPC6-WN 1 ml Day 0 None
Day 14
20 PERC6-SH 1 ml Day 0 None
Day 14
20 PERC6-SH-AIOH 1 ml Day 0 Aluminum hydroxide
Day 14
Reh dra el
20 PERC6-SH-AIPh 1 ml Day 0 Aluminum phosphate
Day 14
Reh dra hos
20 PEPC6-SH-MO 1 ml Day 0 Mineral oil
Day 14
20 PBS lml Day 0
Day 14
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Table XV. Analysis with death taken as endpoint,
intention to treat (safety and efficacy).
N Survival Survival 95% CI
Prob
Mouse Brain MO 60 54 90.0% 79.9 - 95.0%
PER.C6 WN MO 60 53 88.3% 77.8 - 94.2%
PER.C6 WN A10H 60 38 63.3% 50.7 - 74.4%
PER.C6 WN A1Ph 60 41 68.3% 55.8 - 78.7%
PER.C6 WN 60 32 53.3% 40.9 - 65.4%
PER.C6 SH 20 0 0 0 - 16.1%
PER.C6 SH A10H 20 0 0 0 - 16.1%
PER.C6 SH A1Ph 20 0 0 0 - 16.1%
PER.C6 MO 20 1 5.0% 0.9 - 23.6%
PBS 20 0 0 0 - 16.1%
N = number of animals; CI Confiden tiality Interval
Risk difference between:
Mouse Brain-MO and PER.C6-WN-MO:
1.7% (95% CI -9.5 - 12.8) not significant
Mouse Brain-MO and PER.C6-WN-AlOH:
26.7% (95% CI 12.3 - 41.0) significant
Table XVI. Analysis with death or diseased taken as
endpoint (disease-free survival)
N df df Survival 95% CI
Survival Prob
Mouse Brain MO 60 54 90.0% 79.9 - 95.0%
PER.C6 WN MO 60 53 88.3% 77.8 - 94.2%
PER.C6 WN A10H 60 31 51.7% 39.3 - 63.8%
PER.C6 WN A1Ph 60 32 53.3% 40.9 - 65.4%
PER.C6 WN 60 25 41.7% 30.1 - 54.3%
PER.C6 SH 20 0 0 0 - 16.1%
PER.C6 SH A10H 20 0 0 0 - 16.1%
PER.C6 SH A1Ph 20 0 0 0 - 16.1%
PER.C6 MO 20 1 5.0% 0.9 - 23.6%
PBS 20 0 0 0 - 16.1%
Note: figures in italic indicate differences with table XV.
N = number of animals; CI = Confident iality Interval
Risk difference between:
Mouse Brain-MO and PER.C6-WN-MO:
1.7 (95% CI -9.5 - 12.8%) not significant
Mouse Brain-MO and PER.C6-WN-AlOH:
38.3 (95% CI 23.6 - 53.1%) significant
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Table XVII. Analysis with death taken as endpoint among
geese who are challenged with WN (to estimate true
efficacy)
N Survival Survival 95% CI
Prob
Mouse Brain MO 58 54 93.1 83.6 - 97.3%
PER.C6 WN MO 58 53 91.4 81.4 - 96.3%
PER.C6 WN A1OH 59 38 64.4 51.7 - 75.4%
PER.C6 WN A1Ph 56 41 73.2 60.4 - 83.0%
PER.C6 WN 57 32 56.1 43.3 - 68.2%
PER.C6 SH 19 0 0 0 - 16.8%
PER.C6 SH A10H 20 0 0 0 - 16.1%
PER.C6 SH AlPh 20 0 0 0 - 16.1%
PER.C6 MO 20 1 5.0 0.9 - 23.6%
PBS 19 0 0 0 - 16.8%
N = number of animals; CI = Confidentiality Interval
Risk difference between:
Mouse Brain-MO and PER.C6-WN-MO:
1.7 (95% CI -8.0 - 11.5) not significant
Mouse Brain-MO and PER.C6-WN-AlOH:
28.7 (95% CI 14.9 - 42.6) significant
Table XVIII. Analysis with endpoint `death before
challenge' to study the safety of vaccination
N Survival Survival 95% CI
Prob
Mouse Brain MO 60 58 96.7 88.6 - 99.1%
PER.C6 WN MO 60 58 96.7 88.6 - 99.1%
PER.C6 WN A10H 60 59 98.3 91.1 - 99.7%
PER.C6 WN A1Ph 60 56 93.3 84.1 - 97.4%
PER.C6 WN 60 57 95.0 86.3 - 98.3%
PER.C6 SH 20 19 95 76.4 - 99.1%
PER.C6 SH A10H 20 20 100 83.9 - 100%
PER.C6 SH AlPh 20 20 100 83.9 - 100%
PER.C6 MO 20 20 100 83.9 - 100%
PBS 20 19 95 76.4 - 99.1%
N = number of animals; CI = Confidentiality Interval
Risk difference between:
Mouse Brain-MO and PER.C6-WN-MO:
0 (95% CI -6.4 - 6.4) not significant
Mouse Brain-MO and PER.C6-WN-AlOH:
1.7 (95% CI -3.9 - 7.3) not significant
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REFERENCES
Anderson JF, Andreadis TG, Vossbrinck CR, Tirrell S,
Wakem EM, French RA, Garmendia AE and Van Kruiningen HJ
(1999) Isolation of West Nile Virus from mosquitos,
crows, and a Cooper's hawk in Connecticut. Science
286:2331-2333
Arroyo J, Miller CA, Catalan J and Monath TP (2001)
Yellow fever vector live-virus vaccines: West Nile Virus
vaccine development. Trends Mol Med 7:350-354
Bin H, Grossman Z, Pokamunski S, Malkinson M, Weiss L,
Duvdevani P, Banet C, Weisman Y, Annis E, Gandaku D,
Yahalom V, Hindyieh M, Shulman L and Mendelson E (2001)
West Nile fever in Israel 1999-2000: from geese to
humans. Ann N Y Acad Sci 951:127-142
Brinton MA (2002) The molecular biology of West Nile
Virus: A new invader of the Western Hemisphere. Annu Rev
Microbiol 56:371-402
Campbell GL, Marfin AA, Lanciotti RS and Gubler DJ (2002)
West Nile Virus. Lancet Infect Dis 2:519-529
Chambers TJ, Hahn CS, Galler R and Rice CM (1990)
Flavivirus genome organization, expression, and
replication. Annu Rev Microbiol 44:649-688
Chang GJ, Davis BS, Hunt AR, Holmes DA and Kuno G (2001)
Flavivirus DNA vaccines: current status and potential.
Ann NY Acad Sci 951:272-285
Dunster LM, Gibson CA, Stephenson JR, Minor PD and
Barrett AD (1990) Attenuation of virulence of
flaviviruses following passage in HeLa cells. J Gen Virol
71:601-607
Hadfield TL, Turell M, Dempsey MP, David J and Park EJ
(2001) Detection of West Nile Virus in mosquitoes by RT-
PCR. Mol Cell Probes 15:147-150
Hubalek Z and HalouzkaJ (1999) West Nile fever - A
reemerging mosquito-borne viral disease in Europe. Emerg
Infect Dis 5:643-650
Jia XY, Briese T, Jordan I, Rambaut A, Chi HC, Mackenzie
JS, Hall RA, Scherret J and Lipkin WI (1999) Genetic
analysis of West Nile New York 1999 encephalitis virus.
Lancet 354:1971-1972
CA 02501220 2005-04-05
WO 2004/042042 PCT/EP2003/050806
52
Kurane I. Janus J and Ennis FA (1992) Dengue virus
infection of human skin fibroblasts in vitro production
of IFN-beta, IL-6 and GM-CSF. Arch Virol 124:21-30
Lanciotti RS, Roehrig JT, Deubel V, et al. (1999) Origin
of the West Nile Virus responsible for an outbreak of
encephalitis in the northeastern United States. Science
286:2333-2337
Lanciotti RS, Kerst AJ, Nasci RS, et al. (2000) Rapid
detection of West Nile Virus from human clinical
specimens, field-collected mosquitoes, and avian samples
by a TaqMan reverse transcriptase-PCR assay. J Clin
Microbiol 38:4066-4071
Lanciotti RS, Ebel GD, Deubel V, et al. (2002) Complete
genome sequences and phylogenetic analysis of West Nile
Virus strains isolated from the United States, Europe and
the Middle East. Virology 298:96-105
Malkinson M, Banet C, Mahany S, et al. (1998) Virus
encephalomyelitis of geese: some properties of the viral
isolate. Isr J Vet Med 53:44.
Malkinson M. Banet C, Khinich Y, Samina I, Pokamunski S
and Weisman Y (2001) Use of live and inactivated vaccines
in the control of West Nile fever in domestic geese. Ann
N Y Acad Sci 951:255-261
Monath TP (2001) Prospects for development of a vaccine
against the West Nile Virus. Ann NY Acad Sci 951:1-12
Smithburn KC, Hughes TP, Burke AV and Paul JH (1940) A
neurotropic virus isolated from the blood of a native of
Uganda. Am J Trop Med Hyg 20:471-492
Wang T, Anderson JF, Magnarelli LA, Wong SJ, Koski RA and
Fikrig E (2001) Immunization of mice against West Nile
Virus with recombinant envelope protein. J Immunol
167:5273-5277
Wengler G, Wengler G, Nowak T and Castle E (1990)
Description of a procedure which allows isolation of
viral non-structural proteins from BHK vertebrate cells
infected with the West Nile flavivirus in a state which
allows their direct chemical characterization. Virology
177:795-801
Yamshchikov VF, Wengler G. Perelygin AA, Brinton MA and
Compans RW (2001) An infectious clone of the West Nile
Flavivirus. Virology 281:294-304
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WO 2004/042042 PCT/EP2003/050806
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0082W0000RD
Original (for SUBMISSION) - printed on Friday, 07 November, 2003 05:13:28 PM
0-1 Form - PCT/R01134 (EASY)
Indications Relating to Deposited
Microorganism(s) or Other Biological
Material (PCT Rule 13bis)
0-1-1 Prepared using epoline online filing PCT plug-in
(updated 07.11.2003)
0-2 International Application No.
0-3 Applicant's or agent's file reference 0082W0000RD
I The indications made below relate to
the deposited microorganism(s) or
other biological material referred to in
the description on:
1-1 page 4
1-2 line 15
1-3 Identification of Deposit
1-3-1 Name of depositary institution European Collection of Cell Cultures
1-3-2 Address of depositary institution Vaccine Research and Production
Laboratory, Public Health Laboratory
Service, Centre for Applied Microbiology
and Research, Porton Down, Salisbury,
Wiltshire SP4 OJG, United Kingdom
1-3-3 Date of deposit 29 February 1996 (29.02.1996)
1-3-4 Accession Number ECACC 96022940
1-4 Additional Indications NONE
1-5 Designated States for Which all designated States
Indications are Made
1-6 Separate Furnishing of Indications NONE
These indications will be submitted to the
International Bureau later
FOR RECEIVING OFFICE USE ONLY
0-4 This form was received with the
international application: Yes
(yes or no)
0-4-1 Authorized officer
<:~~ C.A.J.A. PASCHE
FOR INTERNATIONAL BUREAU USE ONLY
0-5 This form was received by the
international Bureau on:
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CA 02501220 2005-11-10
53a
SEQUENCE LISTING
<110> Crucell Holland B.V.
<120> Vaccine against West Nile virus
<130> PAT 58976W-1
<140> 2,501,220
<141> 2003-11-07
<150> PCT/NL02/00718
<151> 2002-11-08
<150> PCT/EP03/50129
<151> 2003-04-28
<160> 3
<170> Patentln version 3.1
<210> 1
<211> 21
<212> DNA
<213> Artificial
<220>
<223> Synthetic oligonucleotide WNV 1
<400> 1
ccaccggawg ttgagtagac g 21
<210> 2
<211> 20
<212> DNA
<213> Artificial
<220>
<223> Synthetic oligonucleotide WNV 2
<400> 2
tttgktcacc cagtcctcct 20
<210> 3
<211> 20
<212> DNA
<213> Artificial
<220>
<223> Synthetic oligonucleotide VIC labelled probe
CA 02501220 2005-11-10
53b
<220>
<221> misc_feature
<222> (1)..(1)
<223> VIC label attached
<220>
<221> misc feature
<222> (20)_.(20)
<223> TAMRA label attached
<400> 3
tgctgcctgc grctcaaccc 20
