U.S. patent application number 12/220971 was filed with the patent office on 2009-01-15 for vaccines against west nile virus.
This patent application is currently assigned to Crucell Holland B.V.. Invention is credited to Jaap Goudsmit, Govert Johan Schouten, Alphonsus G.C.M. UytdeHaag.
Application Number | 20090017068 12/220971 |
Document ID | / |
Family ID | 46304386 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090017068 |
Kind Code |
A1 |
UytdeHaag; Alphonsus G.C.M. ;
et al. |
January 15, 2009 |
Vaccines against West Nile Virus
Abstract
Described are vaccines containing (whole-inactivated) West Nile
Viruses and/or West Nile viral proteins derived therefrom, produced
on human cells, wherein the human cells comprise a sequence
encoding at least one early region-1 (E1) gene product of an
adenovirus. The cells are preferably cultured in suspension to very
high densities and under serum-free conditions. Herein, it is
disclosed that use of such cells results in high titers of West
Nile Virus produced.
Inventors: |
UytdeHaag; Alphonsus G.C.M.;
(Vleuten, NL) ; Schouten; Govert Johan;
(Leiderdorp, NL) ; Goudsmit; Jaap; (Amsterdam,
NL) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Assignee: |
Crucell Holland B.V.
Leiden
NL
|
Family ID: |
46304386 |
Appl. No.: |
12/220971 |
Filed: |
July 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11110517 |
Apr 20, 2005 |
7425437 |
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12220971 |
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PCT/EP2003/050806 |
Nov 7, 2003 |
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11110517 |
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09722867 |
Nov 27, 2000 |
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PCT/EP2003/050806 |
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09449854 |
Nov 26, 1999 |
7192759 |
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09722867 |
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Current U.S.
Class: |
424/218.1 ;
435/236; 435/366 |
Current CPC
Class: |
C12N 2770/24134
20130101; A61K 2039/5252 20130101; A61K 2039/55566 20130101; A61K
39/12 20130101; A61P 31/12 20180101; A61K 2039/55505 20130101; C12N
2770/24163 20130101; A61K 2039/552 20130101; C12N 7/00
20130101 |
Class at
Publication: |
424/218.1 ;
435/236; 435/366 |
International
Class: |
A61K 39/12 20060101
A61K039/12; C12N 7/04 20060101 C12N007/04; C12N 5/08 20060101
C12N005/08; A61P 31/12 20060101 A61P031/12 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 8, 2002 |
NL |
PCT/NL02/00718 |
Apr 28, 2003 |
EP |
PCT/EP03/50129 |
Claims
1.-21. (canceled)
22. An inactivated West Nile Virus, said inactivated West Nile
Virus produced by the process of: providing a cell with a nucleic
acid encoding West Nile Virus, wherein the cell expresses at least
one adenoviral E1A peptide; culturing the cell in a suitable medium
under conditions that cause expression of said nucleic acid thus
producing the West Nile Virus; and inactivating the West Nile Virus
thus produced.
23. The inactivated West Nile Virus of claim 22, wherein said
process further comprises: purifying one or more antigenic
components from said inactivated West Nile Virus.
24. A vaccine comprising the inactivated West Nile Virus of claim
22, a pharmaceutically acceptable carrier, and an adjuvant.
25. The inactivated West Nile Virus of claim 22, wherein said
inactivated West Nile Virus is free of non-human mammalian
protein.
26. The inactivated West Nile Virus of claim 23, wherein said
inactivated West Nile Virus is free of non-human mammalian
protein.
27. A vaccine comprising the one or more antigenic components of
claim 23, a pharmaceutically acceptable carrier, and an
adjuvant.
28. The vaccine of claim 24, wherein said vaccine comprises a
lineage II West Nile Virus.
29. An isolated human cell, said isolated human cell comprising: a
nucleic acid sequence encoding an adenoviral E1A gene product; and
a nucleic acid sequence encoding West Nile Virus.
30. The isolated human cell of claim 29, wherein said isolated
human cell is a PER.C6.RTM. cell.
31. A method of protecting a subject from a West Nile Virus
infection, said method comprising: vaccinating the subject with the
vaccine of claim 24.
32. A method of protecting a subject against a West Nile Virus
infection, said method comprising: vaccinating the subject with the
vaccine of claim 27.
33. An inactivated purified West Nile Virus free of non-human
mammalian protein.
34. A vaccine comprising the inactivated purified West Nile Virus
of claim 33, a pharmaceutically acceptable carrier, and an
adjuvant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/110,517, filed Apr. 20, 2005, pending, which
application is a continuation of PCT International Patent
Application No. PCT/EP2003/050806, filed on Nov. 7, 2003,
designating the United States of America, and published, in
English, as PCT International Publication No. WO 2004/042042 A1 on
May 21, 2004, which application claims priority to PCT
International Patent Application No. PCT/NL02/00718, filed Nov. 8,
2002, and to PCT International Patent Application No.
PCT/EP03/50129, filed Apr. 28, 2003. U.S. patent application Ser.
No. 11/110,517 (identified above) is also a continuation-in-part of
pending U.S. patent application Ser. No. 09/722,867 filed on Nov.
27, 2000 which is a continuation-in-part of U.S. patent application
Ser. No. 09/449,854, now U.S. Pat. No. 7,192,759, filed on Nov. 26,
1999. The contents of the entirety of each of the identified
applications and patents are incorporated herein by this
reference.
TECHNICAL FIELD
[0002] The invention relates to the field of medicine. In
particular, it relates to vaccines against flaviviruses and, more
specifically, to West Nile Virus and to methods of producing the
same.
BACKGROUND
[0003] 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 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 United States. Symptoms
vary from fever, headache, skin rash, swollen lymph glands, neck
stiffness, stupor, disorientation, tremors, convulsions, muscle
weakness, pancreatitis, myocarditis, and 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.
[0004] 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; and U.S. Pat. No. 6,306,899, the contents of
the entirety of each of which are incorporated herein by this
reference). 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 subunits (envelope proteins), or poxviruses containing
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; and U.S. Pat. Nos. 6,184,024, 5,514,375,
5,744,140, 5,744,141 6,416,763, 6,432,411, 5,494,671, and
6,258,788, the contents of the entirety of each of which are
incorporated herein by this reference). One veterinary vaccine
containing inactivated West Nile Viruses was approved in August
2001, solely for 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
Israeli authorities in July/August 2001. Numerous disadvantages
exist with the treatments and vaccines mentioned above related to
dosages, ineffectiveness, 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.
SUMMARY OF THE INVENTION
[0005] The 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 the 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 the virus to replicate in the cell or that cause or
allow expression of the nucleic acid coding for the 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.
[0006] Products obtainable by the methods described herein are also
part of the invention and may be used for 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.
[0007] The invention further relates to West Nile Viruses or West
Nile viral proteins for use in a vaccine obtainable by a method or
a use according to the invention, the West Nile Virus or the 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 E1 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.
[0008] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows the Reverse Transcriptase PCR(RT-PCR) products
using samples of cells as deposited under ECACC no. 96022940
infected with West Nile Virus.
[0010] FIG. 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.
[0011] FIG. 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).
[0012] FIG. 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.
[0013] FIG. 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.
[0014] FIG. 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.RTM.-based vaccine including the adjuvant is represented by
line "2," and the PER.C6.RTM.-based vaccine without adjuvant is
represented by line "3."
[0015] FIG. 7 is a diagram showing the time to disease or death of
all geese combined from the three volumes of FIG. 6. Line "1"
(dotted) represents the vaccine with the inactivated West Nile
Virus produced on mouse brain. Line "2" represents the
PER.C6.RTM.-based vaccine including the adjuvant and "3" is the
negative control. The group receiving the PER.C6.RTM.-based vaccine
without adjuvant is not given here.
[0016] FIG. 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 (Aus91); (C) Madagascar 1978 (Mad78); (D)
Kunjin 1960 (Aus60); (E) Cyprus 1968 (Cyp68); (F) Goose Israel 1998
(Isr98).
[0017] FIG. 9 is a graphical representation of part of the data
shown in Table XVII for the Mouse Brain-MO and PER.C6.RTM.-WN-MO
vaccines. Animals that died before challenge (two in both groups,
leaving 58 animals in both groups for challenge) were not included
in this figure.
[0018] FIG. 10 is a graphical representation of part of the data
shown in Table XVII for the different PER.C6.RTM.-based vaccines
with or without aluminum-based adjuvants. Animals that died in
these groups before challenge (one for PER.C6.RTM.-WN-AlOH leaving
59 animals; four in PER.C6.RTM.-WN-AlPh, leaving 56 animals; three
in PER.C6.RTM.-WN, leaving 57 animals) were not included in this
figure.
[0019] FIGS. 11A and 11B show TCID50 titers obtained with West Nile
Virus derived from suspension-growing PER.C6.RTM. cells. The data
depicted in the graph of FIG. 11A was obtained growing the
PER.C6.RTM. cells in VPRO suspension culture media from JRH. The
data depicted in the graph of FIG. 11B was obtained growing the
PER.C6.RTM. cells in APM suspension culture media from Gibco.
Samples were taken at days 2, 3, 4, 5, and 6 post-infection and
assayed for TCID50/ml. Virus dilution is shown on the x-axis and
titer is shown on the y-axis.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Described are vaccines against West Nile Virus and to
methods of producing the 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 protective and that could be produced
in 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.
[0021] 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. These procedures are
not especially suited for the production of vaccines that are to be
used in humans. Vero cells grow on microcarriers, not in
suspension, while HeLa cells are aggressive tumor cells. BHK cells
are not generally found to be safe for the production of
therapeutics. The other systems do not provide a platform for
large-scale production since cells need to grow indefinitely and
should not be derived from a tumor. 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 immortalization through the early region-1 (E1)
from 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.C6.RTM., has been described
to support the growth of influenza virus, measles virus and Herpes
simplex virus type 1 and 2 (WO 01/38362, incorporated in its
entirety by reference herein). 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 of West Nile Virus. The invention discloses that
E1-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.
[0022] The 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 the 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.
[0023] In certain embodiments, described is 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 the 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 the nucleic acid coding for the West Nile Virus,
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. The E1 region of
adenovirus comprises several sub-regions, for example, 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 immortalization to
obtain cell lines. The invention, therefore, also relates to a
method described herein, 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 cells comprises a
functional E1 region of an adenovirus and wherein the E1 region is
stably integrated in the chromosomal genome of the cell.
[0024] Different types of cells can be used to apply the methods
described herein, 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, the cell
is derived from an embryonic retinoblast. Highly preferred are
methods wherein 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.
[0025] The nucleic acid that is administered to the cells in the
methods described herein is preferably RNA wherein the RNA is
preferably delivered to the cell or to the culture of cells by
means of a West Nile Virus.
[0026] 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, the 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 the cells or the culture of
cells.
[0027] Another aspect relates to methods according to the invention
wherein the West Nile Virus is providing the nucleic acid to the
cells in a multiplicity of infection ("moi") ranging from 5 to
5.times.10.sup.-7 plaque-forming units ("PFU") per cell. This range
of PFU is sufficient to obtain high titers of replicating West Nile
Viruses, as can be seen in the examples herein.
[0028] Also described herein are methods 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.
[0029] Inactivation of the produced West Nile Viruses may be
achieved through methods known to a person of ordinary skill in the
art. Examples of inactivation are the use of UV-light or the use of
beta-propiolactone. A preferred inactivation takes place through
the use of formaldehyde (formalin-induced inactivation).
[0030] In certain embodiments, the invention provides methods for
obtaining West Nile Viruses that can be used in subunit 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 the 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 be obtained
after using the methods described herein. Such products are also
encompassed within the claims of the invention.
[0031] 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, the cell comprises stably integrated
into its genome an E1 region from an adenovirus. A highly preferred
adenovirus serotype that is used for providing the E1 region is
adenovirus serotype 5.
[0032] A preferred method utilizes a human cell wherein the 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.
[0033] The invention also relates to the products obtained by the
methods described herein 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, the 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.
[0034] 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 invention.
[0035] 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 E1 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 the human cell is a
derivative of a cell as deposited under ECACC no. 96022940, or a
derivative thereof.
[0036] Also provided is a method of vaccinating an animal or human
subject against West Nile Virus infection, comprising administering
a vaccine according to the invention to the animal or human
subject.
[0037] The 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 E1 region of an adenovirus, with a
nucleic acid encoding the West Nile Virus and/or the West Nile
viral protein; b) culturing the cell obtained in the previous step
in a suitable medium; and c) allowing for expression of the West
Nile Virus and/or the West Nile viral protein in the medium and/or
the cell. Preferably, the 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 invention include (sterile)
filtration, chromatography (e.g., using heparin sulphate),
diafiltration and/or (an)ion exchange chromatography. Also
preferred are methods described herein 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.RTM. 20, TWEEN.RTM. 40, TWEEN.RTM. 60 and/or TWEEN.RTM. 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
inactivated by nucleic acid-disrupting agents such as RNase.
[0038] 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 described herein. 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 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 the subunit is provided
to a cell having at least a sequence encoding at least one gene
product of the E1 region of an adenovirus, by means other than a
West Nile Virus. Therefore, the nucleic acid can be RNA, cDNA and
DNA. Preferably, the nucleic acid is RNA and also preferably, the
nucleic acid delivery vehicle is a West Nile Virus.
[0039] In certain embodiments, the 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, the
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 the
West Nile Virus and/or the West Nile viral protein; b) culturing
the cell obtained in the previous step in a suitable medium; and c)
allowing for expression of the West Nile Virus and/or the West Nile
viral protein in the medium and/or the cell, wherein the cell is
cultured in suspension (non-adherent). Preferably, a suitable
medium for the methods described herein 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 described herein may be
performed by using different large-scale set-ups, such as
fed-batch, perfusion culture and wave bags.
[0040] 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 described herein may be performed by
several methods known in the art, such as single- or multistep
(anion and/or cation) exchange chromatography.
[0041] In certain embodiments, 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 E1 region is present in the chromosomal genome
of the 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 to mean any
such cell that contains (in addition to the E1 region of adenovirus
serotype 5) another heterologous nucleic acid that 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 to mean
descendants of cells as deposited under ECACC no. 96022940 that
have been sub-cultured for a prolonged period, either under
selective pressure and/or mutagenizing 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 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 E1, and
A549 cells transformed with adenovirus E1.
[0042] In certain aspects, provided are 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, the nucleic acid is provided by a West Nile Virus,
which contains (in its wild-type form) a single (+)RNA strand.
[0043] Many strains of West Nile Viruses have been described in the
art (Lanciotti et al. 2002). In one embodiment of the 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 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 described herein. 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 (Aus60 or Aus91 respectively, also referred to as strains MRM
16 or K 6453 respectively, see below), Madagascar 1978, Cyprus 1968
and Israel 1999H are used for the methods described herein. In one
preferred embodiment, West Nile Virus strain NY 1999 hum (a strain
isolated from 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) is produced
using methods described herein. 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; and
U.S. Pat. No. 6,184,024, the contents of the entirety of each of
which are incorporated herein by this reference) by methods
described herein, thereby circumventing possible problems of low
titers, high costs and/or safety issues.
[0044] In a specific embodiment, the 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
("moi") ranging from 5 to 5.times.10.sup.-7 plaque-forming units
per cell. As shown in the examples, the inventors of the invention
were able to show that it is feasible to obtain titers of 10.sup.9
pfu/ml after three days following an moi that was as low as 0.005
pfu/cell, using methods described herein, while titers obtained
with a higher moi, were most likely even higher.
[0045] In one embodiment, the invention relates to the use of a
human cell having a sequence encoding at least one E1 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, the human cell is
derived from a primary retinoblast and, even more preferred, are
uses according to the invention, wherein the human cell is a cell
as deposited under ECACC no. 96022940, or a derivative thereof.
[0046] In certain embodiments, the 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, the West Nile Virus or the 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, the vaccine also comprises an adjuvant.
Preferably, the human vaccine comprises an adjuvant reagent that is
acceptable for use in humans, such as "Alum" or aluminum hydroxide,
which is an adjuvant known to persons skilled in the art. Another
adjuvant that may be applied is aluminum 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 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 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 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.
[0047] In certain embodiments, the invention relates to a human
cell having a sequence encoding at least one E1 gene product of an
adenovirus in its genome and having a nucleic acid encoding a West
Nile Virus. Preferably, the human cell having a sequence encoding
at least one E1 gene product of an adenovirus in its genome and
having a nucleic acid encoding a West Nile Virus is the cell line
as deposited under ECACC no. 96022940, or a derivative thereof.
EXAMPLES
Example 1
Infection of Cells with Strain West Nile B956
[0048] 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.C6.RTM., were banked and cultured as described (WO 01/38362). A
series of cells were cultured in T80 culture flasks with 10.sup.7
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 5.times.10.sup.8
plaque-forming units per milliliter (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.RTM. PCR analysis (Applied
Biosystems, CA) (Lanciotti et al. 2000) to determine the titer of
the virus produced, for Reverse Transcriptase (RT) PCR, and for
Immune Fluorescence. RT-PCR and TAQMAN.RTM. PCR was performed using
West Nile Virus specific primers (see below).
[0049] The input virus was diluted in DMEM/5% FBS nHI and incubated
for one hour with the PER.C6.RTM. cells in eight serial dilutions
(10.sup.-1 to 10.sup.-8, with an moi of 5 to 5.times.10.sup.-7
pfu/cell). After incubation of the virus with the cells, the
culture medium was discarded and replaced with fresh DMEM/5% FBS
nHI (not-Heat Inactivated). Cells were cultured at 37.degree.
C./10% CO.sub.2 at all times. Cytopathological effect (CPE) was
scored visually after 24 hours, 48 hours and 72 hours
post-infection. Results are shown in Table II. No CPE was detected
at 24 hours post-infection. However, clear CPE was visible at the
highest occurrences moi after 48 hours, while in most cases
significant CPE was detected at 72 hours post-infection. These
results show clearly that E1-transformed human cells, such as
PER.C6.RTM. 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
[0050] Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was
performed on the mock-transfected cells as deposited under ECACC
no. 96022940 and on the 24-hour and 48-hour post-infection samples
of such cells infected with an moi of 5.times.10.sup.-1 and
5.times.10.sup.-4 pfu/cell. RNA samples were diluted 1:10, 1:100
and 1:1000. RT-PCR was performed by using the QIAGEN.RTM. One-Step
RT-PCR Kit (QIAGEN.RTM.) 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 solely contained water,
while the positive control contained input virus from the BHK
supernatant. The PCR program that was used was as follows: 30
minutes at 50.degree. C., 15 minutes at 95.degree. C., followed by
35 cycles of 30 seconds at 94.degree. C., 30 seconds at 50.degree.
C. and one minute at 72.degree. C. and a final step of ten minutes
at 72.degree. C. Obtained amplified nucleic acid was loaded on a
gel and visualized. Results show that West Nile viral RNA is
present in all samples of cells that were infected with West Nile
Virus, while no positive signal could be detected in the mock
infections (FIG. 1). The amount increases from 24 hours to 48 hours
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
[0051] Samples were obtained 48 hours post-infection from the cells
as deposited under ECACC no. 96022940 that were mock-infected and
that were infected with an moi of 5.times.10.sup.-3 (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 also recognizes the envelope of the West
Nile Virus. The serum was obtained after injection of Yellow Virus
in a monkey and recovery of the serum. Fixing with acetone 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 FIG. 2. These results indicate
that West Nile Virus is able to infect human adenovirus
E1-transformed cells, such as PER.C6.RTM. cells.
Example 4
Real-Time Taqman.RTM. PCR for the Detection and Quantification of
West Nile Virus RNA in infected PER.C6.RTM. cells
[0052] To determine the titers of the West Nile Virus that were
produced by the cells as deposited under ECACC no. 96022940, a
real-time reverse transcriptase TAQMAN.RTM. PCR was performed on
all samples, except for the 72-hour post-infection samples of the
three highest occurrences of moi as shown in Table II.
[0053] RNA was extracted using the spin protocol of the QIAamp
Viral RNA Mini Kit (QIAGEN.RTM.) following the description provided
by the manufacturer. 200 .mu.l cell culture supernatant was mixed
with 60 .mu.l elution volume. Amplification and real-time detection
was performed using the MASTERMIX.TM. without UNG with the RNase
Inhibitor Mix (Applied Biosystems) and the QUANTITECT.RTM. Probe
RT-PCR Kit (QIAGEN.RTM.) using the forward primer WNV 1 and reverse
primer WNV 2, and with the VIC-labeled 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-labeled probe
100 nM.
[0054] Results are shown in Table III. Clearly, PER.C6.RTM. cells
are able to sustain growth of at least a titer of 1.times.10.sup.9
pfu/ml using an input moi of 5.times.10.sup.-3 pfu/cell, 72 hours
post-infection, while probably even higher titers were obtained in
lower dilutions. This indicates that PER.C6.RTM. is a very useful
tool for the production of West Nile Viruses. Since it has been
shown that PER.C6.RTM. cells can grow to very high densities in
serum-free medium in suspension and in large incubators (>1000
liter), 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 or to purify separate subunits from the
disrupted virus, such as envelope proteins for use in the so-called
subunit 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 E1-Transformed Cells for an Animal-Challenge Model in
Geese
[0055] 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 moi of 0.001 virus particles ("vp") per 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 10.sup.10.66 or 4.57.times.10.sup.10
CCID.sub.50/ml (Cell Culture Infectious Dose 50 per ml) for the
virus produced on cells as deposited under ECACC no. 96022940 and
10.sup.10.83 or 6.76.times.10.sup.10 CCID.sub.50/ml for the virus
produced on mouse brain. The results of the titration on Vero cells
are given in FIG. 4.
[0056] 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 ten-fold serial dilutions of the culture
supernatant. Mice were observed for mortality over a nine-day
period. The mortality titer for the PER.C6.RTM.-produced viruses
was found to be 10.sup.9.76 MLD.sub.50/0.1 ml (mouse lethal dose 50
per 0.1 ml), while the titer of the virus preparation from mouse
brain was found to be 10.sup.9.63 MLD.sub.50/0.1 ml, as shown in
FIG. 5.
[0057] The virus preparation was inactivated by adding 1 ml
formaldehyde (stock solution of 4%) to 100 ml virus-containing
PER.C6.RTM. supernatant and by stirring this solution at 4.degree.
C. for four 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, which were
subsequently observed for 14 days for mortality. No mortality was
observed in any of the animals. Thus, it is shown here that
E1-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.C6.RTM.-Produced
West Nile Vaccine in Comparison with a Mouse Brain-Produced
Vaccine
[0058] Two-week-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 the Mouse brain vaccine (serving as
a positive control) was produced as also 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.RTM. 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 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 aluminum
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 invention.
[0059] 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 three 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 three weeks.
[0060] The results of these experiments are shown in Tables V and
VI and clearly indicate that the PER.C6.RTM.-produced West Nile
Viruses (in an inactivated form and in the combination with an
adjuvant) are as potent as the vaccine produced on Mouse Brain. The
survival rate in the animal groups receiving PER.C6.RTM.-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 is significant between the rates found with the
PER.C6.RTM. versus the Mouse Brain-based vaccine 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 groups that
received a vaccine; all animals in the negative control group died
within six days upon challenge. The results are also depicted
graphically in FIG. 6 for the three separate volumes used (FIG. 6A:
0.25 ml; FIG. 6B: 0.5 ml; and FIG. 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.RTM.-based vaccine+adjuvant is
indicated with line 2, the group receiving the PER.C6.RTM.-based
vaccine minus adjuvant is indicated with line 3, and the negative
control group is indicated with line 4. FIG. 7 shows the results
summarized in Table VI for the three volume groups taken together
and also clearly indicates that the PER.C6.RTM.-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
[0061] 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 E1-transformed cells such as PER.C6.RTM.. 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
LD.sub.50 (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 LD.sub.50 dose of
more than 10,000 pfu when administered i.p. If the viruses are
administered intra-cranially, no large difference in neurovirulence
is found. These aspects of these strains are known in the art.
[0062] 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.RTM. and were
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 previously outlined. 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.
[0063] Infection of cells as deposited under ECACC no. 96022940
with the different West Nile Virus strains was generally performed
as follows. Cells were trypsinized, counted, and seeded in 25
cm.sup.2 flasks using 5.times.10.sup.6 cells per 5 ml DMEM, 5% FBS
medium per flask. Cells were incubated for 24 hours at 37.degree.
C. under 10% CO.sub.2. 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. The viruses
were then added and incubated with the cells at 37.degree. C. 10%
CO.sub.2 for several days. When full cpe had occurred, cells and
medium were harvested and supernatant was clarified by
centrifugation, ten minutes at 2000 rpm. Subsequently, virus titers
were determined for different virus dilutions. Results regarding
cpe are depicted in Tables VIII to XIII, while the TCID50 titers
obtained using the different virus dilutions are given in FIGS. 8A
to F. The highest titers in TCID50/ml for each strain were (with
the specific input moi between brackets):
[0064] USA99b: 1.times.10.sup.9 (5.times.10.sup.-7)
[0065] Isr98: 5.6.times.10.sup.9 (8.9.times.10.sup.-6)
[0066] Mad78: 1.times.10.sup.7 (1.6.times.10.sup.-4)
[0067] Cyp68: 5.6.times.10.sup.8 (5.times.10.sup.-4)
[0068] Aus60: 3.2.times.10.sup.7 (1.6.times.10.sup.-2)
[0069] Aus91: 1.times.10.sup.8 (5.times.10.sup.-3).
[0070] These results show that all tested West Nile Virus strains
grow on PER.C6.RTM. to significant titers using a relatively low
moi, 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.RTM. in Combination with Different
Adjuvant Compounds
[0071] A double-blind vehicle-controlled study to assess the effect
of aluminum hydroxide and aluminum phosphate adjuvants on the
efficacy of a PER.C6.RTM.-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.RTM.-based
experimental WN vaccine was found using mineral oil as adjuvant
(see Example 6). However, mineral oil cannot be used in vaccines
for human applications. This implicates the requirement of testing
alternative adjuvants that were previously approved for human
use.
[0072] Aluminum-based adjuvants have been used for decades to
increase the immune response of vaccines for human and animal use.
Two types of aluminum-based adjuvants, REHYDRAGEL.RTM. aluminum
hydroxide and REHYDRAPHOS.RTM. aluminum phosphate were tested for
the immune potentiating effect in PER.C6.RTM.-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.
[0073] The bulk of the 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 10.sup.10.25 TCID50/ml and referred to as Mouse
Brain-MV.
[0074] The PER.C6.RTM.-based West Nile Virus bulk was produced by
inoculating sub-confluent (80%) monolayers of PER.C6.RTM. cells,
grown in T175 flasks in DMEM with FBS, with strain Goose Israel
1998 (Mouse brain-derived) at an moi of 10.sup.-3. The
virus-containing culture supernatant was harvested at day 6
post-infection and clarified by centrifugation at 1000 RPM for 20
minutes. The resulting virus preparation was titrated on VERO cells
seeded in 96-well plates. The titer was 10.sup.10.66 TCID50/ml. The
virus preparation was subsequently titrated in suckling mice by
intra-cranial injection of 30 .mu.l, ten-fold serial dilutions of
the culture supernatant. Mice were observed for mortality over a
nine-day period. The titer was 10.sup.9.76 MLD50/ml and referred to
as PER.C6.RTM.-WN.
[0075] A batch of supernatant from PER.C6.RTM. cells was produced
under similar conditions as for PER.C6.RTM. 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.RTM.-SH.
[0076] The West Nile Virus bulk preparations from PER.C6.RTM. and
the supernatant of Sham-infected PER.C6.RTM. cells were inactivated
by adding 1 ml formaldehyde (stock solution of 4%) to 100 ml
virus-containing PER.C6.RTM. supernatant and by stirring at
4.degree. C. for four 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 .mu.l of ten-fold serial dilutions
(10.sup.4.0-10.sup.10.0) were injected intra-cranial in suckling
mice. The animals were subsequently observed for mortality over
nine days. The resulting titers were sufficiently low; no live
harmful virus was detectable using this animal test assay.
[0077] PER.C6.RTM.-WN was formulated with REHYDRAGEL.RTM. LV
containing 2% A1.sub.2O.sub.3 (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 REHYDRAGEL.RTM. LV was added. The solution
was manually swirled and then set on an orbital shaker set at 90
rpm for one hour 25 minutes at 20.degree. C. The formulated
vaccine, referred to as PER.C6.RTM.-WN-A1OH, was then aliquoted
aseptically at 23 ml/vial (six vials) and stored at 4.degree.
C.
[0078] PER.C6.RTM.-WN was formulated with REHYDRAPHOS.RTM.
containing 2% solid aluminum 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.RTM. was added. The
solution was manually swirled and then set on an orbital shaker set
at 90 rpm for one hour 25 minutes at 20.degree. C. The formulated
vaccine, referred to as PER.C6-WN-A1Ph, was then aliquoted
aseptically at 23 ml/vial (six vials) and stored at 4.degree.
C.
[0079] PER.C6.RTM.-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 one hour 25 minutes at 20.degree.
C. The vaccine was then aliquoted aseptically at 23 ml/vial (six
vials) and stored at 4.degree. C.
[0080] PER.C6.RTM.-SH was formulated with REHYDRAGEL.RTM. 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 100 ml
bottle. Subsequently, 2.15 ml of REHYDRAGEL.RTM. LV was added. The
solution was manually swirled and then set on an orbital shaker set
at 90 rpm for one hour 25 minutes at 20.degree. C. The vaccine
referred to as PER.C6.RTM.-SH-A1OH was then aliquoted aseptically
at 24 ml/vial (two vials) and stored at 4.degree. C.
[0081] PER.C6.RTM.-SH was formulated with REHYDRAPHOS.RTM. 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 100 ml
bottle. Subsequently, 2.15 ml of REHYDRAPHOS.RTM. was added. The
solution was manually swirled and then set on an orbital shaker set
at 90 rpm for one hour 25 minutes at 20.degree. C. The vaccine
referred to as PER.C6.RTM.-SH-A1Ph was then aliquoted aseptically
at 24 ml/vial (two vials) and stored at 4.degree. C.
[0082] PER.C6.RTM.-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 one hour 25 minutes at
20.degree. C. The vaccine was then aliquoted aseptically at 24
ml/vial (two vials) and stored at 4.degree. C.
[0083] Mouse Brain-WN, PER.C6.RTM.-WN and PER.C6.RTM.-SH vaccines
were formulated with Mineral Oil using methods known to persons
skilled in the art of adjuvantizing vaccines. These preparations
were referred to as Mouse Brain-WN-MO, PER.C6.RTM.-WN-MO and
PER.C6.RTM.-SH-MO, respectively.
[0084] The study enrolled 400 geese (Anser anser, females and
males, three 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.
[0085] 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
(three weeks after the second vaccination), 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 aliquoted and stored
at -70.degree. C. The virus was inoculated intra-cranially into the
geese at a dose of 10.sup.2.0 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 in the PER.C6.RTM.-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 FIGS. 9
and 10.
[0086] Clearly, the data indicate that a West Nile Virus vaccine
produced on PER.C6.RTM. 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.RTM.-based vaccine plus mineral oil
(PER.C6.RTM.-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.RTM.-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. However, since the number of
surviving animals in the groups that received a PER.C6.RTM.-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.
[0087] 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 toward
the most suitable adjuvant for human use in combination with the
PER.C6.RTM.-based West Nile Virus vaccine are to be performed. We
predict that a vaccine preparation with Alum (aluminum hydroxide or
aluminum 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.RTM.
Cells
[0088] 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 cultures, it
is preferred that cells grow in suspension instead of in attached
settings. PER.C6.RTM. is a cell line that has proven to grow well
in both settings. An experiment was performed to see whether
suspension-growing PER.C6.RTM. 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.RTM. cells and that these cells
(in attached setting) were able to sustain growth to significant
titers. For the suspension growth, two different available
suspension culture media were used: VPRO (JRH) and APM (Gibco).
[0089] The experiment was performed as follows: six-well plates
containing 1.times.10.sup.6 cells per ml were inoculated with
different input virus moi. Samples were taken on days 2, 3, 4, 5
and 6 post-infection and TCID50/ml titers were determined. The
results are depicted in FIG. 11A (VPRO medium) and FIG. 11B (APM
medium).
[0090] The results indicate that suspension-growing PER.C6.RTM.
cells are capable of sustaining growth of West Nile Virus to high
titers. Thus, this indicates that PER.C6.RTM. 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.
TABLE-US-00001 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
Senegal 1990 Israel 1952 Uganda 1937 Romania 1996 M C. Afr. Rep
1972a Kenya 1998 C. Afr. Rep 1972b Senegal 1993 C. Afr. 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. Afr. Rep 1967 Ivory
Coast 1981 Kunjin (strains 1960, 1973, 1984b, 1991, 1984a, 1966 and
1994) India (strains 1955a, 1980, 1958, 1955b)
TABLE-US-00002 TABLE II CPE score (in %) on PER.C6 .RTM. cells
infected with serial dilutions of West Nile Virus B956 (moi ranges
from 5 to 5 .times. 10.sup.-7 pfu/cell). % CPE 24 hours 48 hours 72
hours Dilution moi Input virus post- post- post- (v/v) (pfu/cell)
(pfu/flask) infection infection infection mock 0 0 0 0 0 10.sup.-1
5 5 .times. 10.sup.7 0 100 nd 10.sup.-2 5 .times. 10.sup.-1 5
.times. 10.sup.6 0 100 nd 10.sup.-3 5 .times. 10.sup.-2 5 .times.
10.sup.5 0 50 100 10.sup.-4 5 .times. 10.sup.-3 5 .times. 10.sup.4
0 5 100 10.sup.-5 5 .times. 10.sup.-4 5 .times. 10.sup.3 0 0 80
10.sup.-6 5 .times. 10.sup.-5 5 .times. 10.sup.2 0 0 50 10.sup.-7 5
.times. 10.sup.-6 50 0 0 0 10.sup.-8 5 .times. 10.sup.-7 5 0 0 0
CPE was scored 24 hours, 48 hours and 72 hours post-infection.
Input virus titer was 5 .times. 10.sup.8 pfu/ml as determined in
plaque assays using Vero cells and 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 hours
post-infection.
TABLE-US-00003 TABLE III West Nile Virus titers as determined by
real-time RT-TAQMAN .RTM. PCR. 24 hours 48 hours 72 hours moi Input
virus post- post- post- Dilution pfu/cell pfu/flask infection
infection infection 10.sup.-1 5 5 .times. 10.sup.7 3.6 .times.
10.sup.7 5.9 .times. 10.sup.7 nd 10.sup.-2 5 .times. 10.sup.-1 5
.times. 10.sup.6 6.5 .times. 10.sup.6 2.7 .times. 10.sup.7 nd
10.sup.-3 5 .times. 10.sup.-2 5 .times. 10.sup.5 1.9 .times.
10.sup.6 6.1 .times. 10.sup.7 nd 10.sup.-4 5 .times. 10.sup.-3 5
.times. 10.sup.4 2.5 .times. 10.sup.5 1.9 .times. 10.sup.7 1.0
.times. 10.sup.9 10.sup.-5 5 .times. 10.sup.-4 5 .times. 10.sup.3
2.2 .times. 10.sup.4 5.7 .times. 10.sup.6 4.3 .times. 10.sup.8
10.sup.-6 5 .times. 10.sup.-5 5 .times. 10.sup.2 5.6 .times.
10.sup.3 7.1 .times. 10.sup.5 1.5 .times. 10.sup.8 10.sup.-7 5
.times. 10.sup.-6 50 3.4 .times. 10.sup.3 7.9 .times. 10.sup.4 9.8
.times. 10.sup.7 10.sup.-8 5 .times. 10.sup.-7 5 5.5 .times.
10.sup.3 4.2 .times. 10.sup.3 1.2 .times. 10.sup.6 Titers are given
in pfu/ml (right columns depicted as 24 hours, 48 hours and 72
hours post-infection).
TABLE-US-00004 TABLE IV Vaccination scheme using PER.C6 .RTM.-based
West Nile vaccine and a Mouse brain-produced West Nile vaccine in
two-week-old geese. Group (n =) Material Volume Vaccination
Adjuvant PART A 1. 20 WN vaccine 0.25 ml Day 0 None PER.C6 .RTM.
Day 14 2 22 WN vaccine 0.5 ml Day 0 None PER.C6 .RTM. Day 14 3. 23
WN vaccine 1 ml Day 0 None PER.C6 .RTM. Day 14 PART B 1. 25 WN
vaccine 0.25 ml Day 0 Mineral oil PER.C6 .RTM. Day 14 2. 25 WN
vaccine 0.5 ml Day 0 Mineral oil PER.C6 .RTM. Day 14 3. 28 WN
vaccine 1.0 ml Day 0 Mineral oil PER.C6 .RTM. Day 14 PART C 1. 19
Mouse brain 0.25 ml Day 0 Mineral oil vaccine Day 14 2. 20 Mouse
brain 0.5 ml Day 0 Mineral oil vaccine Day 14 3. 18 Mouse brain 1.0
ml Day 0 Mineral oil vaccine Day 14 4. 13 No Vaccine
TABLE-US-00005 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 .RTM. cells or using mouse
brains (MB) and challenged with West Nile Virus strain Goose Israel
1998. Disease/ Disease free/ Vaccine Volume Adjuvant N Death
survival WNV PER.C6 .RTM. 1.0 ml None 23 15 34.8% WNV PER.C6 .RTM.
0.5 ml None 22 10 54.6% WNV PER.C6 .RTM. 0.25 ml None 20 11 45.0%
WNV PER.C6 .RTM. 1.0 ml Mineral Oil 28 2 92.9% WNV PER.C6 .RTM. 0.5
ml Mineral Oil 25 3 88.0% WNV PER.C6 .RTM. 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% 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 represents the number of
animals per group. The control group of 13 animals did not receive
any vaccine and all died upon challenge.
TABLE-US-00006 TABLE VI The results of Table V taken together for
the geese groups that received the Mouse Brain-derived vaccine and
the PER.C6 .RTM.-derived vaccine (all with Mineral Oil as adjuvant)
as compared to the control group that did not receive vaccination.
Disease free/ Vaccine Adjuvant N Disease/Death survival WNV PER.C6
.RTM. Mineral Oil 78 8 89.7% WNV MB Mineral Oil 57 4 93.0% Control
-- 13 13 0%
TABLE-US-00007 TABLE VII Selection of Lineage I and II West Nile
Virus strains used in a cross- protection assay in geese. 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 (Aus91) >10,000 3.2 II Cyprus 1968 (Cyp68)
>10,000 0.5 II Madagascar 1978 (Mad78) >10,000 n.d. Viruses
of the different strains were produced in PER.C6 .RTM.. 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 LD.sub.50 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.
TABLE-US-00008 TABLE VIII CPE scores after infection of PER.C6
.RTM. cells with West Nile Virus New York 1999 (NY99 isolated from
a Snowy Owl, The Bronx Zoo, New York) USA99b dilutions day 2 day 3
day 4 day 5 day 6 10.sup.-3 + (<1%) + (75%) full cpe na na
10.sup.-4 - + (25%) + (75%) full cpe na 10.sup.-5 - + (1%) + (1%) +
(10%) + (75%) 10.sup.-6 - - + (<1%) + (<1%) + (25-50%)
10.sup.-7 - - + (<1%) + (<1%) + (5%) 10.sup.-8 - - - - -
TABLE-US-00009 TABLE IX CPE scores after infection of PER.C6 .RTM.
cells with West Nile Virus Aus91 (strain Kunjin 1991 [K 6453],
isolated in Australia from Culex annulirostris mosquitoes in 1991)
AUS91 dilutions day 2 day 3 day 4 day 5 day 6 10.sup.-3 - + (1%) +
(1-5%) + (10%) + (1-5%) 10.sup.-4 - - + (<1%) + (1%) + (1%)
10.sup.-5 - - - - - 10.sup.-6 - - - - - 10.sup.-7 - - - - -
10.sup.-8 - - - - -
TABLE-US-00010 TABLE X CPE scores after infection of PER.C6 .RTM.
cells with West Nile Virus Mad78 (strain Madagascar 1998) MAD78
dilutions day 2 day 3 day 4 day 5 day 6 10.sup.-3 - - - + (1%) +
(1%) 10.sup.-4 - - - - + (<1%) 10.sup.-5 - - - - - 10.sup.-6 - -
- - - 10.sup.-7 - - - - - 10.sup.-8 - - - - -
TABLE-US-00011 TABLE XI CPE scores after infection of PER.C6 .RTM.
cells with West Nile Virus Aus60 (strain Kunjin 1960 [MRM 16],
isolated in Australia from Culex annulirostris mosquitoes in 1960)
AUS60 dilutions day 2 day 3 day 4 day 5 day 6 10.sup.-3 - - + (25%)
+ (50-75%) + (75%) 10.sup.-4 - - + (1-5%) + (1-5%) + (10%)
10.sup.-5 - - - + (<1%) + (1%) 10.sup.-6 - - - + (<1%) -
10.sup.-7 - - - - - 10.sup.-8 - - - - -
TABLE-US-00012 TABLE XII CPE scores after infection of PER.C6 .RTM.
cells with West Nile Virus Cyp68 (strain Cyprus 1968) CYP68
dilutions day 2 day 3 day 4 day 5 day 6 10.sup.-3 - + (1%) + (1%) +
(10%) + (25%) 10.sup.-4 - - - + (1%) + (1%) 10.sup.-5 - - - - -
10.sup.-6 - - - - - 10.sup.-7 - - - - - 10.sup.-8 - - - - -
TABLE-US-00013 TABLE XIII CPE scores after infection of PER.C6
.RTM. cells with West Nile Virus Isr98 (strain Goose Israel 1998)
ISR98 dilutions day 2 day 3 day 4 day 5 day 6 10.sup.-3 + (<1%)
+ (50%) full cpe na na 10.sup.-4 - + (25%) + (75%) full cpe na
10.sup.-5 - + (1%) + (1-5%) + (50-75%) full cpe 10.sup.-6 - +
(<1%) + (<1%) + (25%) + (50%) 10.sup.-7 - - - - + (<1%)
10.sup.-8 - - - - -
TABLE-US-00014 TABLE XIV Vaccination scheme of geese using West
Nile Virus vaccines comprising different adjuvant compounds. Group
Vol- Injec- size Material ume tions Adjuvant 3 .times. 20 Mouse
Brain WN-MO 1 ml Day 0 Mineral oil Day 14 3 .times. 20 PER.C6
.RTM.-WN-MO 1 ml Day 0 Mineral oil Day 14 3 .times. 20 PER.C6
.RTM.-WN-AlOH 1 ml Day 0 Aluminum Day 14 hydroxide REHYDRAGEL .RTM.
3 .times. 20 PER.C6 .RTM.-WN-AlPh 1 ml Day 0 Aluminum Day 14
phosphate REHYDRAPHOS .RTM. 3 .times. 20 PER.C6 .RTM.-WN 1 ml Day 0
None Day 14 20 PER.C6 .RTM.-SH 1 ml Day 0 None Day 14 20 PER.C6
.RTM.-SH-AlOH 1 ml Day 0 Aluminum Day 14 hydroxide REHYDRAGEL .RTM.
20 PER.C6 .RTM.-SH-AlPh 1 ml Day 0 Aluminum Day 14 phosphate
REHYDRAPHOS .RTM. 20 PER.C6 .RTM.-SH-MO 1 ml Day 0 Mineral oil Day
14 20 PBS 1 ml Day 0 Day 14
TABLE-US-00015 TABLE XV Analysis with death taken as endpoint,
intention to treat (safety and efficacy). N Survival Survival Prob
95% CI Mouse Brain MO 60 54 90.0% 79.9-95.0% PER.C6 .RTM. WN MO 60
53 88.3% 77.8-94.2% PER.C6 .RTM. WN AlOH 60 38 63.3% 50.7-74.4%
PER.C6 .RTM. WN AlPh 60 41 68.3% 55.8-78.7% PER.C6 .RTM. WN 60 32
53.3% 40.9-65.4% PER.C6 .RTM. SH 20 0 0 0-16.1% PER.C6 .RTM. SH
AlOH 20 0 0 0-16.1% PER.C6 .RTM. SH AlPh 20 0 0 0-16.1% PER.C6
.RTM. MO 20 1 5.0% 0.9-23.6% PBS 20 0 0 0-16.1% N = number of
animals; CI = Confidentiality Interval Risk difference between:
Mouse Brain-MO and PER.C6 .RTM.-WN-MO: 1.7% (95% CI -9.5-12.8) not
significant Mouse Brain-MO and PER.C6 .RTM.-WN-AlOH: 26.7% (95% CI
12.3-41.0) significant
TABLE-US-00016 TABLE XVI Analysis with death or diseased taken as
endpoint (disease-free survival) df Survival N df Survival Prob 95%
CI Mouse Brain MO 60 54 90.0% 79.9-95.0% PER.C6 .RTM. WN MO 60 53
88.3% 77.8-94.2% PER.C6 .RTM. WN AlOH 60 31 57.7% 39.3-63.8% PER.C6
.RTM. WN AlPh 60 32 53.3% 40.9-65.4% PER.C6 .RTM. WN 60 25 47.7%
30.1-54.3% PER.C6 .RTM. SH 20 0 0 0-16.1% PER.C6 .RTM. SH AlOH 20 0
0 0-16.1% PER.C6 .RTM. SH AlPh 20 0 0 0-16.1% PER.C6 .RTM. MO 20 1
5.0% 0.9-23.6% PBS 20 0 0 0-16.1% Note: Numbers in italic indicate
differences with table XV. N = number of animals; CI =
Confidentiality Interval Risk difference between: Mouse Brain-MO
and PER.C6 .RTM.-WN-MO: 1.7 (95% CI -9.5-12.8%) not significant
Mouse Brain-MO and PER.C6 .RTM.-WN-AlOH: 38.3 (95% CI 23.6-53.1%)
significant
TABLE-US-00017 TABLE XVII Analysis with death taken as endpoint
among geese that were challenged with WN (to estimate true
efficacy) N Survival Survival Prob 95% CI Mouse Brain MO 58 54 93.1
83.6-97.3% PER.C6 .RTM. WN MO 58 53 91.4 81.4-96.3% PER.C6 .RTM. WN
AlOH 59 38 64.4 51.7-75.4% PER.C6 .RTM. WN AlPh 56 41 73.2
60.4-83.0% PER.C6 .RTM. WN 57 32 56.1 43.3-68.2% PER.C6 .RTM. SH 19
0 0 0-16.8% PER.C6 .RTM. SH AlOH 20 0 0 0-16.1% PER.C6 .RTM. SH
AlPh 20 0 0 0-16.1% PER.C6 .RTM. 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 .RTM.-WN-MO: 1.7 (95%
CI -8.0-11.5) not significant Mouse Brain-MO and PER.C6
.RTM.-WN-AlOH: 28.7 (95% CI 14.9-42.6) significant
TABLE-US-00018 TABLE XVIII Analysis with endpoint "death before
challenge" to study the safety of vaccination N Survival Survival
Prob 95% CI Mouse Brain MO 60 58 96.7 88.6-99.1% PER.C6 .RTM. WN MO
60 58 96.7 88.6-99.1% PER.C6 .RTM. WN AlOH 60 59 98.3 91.1-99.7%
PER.C6 .RTM. WN AlPh 60 56 93.3 84.1-97.4% PER.C6 .RTM. WN 60 57
95.0 86.3-98.3% PER.C6 .RTM. SH 20 19 95 76.4-99.1% PER.C6 .RTM. SH
AlOH 20 20 100 83.9-100% PER.C6 .RTM. SH AlPh 20 20 100 83.9-100%
PER.C6 .RTM. 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 .RTM.-WN-MO: 0 (95% CI -6.4-6.4)
not significant Mouse Brain-MO and PER.C6 .RTM.-WN-AlOH: 1.7 (95%
CI -3.9-7.3) not significant
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Sequence CWU 1
1
3121DNAArtificialSynthetic oligonucleotide WNV 1 1ccaccggawg
ttgagtagac g 21220DNAArtificialSynthetic oligonucleotide WNV 2
2tttgktcacc cagtcctcct 20320DNAArtificialSynthetic oligonucleotide
VIC labelled probe 3tgctgcctgc grctcaaccc 20
* * * * *