U.S. patent application number 12/325864 was filed with the patent office on 2009-07-30 for flavivirus vaccines.
Invention is credited to Juan Arroyo, Farshad Guirakhoo, Thomas P. Monath, Konstantin Pugachev.
Application Number | 20090191240 12/325864 |
Document ID | / |
Family ID | 29739394 |
Filed Date | 2009-07-30 |
United States Patent
Application |
20090191240 |
Kind Code |
A1 |
Monath; Thomas P. ; et
al. |
July 30, 2009 |
Flavivirus Vaccines
Abstract
The invention provides flavivirus vaccines and methods of making
and using these vaccines.
Inventors: |
Monath; Thomas P.; (Harvard,
MA) ; Guirakhoo; Farshad; (Melrose, MA) ;
Arroyo; Juan; (S.Weymouth, MA) ; Pugachev;
Konstantin; (Natick, MA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
29739394 |
Appl. No.: |
12/325864 |
Filed: |
December 1, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10345036 |
Jan 15, 2003 |
7459160 |
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12325864 |
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60348949 |
Jan 15, 2002 |
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60385281 |
May 31, 2002 |
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Current U.S.
Class: |
424/218.1 ;
435/235.1; 435/5 |
Current CPC
Class: |
Y02A 50/386 20180101;
C12N 7/00 20130101; C07K 14/005 20130101; C12N 2770/24134 20130101;
C12N 2770/24162 20130101; Y02A 50/39 20180101; A61P 31/14 20180101;
Y02A 50/388 20180101; A61K 2039/5254 20130101; Y02A 50/30 20180101;
C12N 2770/24122 20130101; A61K 39/12 20130101; Y02A 50/394
20180101 |
Class at
Publication: |
424/218.1 ;
435/235.1; 435/5 |
International
Class: |
A61K 39/12 20060101
A61K039/12; C12N 7/00 20060101 C12N007/00; C12Q 1/70 20060101
C12Q001/70 |
Claims
1. a flavivirus comprising a hinge region mutation that reduces
viscerotropism of said flavivirus.
2. The flavivirus of claim 1, wherein said flavivirus comprises a
yellow fever virus vaccine strain.
3. The flavivirus of claim 1, wherein said flavivirus is a
viscerotropic flavivirus selected from the group consisting of
Dengue virus, West Nile virus, Wesselsbron virus, Kyasanur Forest
Disease virus, and Omsk Hemorrhagic fever virus.
4. The flavivirus of claim 1, wherein said flavivirus is a chimeric
flavivirus.
5. The flavivirus of claim 4, wherein said chimeric flavivirus
comprises the capsid and non-structural proteins of a first
flavivirus virus and the pre-membrane and envelope proteins of a
second flavivirus comprising an envelope protein mutation that
decreases viscerotropism of said chimeric flavivirus.
6. The flavivirus of claim 5, wherein said second flavivirus is a
Japanese encephalitis virus.
7. The flavivirus of claim 5, wherein said second flavivirus is a
Dengue virus.
8. The flavivirus of claim 7, wherein said Dengue virus is
Dengue-1, Dengue-2, Dengue-3, or Dengue-4 virus.
9. The flavivirus of claim 7, wherein said mutation is in the
lysine at Dengue envelope amino acid position 202 or 204.
10. The flavivirus of claim 9, wherein said mutation is a
substitution of said lysine.
11. The flavivirus of claim 10, wherein said lysine is substituted
with arginine.
12. A vaccine composition comprising the flavivirus of claim 1 and
a pharmaceutically acceptable carrier or diluent.
13. A method of inducing an immune response to a flavivirus in a
patient, said method comprising administering to said patient the
vaccine composition of claim 12.
14. The method of claim 13, wherein said patient does not have, but
is at risk of developing, said flavivirus infection.
15. The method of claim 13, wherein said patient has said
flavivirus infection.
16. A method of producing a flavivirus vaccine, said method
comprising introducing into said flavivirus a mutation that results
in decreased viscerotropism.
17. A method of identifying a flavivirus vaccine candidate, said
method comprising the steps of: introducing a mutation into the
hinge region of said flavivirus; and determining whether the
flavivirus comprising said hinge region mutation has decreased
viscerotropism, as compared with a flavivirus virus lacking the
mutation.
18. The method of claim 17, wherein said flavivirus is a yellow
fever virus.
19. The method of claim 17, wherein said flavivirus is a chimeric
flavivirus.
Description
[0001] This application is a continuation of U.S. Ser. No.
10/345,036, filed Jan. 15, 2003, which claims priority from U.S.
Provisional Patent Application Ser. Nos. 60/348,949, filed Jan. 15,
2002, and 60/385,281, filed May 31, 2002, the contents of each of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to flavivirus vaccines.
BACKGROUND OF THE INVENTION
[0003] Flavivirises are small, enveloped, positive-strand RNA
viruses, several of which pose current or potential threats to
global public health. Yellow fever virus, for example, has been the
cause of epidemics in certain jungle locations of sub-Saharan
Africa, as well as in some parts of South America. Although many
yellow fever infections are mild, the disease can also cause
severe, life-threatening illness. The disease state has two phases.
The initial or acute phase is normally characterized by high fever,
chills, headache, backache, muscle aches, loss of appetite, nausea,
and vomiting. After three to four days, these symptoms disappear.
In some patients, symptoms then reappear, as the disease enters its
so-called toxic phase. During this phase, high fever reappears and
can lead to shock, bleeding (e.g., bleeding from the mouth, nose,
eyes, and/or stomach), kidney failure, and liver failure. Indeed,
liver failure causes jaundice, which is yellowing of the skin and
the whites of the eyes, and thus gives "yellow fever" its name.
About half of the patients who enter the toxic phase die within 10
to 14 days. However, persons that recover from yellow fever have
lifelong immunity against reinfection. The number of people
infected with yellow fever virus over the last two decades has been
increasing, with there now being about 200,000 yellow fever cases,
with about 30,000 deaths, each year. The re-emergence of yellow
fever virus thus presents a serious public health concern.
[0004] Dengue (DEN) virus is another example of a flavivirus.
Dengue viruses are transmitted to humans by mosquitoes (mainly by
Aedes aegypti) and are the cause of a growing public health problem
worldwide. Fifty to one hundred million persons are infected by
Dengue virus annually, and rates of infection as high as 6% have
been observed in some areas (Gubler, "Dengue and Dengue Hemorrhagic
Fever," CABI Publ., New York, Chapter 1, pp. 1-22, 1997; Burke et
al., Am. J. Trop. Med. Hyg. 38:172-180, 1988). Four serotypes of
Dengue virus (dengue types 1-4) circulate in the Caribbean, Asia,
and the Americas. The severe, potentially lethal form of DEN
infection [dengue hemorrhagic fever/dengue shock syndrome
(DHF/DSS)] is an immunopathological disease occurring in
individuals who have sustained sequential infections with different
DEN serotypes. Over 3.6 million cases of DHF and 58,000 deaths
caused by DHF were reported between 1980 and 1995 (Halstead,
"Dengue and Dengue Hemorrhagic Fever," CABI Publ., New York,
Chapter 2, pp. 23-44, 1997). Because of the pathogenesis of
DHF/DSS, it is generally thought that a optimal dengue vaccine may
need to immunize against all four serotypes of Dengue virus
simultaneously and induce long-lasting immunity. Despite the
extensive efforts that have made towards developing an effective
Dengue vaccine since World War II, there is currently no approved
dengue vaccine available.
[0005] Flaviviruses, including yellow fever virus and dengue virus,
have two principal biological properties responsible for their
induction of disease states in humans and animals. The first of
these two properties is neurotropism, which is the propensity of
the virus to invade and infect nervous tissue of the host.
Neurotropic flavivirus infection can result in inflammation and
injury of the brain and spinal cord (i.e., encephalitis), impaired
consciousness, paralysis, and convulsions. The second biological
property of flaviviruses is viscerotropism, which is the propensity
of the virus to invade and infect vital visceral organs, including
the liver, kidney, and heart. Viscerotropic flavivirus infection
can result in inflammation and injury of the liver (hepatitis),
kidney (nephritis), and cardiac muscle (myocarditis), leading to
failure or dysfunction of these organs. Neurotropism and
viscerotropism appear to be distinct and separate properties of
flaviviruses.
[0006] Some flaviviruses are primarily neurotropic (such as West
Nile virus), others are primarily viscerotropic (e.g., yellow fever
virus and dengue virus), and still others exhibit both properties
(such as Kyasanur Forest disease virus). However, both neurotropism
and viscerotropism are present to some degree in all flaviviruses.
Within the host, an interaction between viscerotropism and
neurotropism is likely to occur, because infection of viscera
occurs before invasion of the central nervous system. Thus,
neurotropism depends on the ability of the virus to replicate in
extraneural organs (viscera). This extraneural replication produces
viremia, which in turn is responsible for invasion of the brain and
spinal cord.
[0007] One approach to developing vaccines against flaviviruses is
to modify their virulence properties, so that the vaccine virus has
lost its neurotropism and viscerotropism for humans or animals. In
the case of yellow fever virus, two vaccines (yellow fever 17D and
the French neurotropic vaccine) have been developed (Monath,
"Yellow Fever," In Plotkin and Orenstein, Vaccines, 3.sup.rd ed.,
1999, Saunders, Philadelphia, pp. 815-879). The yellow fever 17D
vaccine was developed by serial passage in chicken embryo tissue,
and resulted in a virus with significantly reduced neurotropism and
viscerotropism. The French neurotropic vaccine was developed by
serial passages in mouse brain tissue, and resulted in loss of
viscerotropism, but retained neurotropism. A high incidence of
neurological accidents (post-vaccinal encephalitis) was associated
with the use of the French vaccine. Approved vaccines are not
currently available for many medically important flaviviruses
having viscerotropic properties, such as dengue, West Nile, and
Omsk hemorrhagic fever viruses, among others.
[0008] Fully processed, mature virions of flaviviruses contain
three structural proteins, capsid (C), membrane (M), and envelope
(E), and seven non-structural proteins. Immature flavivirions found
in infected cells contain pre-membrane (prM) protein, which is a
precursor to the M protein. The flavivirus proteins are produced by
translation of a single, long open reading frame to generate a
polyprotein, followed by a complex series of post-translational
proteolytic cleavages of the polyprotein, to generate mature viral
proteins (Amberg et al., J. Virol. 73:8083-8094, 1999; Rice,
"Flaviviridae," In Virology, Fields (ed.), Raven-Lippincott, New
York, 1995, Volume I, p. 937). The virus structural proteins are
arranged in the polyprotein in the order C-prM-E.
SUMMARY OF THE INVENTION
[0009] The invention provides flaviviruses including one or more
hinge region mutations that reduce viscerotropism of the
flaviviruses. These flaviviruses can be, for example, yellow fever
virus (e.g., a yellow fever virus vaccine strain); a viscerotropic
flavivirus selected from the group consisting of Dengue virus, West
Nile virus, Wesselsbron virus, Kyasanur Forest Disease virus, and
Omsk Hemorrhagic fever virus; or a chimeric flavivirus. In one
example of a chimeric flavivirus, the chimera includes the capsid
and non-structural proteins of a first flavivirus virus (e.g., a
yellow fever virus) and the pre-membrane and envelope proteins of a
second flavivirus (e.g., a Japanese encephalitis virus or a Dengue
virus (e.g., Dengue virus 1, 2, 3, or 4)) including an envelope
protein mutation that decreases viscerotropism of the chimeric
flavivirus. In the case of Dengue virus, the mutation can be, for
example, in the lysine at Dengue envelope amino acid position 202
or 204. This amino acid can be substituted by, for example,
arginine.
[0010] The invention also provides vaccine compositions that
include any of the viruses described herein and a pharmaceutically
acceptable carrier or diluent, as well as methods of inducing an
immune response to a flavivirus in a patient by administration of
such a vaccine composition to the patient. Patients treated using
these methods may not have, but be at risk of developing, the
flavivirus infection, or may have the flavivirus infection.
[0011] Also included in the invention are methods of producing
flavivirus vaccines, involving introducing into a flavivirus a
mutation that results in decreased viscerotropism. Further, the
invention includes methods of identifying flavivirus (e.g., yellow
fever virus or chimeric flavivirus) vaccine candidates, involving
(i) introducing a mutation into the hinge region of a flavivirus;
and (ii) determining whether the flavivirus including the hinge
region mutation has decreased viscerotropism, as compared with a
flavivirus virus lacking the mutation.
[0012] The flaviviruses of the invention are advantageous because,
in having decreased viscerotropism, they provide an additional
level of safety, as compared to their non-mutated counterparts,
when administered to patients. Additional advantages of these
viruses are provided by the fact that they can include sequences of
yellow fever virus strain YF17D (e.g., sequences encoding capsid
and non-structural proteins), which (i) has had its safety
established for >60 years, during which over 350 million doses
have been administered to humans, (ii) induces a long duration of
immunity after a single dose, and (iii) induces immunity rapidly,
within a few days of inoculation. In addition, the vaccine viruses
of the invention cause an active infection in the treated patients.
As the cytokine milieu and innate immune response of immunized
individuals are similar to those in natural infection, the
antigenic mass expands in the host, properly folded conformational
epitopes are processed efficiently, the adaptive immune response is
robust, and memory is established. Moreover, in certain chimeras of
the invention, the prM and E proteins derived from the target virus
contain the critical antigens for protective humoral and cellular
immunity.
[0013] Other features and advantages of the invention will be
apparent from the following detailed description, the drawings, and
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows plaque size variants produced by
ChimeriVax.TM.-JE FRhL.sub.3 (large plaque, Panel A) and FRhL.sub.5
(small plaque, Panel B). Plaques were stained using rabbit anti-JE
antiserum followed by anti-rabbit IgG-horseradish peroxidase.
[0015] FIG. 2 is a series of graphs showing survival distributions
of YF-VAX.RTM. and ChimeriVax.TM.-JE constructs, with and without a
mutation at E279 (M.fwdarw.K). Four day-old suckling mice
inoculated by the intracerebral route with (FIG. 2A) approximately
0.7 log.sub.10 PFU; (FIG. 2B) approximately 1.7 log.sub.10 PFU; and
(FIG. 2C) .about.2.7 log.sub.10 PFU.
[0016] FIG. 3 is a graph of regression analysis, mortality vs.
virus dose, showing similar slopes and parallel lines for viruses
with (FRhL.sub.5) and without (FRhL.sub.3) the Met to Lys
reversion, allowing statistical comparison. The FRhL.sub.5 virus
was 18.52 times more potent (virulent) than FRhL.sub.3
(p<0.0001).
[0017] FIG. 4 shows the results of independent RNA transfection and
passage series of ChimeriVax.TM.-JE virus in FRhL and Vero cells.
The emergence of mutations in the prME genes by passage level is
shown.
[0018] FIG. 5 is a three-dimensional model of the flavivirus
envelope glycoprotein ectodomain showing locations of mutations in
the hinge region occurring with adaptation in FRhL or Vero cells.
The sequence of the JE envelope glycoprotein (strain JaOArS982;
Sumiyoshi et al., Virology 161:497-510, 1987) was aligned to one of
the TBE structural templates (Rey et al., Nature 375:291-298, 1995)
as an input for automated homology modeling building by the method
of SegMod (Segment Match Modeling) using LOOK software (Molecular
Application Group, Palo Alto, Calif.).
DETAILED DESCRIPTION
[0019] The invention provides flaviviruses (e.g., yellow fever
viruses and chimeric flaviviruses) having one or more mutations
that result in decreased viscerotropism, methods for making such
flaviviruses, and methods for using these flaviviruses to prevent
or to treat flavivirus infection. The mutation (or mutations) in
the flaviviruses of the invention is present in the hinge region of
the envelope protein, which we have shown plays a role in
determining viscerotropism. The viruses and methods of the
invention are described further, as follows.
[0020] One example of a flavivirus that can be used in the
invention is yellow fever virus. Mutations can be made in the hinge
region of the envelope of a wild-type infectious clone, e.g., the
Asibi infectious clone or an infectious clone of another wild-type,
virulent yellow fever virus, and the mutants can then be tested in
an animal model system (e.g., in hamster and/or monkey model
systems) to identify sites affecting viscerotropism. Reduction in
viscerotropism is judged by, for example, detection of decreased
viremia and/or liver injury in the model system (see below for
additional details). One or more mutations found to decrease
viscerotropism of the wild-type virus are then introduced into a
vaccine strain (e.g., YF17D), and these mutants are tested in an
animal model system (e.g., in a hamster and/or a monkey model
system) to determine whether the resulting mutants have decreased
viscerotropism. Mutants that are found to have decreased
viscerotropism can then be used as new vaccine strains that have
increased safety, due to decreased levels of viscerotropism.
[0021] Additional flaviviruses that can be used in the invention
include other mosquito-borne flaviviruses, such as Japanese
encephalitis, Dengue (serotypes 1-4), Murray Valley encephalitis,
St. Louis encephalitis, West Nile, Kunjin, Rocio encephalitis, and
Ilheus viruses; tick-borne flaviviruses, such as Central European
encephalitis, Siberian encephalitis, Russian Spring-Summer
encephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever,
Louping ill, Powassan, Negishi, Absettarov, Hansalova, Apoi, and
Hypr viruses; as well as viruses from the Hepacivirus genus (e.g.,
Hepatitis C virus). All of these viruses have some propensity to
infect visceral organs. The viscerotropism of these viruses may not
cause dysfunction of vital visceral organs, but the replication of
virus in these organs can cause viremia and thus contribute to
invasion of the central nervous system. Decreasing the
viscerotropism of these viruses by mutagenesis can thus reduce
their abilities to invade the brain and to cause encephalitis.
[0022] In addition to the viruses listed above, as well as other
flaviviruses, chimeric flaviviruses that include one or more
mutations that decrease viscerotropism are included in the
invention. These chimeras can consist of a flavivirus (i.e., a
backbone flavivirus) in which a structural protein (or proteins)
has been replaced with a corresponding structural protein (or
proteins) of a second virus (i.e., a test or a predetermined virus,
such as a flavivirus). For example, the chimeras can consist of a
backbone flavivirus (e.g., a yellow fever virus) in which the prM
and E proteins of the flavivirus have been replaced with the prM
and E proteins of the second, test virus (e.g., a dengue virus
(1-4), Japanese encephalitis virus, West Nile virus, or another
virus, such as any of those mentioned herein) (the E protein of
which has a hinge region mutation as described herein). The
chimeric viruses can be made from any combination of viruses.
Preferably, the virus against which immunity is sought is the
source of the inserted structural protein(s).
[0023] A specific example of a chimeric virus that can be included
in the vaccines of the invention is the yellow fever human vaccine
strain, YF17D, in which the prM protein and the E protein have been
replaced with the prM protein and the E protein (including a hinge
mutation as described herein) of another flavivirus, such as a
Dengue virus (serotype 1, 2, 3, or 4), Japanese encephalitis virus,
West Nile virus, St. Louis encephalitis virus, Murray Valley
encephalitis virus, or any other flavivirus, such as one of those
listed above. For example, the following chimeric flaviviruses,
which were deposited with the American Type Culture Collection
(ATCC) in Manassas, Va., U.S.A. under the terms of the Budapest
Treaty and granted a deposit date of Jan. 6, 1998, can be used to
make viruses of the invention: Chimeric Yellow Fever 17D/Dengue
Type 2 Virus (YF/DEN-2; ATCC accession number ATCC VR-2593) and
Chimeric Yellow Fever 17D/Japanese Encephalitis SA14-14-2 Virus
(YF/JE A1.3; ATCC accession number ATCC VR-2594). Details of making
chimeric viruses that can be used in the invention are provided,
for example, in International applications PCT/US98/03894 and
PCT/US00/32821; and Chambers et al., J. Virol. 73:3095-3101, 1999,
each of which is incorporated by reference herein in its
entirety.
[0024] As is noted above, mutations that are included in the
viruses of the present invention decrease viscerotropism. In one
example, these mutations are present in the hinge region of the
flavivirus envelope protein. The polypeptide chain of the envelope
protein folds into three distinct domains: a central domain (domain
I), a dimerization domain (domain II), and an immunoglobulin-like
module domain (domain III). The hinge region is present between
domains I and II and, upon exposure to acidic pH, undergoes a
conformational change (hence the designation "hinge") involved in
the fusion of viral and endosomal membranes, after virus uptake by
receptor-mediated endocytosis. Numerous envelope amino acids are
present in the hinge region including, for example, amino acids
48-61, 127-131, and 196-283 of yellow fever virus (Rey et al.,
Nature 375:291-298, 1995). Any of these amino acids, or closely
surrounding amino acids (and corresponding amino acids in other
flavivirus envelope proteins), can be mutated according to the
invention, and tested for a resulting decrease in viscerotropism.
Mutations can be made in the hinge region using standard methods,
such as site-directed mutagenesis. One example of the type of
mutation present in the viruses of the invention is substitutions,
but other types of mutations, such as deletions and insertions, can
be used as well. In addition, as is noted above, the mutations can
be present singly or in the context of one or more additional
mutations.
[0025] The viruses (including chimeras) of the present invention
can be made using standard methods in the art. For example, an RNA
molecule corresponding to the genome of a virus can be introduced
into primary cells, chick embryos, or diploid cell lines, from
which (or the supernatants of which) progeny virus can then be
purified. Another method that can be used to produce the viruses
employs heteroploid cells, such as Vero cells (Yasumura et al.,
Nihon Rinsho 21, 1201-1215, 1963). In this method, a nucleic acid
molecule (e.g., an RNA molecule) corresponding to the genome of a
virus is introduced into the heteroploid cells, virus is harvested
from the medium in which the cells have been cultured, harvested
virus is treated with a nuclease (e.g., an endonuclease that
degrades both DNA and RNA, such as Benzonase.TM.; U.S. Pat. No.
5,173,418), the nuclease-treated virus is concentrated (e.g., by
use of ultrafiltration using a filter having a molecular weight
cut-off of, e.g., 500 kDa), and the concentrated virus is
formulated for the purposes of vaccination. Details of this method
are provided in U.S. Patent Application Ser. No. 60/348,565, filed
Jan. 15, 2002, which is incorporated herein by reference.
[0026] The viruses of the invention can be administered as primary
prophylactic agents in adults or children at risk of infection, or
can be used as secondary agents for treating infected patients. For
example, in the case of yellow fever/dengue chimeras, the vaccines
can be used in adults or children at risk of Dengue infection, or
can be used as secondary agents for treating Dengue-infected
patients. Examples of patients who can be treated using the
dengue-related vaccines and methods of the invention include (i)
children in areas in which Dengue is endemic, such as Asia, Latin
America, and the Caribbean, (ii) foreign travelers, (iii) military
personnel, and (iv) patients in areas of a Dengue epidemic.
Moreover, inhabitants of regions into which the disease has been
observed to be expanding (e.g., Argentina, Chile, Australia, parts
of Africa, southern Europe, the Middle East, and the southern
United States), or regions in which it may be observed to expand in
the future (e.g., regions infested with Aedes aegypti), can be
treated according to the invention.
[0027] Formulation of the viruses of the invention can be carried
out using methods that are standard in the art. Numerous
pharmaceutically acceptable solutions for use in vaccine
preparation are well known and can readily be adapted for use in
the present invention by those of skill in this art. (See, e.g.,
Remington's Pharmaceutical Sciences (18.sup.th edition), ed. A.
Gennaro, 1990, Mack Publishing Co., Easton, Pa.) In two specific
examples, the viruses are formulated in Minimum Essential Medium
Earle's Salt (MEME) containing 7.5% lactose and 2.5% human serum
albumin or MEME containing 10% sorbitol. However, the viruses can
simply be diluted in a physiologically acceptable solution, such as
sterile saline or sterile buffered saline. In another example, the
viruses can be administered and formulated, for example, in the
same manner as the yellow fever 17D vaccine, e.g., as a clarified
suspension of infected chicken embryo tissue, or a fluid harvested
from cell cultures infected with the chimeric yellow fever
virus.
[0028] The vaccines of the invention can be administered using
methods that are well known in the art, and appropriate amounts of
the vaccines administered can be readily be determined by those of
skill in the art. For example, the viruses of the invention can be
formulated as sterile aqueous solutions containing between 10.sup.2
and 10.sup.7 infectious units (e.g., plaque-forming units or tissue
culture infectious doses) in a dose volume of 0.1 to 1.0 ml, to be
administered by, for example, intramuscular, subcutaneous, or
intradermal routes. In addition, because flaviviruses may be
capable of infecting the human host via the mucosal routes, such as
the oral route (Gresikova et al., "Tick-borne Encephalitis," In The
Arboviruses, Ecology and Epidemiology, Monath (ed.), CRC Press,
Boca Raton, Fla., 1988, Volume IV, 177-203), the viruses can be
administered by mucosal routes as well. Further, the vaccines of
the invention can be administered in a single dose or, optionally,
administration can involve the use of a priming dose followed by a
booster dose that is administered, e.g., 2-6 months later, as
determined to be appropriate by those of skill in the art.
[0029] Optionally, adjuvants that are known to those skilled in the
art can be used in the administration of the viruses of the
invention. Adjuvants that can be used to enhance the immunogenicity
of the viruses include, for example, liposomal formulations,
synthetic adjuvants, such as (e.g., QS21), muramyl dipeptide,
monophosphoryl lipid A, or polyphosphazine. Although these
adjuvants are typically used to enhance immune responses to
inactivated vaccines, they can also be used with live vaccines. In
the case of a virus delivered via a mucosal route, for example,
orally, mucosal adjuvants such as the heat-labile toxin of E. coli
(LT) or mutant derivations of LT can be used as adjuvants. In
addition, genes encoding cytokines that have adjuvant activities
can be inserted into the viruses. Thus, genes encoding cytokines,
such as GM-CSF, IL-2, IL-12, IL-13, or IL-5, can be inserted
together with foreign antigen genes to produce a vaccine that
results in enhanced immune responses, or to modulate immunity
directed more specifically towards cellular, humoral, or mucosal
responses.
[0030] In the case of Dengue virus, against which optimal
vaccination can involve the induction of immunity against all four
of the dengue serotypes, the chimeric viruses of the present
invention can be used in the formulation of tetravalent vaccines.
Any or all of the chimeras used in such tetravalent formulations
can include a mutation that decreases viscerotropism, as is
described herein. The chimeras can be mixed to form tetravalent
preparations at any point during formulation, or can be
administered in series. In the case of a tetravalent vaccine, to
reduce the possibility of viral interference and thus to achieve a
balanced immune response, the amounts of each of the different
chimeras present in the administered vaccines can vary. Briefly, in
one example of such a formulation, at least 5 fold less of the
Dengue-2 chimera (e.g., 10, 50, 100, 200, or 500 fold less) is used
relative to the other chimeras. In this example, the amounts of the
Dengue-1, Dengue-3, and Dengue-4 chimeras can be equivalent or can
vary. In another example, the amounts of Dengue-4 and/or Dengue 1
virus can be decreased as well. For example, in addition to using
less Dengue-2 chimera, at least 5 fold less of the Dengue-4 chimera
(e.g., 10, 50, 100, 200, or 500 fold less) can be used relative to
the Dengue-1 and Dengue-3 chimeras; at least 5 fold less of the
Dengue-1 chimera (e.g., 10, 50, 100, 200, or 500 fold less) can be
used relative to the Dengue-3 and Dengue-4 chimeras; or at least 5
fold less of the Dengue-1 and Dengue-4 chimeras can be used
relative to the Dengue-3 chimera. It may be particularly desirable,
for example, to decrease the amount of Dengue-1 chimera relative to
the amounts of Dengue-3 and/or Dengue-4 chimeras when the E204/E202
mutation described herein is not included in the chimera.
[0031] Details of the characterization of one example of a mutation
included in the invention, which occurs at position 279 of the
envelope protein of a yellow fever/Japanese encephalitis chimera,
are provided below. Also provided below are details concerning
yellow fever/dengue virus chimeras, in which dengue virus envelope
proteins include one or more mutations that decrease
viscerotropism. In one example of such a mutation, the lysine at
position 204 of the envelope protein of Dengue-1, Dengue-2, or
Dengue-4, or the lysine at position 202 of the envelope protein of
Dengue-3, which is two amino acids shorter than the envelope
proteins of the other Dengue serotypes, is substituted or deleted.
This lysine can be, for example, substituted with arginine. Other
residues near envelope amino acid 204 (202 for Dengue-3) can also
be mutated to achieve decreased viscerotropism. For example, any of
amino acids 200-208 or combinations of these amino acids can be
mutated. Specific examples include the following: position 202 (K)
of Dengue-1; position 202 (E) of Dengue-2; position 200 of Dengue-3
(K); and positions 200 (K), 202 (K), and 203 (K) of Dengue-4. These
residues can be substituted with, for example, arginine.
EXPERIMENTAL RESULTS
I. Yellow Fever/Japanese Encephalitis Chimera Including a Hinge
Region Mutation
Summary
[0032] A chimeric yellow fever (YF)-Japanese encephalitis (JE)
vaccine (ChimeriVax.TM.-JE) was constructed by insertion of the
prM-E genes from the attenuated JE SA14-14-2 vaccine strain into a
full-length cDNA clone of YF 17D virus. Passage in fetal rhesus
lung (FRhL) cells led to the emergence of a small-plaque virus
containing a single Met.fwdarw.Lys amino acid mutation at E279,
reverting this residue from the SA14-14-2 to the wild-type amino
acid. A similar virus was also constructed by site-directed
mutagenesis. The E279 mutation is located in a beta-sheet in the
hinge region of the E protein, which is responsible for a
pH-dependent conformational change during virus penetration from
the endosome into the cytoplasm of an infected cell. In independent
transfection-passage studies in FRhL or Vero cells, mutations
appeared most frequently in hinge 4 (bounded by amino acids E266 to
E284), reflecting genomic instability in this functionally
important region. The E279 reversion caused a significant increase
in neurovirulence, as determined by LD50 and survival distribution
in suckling mice and by histopathology in rhesus monkeys. Based on
sensitivity and comparability of results with monkeys, the suckling
mouse is an appropriate host for safety testing of flavivirus
vaccine candidates for neurotropism. The E279 Lys virus was
restricted with respect to extraneural replication in monkeys, as
viremia and antibody levels (markers of viscerotropism) were
significantly reduced as compared to E279 Met virus.
Background
[0033] The study of chimeric viruses has afforded new insights into
the molecular basis of virulence and new prospects for vaccine
development. For example, molecular clones of positive-strand
alphaviruses (Morris-Downes et al., Vaccine 19:3877-3884, 2001;
Xiong et al., Science 243:1188-1191, 1991) and flaviviruses (Bray
et al., Proc. Natl. Acad. Sci. U.S.A. 88:10342-10346, 1991;
Chambers et al., J. Virol. 73:3095-3101, 1999; Guirakhoo et al., J.
Virol. 75:7290-7304, 2001; Huang et al., J. Virol. 74:3020-3028,
2000) have been modified by insertion of structural genes encoding
the viral envelope and determinants involved in neutralization,
cell attachment, fusion, and internalization. The replication of
these chimeric viruses is controlled by nonstructural proteins and
the non-coding termini expressed by the parental strain, while the
structural proteins from the donor genes afford specific immunity.
The biological characteristics of chimeric viruses are determined
by both the donor and recipient virus genes. By comparing
constructs with nucleotide sequence differences across the donor
genes, it is possible to dissect out the functional roles of
individual amino acid residues in virulence and attenuation.
[0034] Using a chimeric yellow fever (YF) virus that incorporated
the prM-E genes from an attenuated strain (SA14-14-2) of Japanese
encephalitis (JE), a detailed examination was made of the role of
10 amino acid mutations that distinguished the attenuated JE virus
from virulent, wild-type JE Nakayama virus (Arroyo et al., J.
Virol. 75:934-942, 2001). The virulence factors were defined by
reverting each mutation singly or as clusters to the wild-type
sequence and determining the effects on neurovirulence for young
adult mice inoculated by the intracerebral (IC) route with 10.sup.4
plaque-forming units (PFU). All of the single-site revertant
viruses remained highly attenuated, and reversions at 3 or 4
residues were required to restore a neurovirulent phenotype. Only
one single-site revertant (E279 Met.fwdarw.Lys) showed any evidence
of a change in virulence, with 1 of 8 animals succumbing after IC
inoculation.
[0035] In order to explore further the functional role of the E279
determinant, we compared chimeric YF/JE viruses that differed at
this amino acid residue for their abilities to cause encephalitis
in suckling mice and monkeys. IC inoculation of monkeys is
routinely used as a test for safety of flavivirus and other live
vaccines, and quantitative pathological examination of brain and
spinal cord tissue provides a sensitive method for distinguishing
strains of the same virus with subtle differences in neurovirulence
(Levenbook et al., J. Biol. Stand. 15: 305-313, 1987). Suckling
mice provide a more sensitive model than older animals, since
susceptibility to neurotropic flaviviruses is age-dependent (Monath
et al., J. Virol. 74:1742-1751, 2000). The results confirmed that
the single Met.fwdarw.Lys amino acid mutation at E279 conferred an
increase in neurovirulence. This mutation is located in the `hinge`
region of the E protein, which is responsible for a pH-dependent
conformational change during virus penetration from the endosome
into the cytoplasm of an infected cell (Reed et al., Am. J. Hyg.
27:493-497, 1938). Importantly, the suckling mouse was shown to
predict the virulence profile in rhesus monkeys. Based on the
detection of a change in neurovirulence conferred by a point
mutation, we propose that the suckling mouse is an appropriate host
for safety testing of flavivirus vaccine candidates for
neurotropism.
[0036] While enhancing neurovirulence, the E279 mutation appeared
to have the opposite effect on viscerotropism, as measured by
decreased viremia and antibody response in monkeys, accepted
markers of this viral trait (Wang et al., J. Gen. Virol.
76:2749-2755, 1995).
Materials and Methods
Viruses
[0037] Development of the ChimeriVax.TM.-JE vaccine began by
cloning a cDNA copy of the entire 11-kilobase genome of YF 17D
virus (Chambers et al., J. Virol. 73:3095-3101, 1999). To
accomplish this, YF 17D genomic sequences were propagated in two
plasmids, which encode the YF sequences from nucleotide (nt) 1-2276
and 8279-10,861 (plasmid YF5'3'IV), and from 1373-8704 (plasmid
YFM5.2), respectively. Full-length cDNA templates were generated by
ligation of appropriate restriction fragments derived from these
plasmids. YF sequences within the YF 5'3'IV and YFM5.2 plasmids
were replaced by the corresponding JE (SA14-14-2) pr-ME sequences,
resulting in the generation of YF5'3'IV/JE (prM-E') and YFM5.2/JE
(E'-E) plasmids. These plasmids were digested sequentially with
restriction endonucleases NheI and BspEI. Appropriate fragments
were ligated with T4 DNA ligase, cDNA was digested with XhoI enzyme
to allow transcription, and RNA was produced from an Sp6 promoter.
Transfection of diploid fetal rhesus lung (FRhL) cells with
full-length RNA was performed by electroporation. Supernatant
containing virus was harvested when cytopathic effect was observed
(generally day 3), clarified by low-speed centrifugation and
sterile-filtered at 0.22 .mu.m. Fetal bovine serum (FBS) 50% v/v
final concentration was added as a stabilizer. The virus was
titrated by plaque assay in Vero cells, as previously described
(Monath et al., Vaccine 17:1869-1882, 1999). The chimeric virus was
sequentially passed in FRhL or Vero cells (Vero-PM, Aventis
Pasteur, Marcy l'Etoile, France) at a multiplicity of infection of
approximately 0.001. Commercial yellow fever 17D vaccine
(YF-VAX.RTM.) was obtained from Aventis-Pasteur (formerly
Pasteur-Merieux-Connaught), Swiftwater, Pa.
Site-Directed Mutagenesis
[0038] Virus containing a single-site Met.fwdarw.Lys reversion at
residue E279 was generated by oligo-directed mutagenesis as
described (Arroyo et al., J. Virol. 75:934-942, 2001). Briefly, a
plasmid (pBS/JE SA14-14-2) containing the JE SA-14-14-2 E gene
region from nucleotides 1108 to 2472 (Cecilia et al., Virology
181:70-77, 1991) was used as template for site-directed
mutagenesis. Mutagenesis was performed using the Transformer
site-directed mutagenesis kit (Clontech, Palo Alto, Calif.) and
oligonucleotide primers synthesized at Life Technologies (Grand
Island, N.Y.). Plasmids were sequenced across the E region to
verify that the only change was the engineered mutation. A region
encompassing the E279 mutation was then subcloned from the pBS/JE
plasmid into pYFM5.2/JE SA14-14-2 (Cecilia et al., Virology
181:70-77, 1991) using the NheI and EheI (Kas I) restriction sites.
Assembly of full-length DNA and SP6 transcription were performed as
described above; however, RNA transfection of Vero cells was
performed using Lipofectin (Gibco/BRL).
Sequencing
[0039] RNA was isolated from infected monolayers by Trizol.RTM.
(Life Technologies). Reverse transcription was performed with
Superscript II Reverse Transcriptase (RT) and a long-RT protocol
(Life Technologies), followed by RNaseH treatment (Promega) and
long-PCR (XL PCR, Perkin-Elmer/ABI). RT, PCR, and sequencing
primers were designed using YF17D strain sequence (GeneBank
Accession number K02749) and JE-SA14-14-2 strain sequence (GeneBank
Accession number D90195) as references. PCR products were
gel-purified (Qiaquick gel-extraction kit from Qiagen) and
sequenced using Dye-Terminator dRhodamine sequencing reaction mix
(Perkin-Elmer/ABI). Sequencing reactions were analyzed on a model
310 Genetic Analyzer (Perkin-Elmer/ABI) and DNA sequences were
evaluated using Sequencher 3.0 (GeneCodes) software.
Plaque Assays and Neutralization Tests
[0040] Plaque assays were performed in 6 well plates of monolayer
cultures of Vero cells. After adsorption of virus for 1 hour
incubation at 37.degree. C., the cells were overlaid with agarose
in nutrient medium. On day 4, a second overlay was added containing
3% neutral red. Serum-dilution, plaque-reduction neutralization
tests were performed as previously described (Monath et al.,
Vaccine 17:1869-1882, 1999).
Weaned Mouse Model
[0041] Groups of 8 to 10 female 4 week old ICR mice (Taconic Farms,
Inc. Germantown, N.Y.) were inoculated IC with 30 .mu.L of chimeric
YF/JE SA14-14-2 (ChimeriVax.TM.-JE) constructs with (dose 4.0
log.sub.10 PFU in) or without (3.1 log.sub.10 PFU) the E279
mutation. An equal number of mice were inoculated with YF-VAX.RTM.
or diluent. Mice were followed for illness and death for 21
days.
Suckling Mouse Model
[0042] Pregnant female ICR mice (Taconic Farms) were observed
through parturition in order to obtain litters of suckling mice of
exact age. Suckling mice from multiple litters born within a 48
hour interval were pooled and randomly redistributed to mothers in
groups of up to 121 mice. Litters were inoculated IC with 20 .mu.L
of serial tenfold dilutions of virus and followed for signs of
illness and death for 21 days. The virus inocula were
back-titrated. 50% lethal dose (LD.sub.50) values were calculated
by the method of Reed and Muench (Morris-Downes et al., Vaccine
19:3877-3884, 2001). Univariate survival distributions were plotted
and compared by log rank test.
Monkey Model
[0043] The monkey neurovirulence test was performed as described by
Levenbook et al. (Levenbook et al., J. Biol. Stand. 15: 305-313,
1987) and proscribed by WHO regulations for safety testing YF 17D
seed viruses (Wang et al., J. Gen. Virol. 76:2749-2755, 1995). This
test has previously been applied to the evaluation of
ChimeriVax.TM.-JE vaccines, and results of tests on FRhL.sub.3
virus were described (Monath et al., Curr. Drugs-Infect. Dis.,
1:37-50; 2001; Monath et al., Vaccine 17:1869-1882, 1999). Tests
were performed at Sierra Biomedical Inc. (Sparks, Nev.), according
to the U.S. Food and Drug Administration Good Laboratory Practice
(GLP) regulations (21 C.F.R., Part 58). On Day 1, ten (5 male, 5
female) rhesus monkeys weighing 3.0-6.5 kg received a single
inoculation of 0.25 mL undiluted ChimeriVax.TM.-JE virus with or
without the E279 Met.fwdarw.Lys mutation or YF-VAX.RTM. into the
frontal lobe of the brain. Monkeys were evaluated daily for
clinical signs and scored as 0 (no signs), 1 (rough coat, not
eating), 2 (high-pitched voice, inactive, slow moving, 3 (shaky
movements, tremors, incoordination, limb weakness), and 4
(inability to stand, limb paralysis, death). The clinical score for
each monkey is the mean of the animal's daily scores, and the
clinical score for the treatment group is the arithmetic mean of
the individual clinical scores. Viremia levels were measured by
plaque assay in Vero cells using sera collected on days 2-10. On
day 31, animals were euthanized, perfused with isotonic saline-5%
acetic acid followed by neutral-buffered 10% formalin, and
necropsies were performed. Brains and spinal cords were fixed,
sectioned and stained with gallocyanin. Neurovirulence was assessed
by the presence and severity of lesions in various anatomical
formations of the central nervous system. Severity was scored
within each tissue block using the scale specified by WHO (Wang et
al., J. Gen. Virol. 76:2749-2755, 1995):
[0044] Grade 1: Minimal: 1-3 small focal inflammatory infiltrates.
A few neurons may be changed or lost.
[0045] Grade 2: Moderate: more extensive focal inflammatory
infiltrates. Neuronal changes or loss affects not more than
one-third of neurons.
[0046] Grade 3: Severe: neuronal changes or loss affecting 33-90%
of neurons; moderate focal or diffuse inflammatory changes
[0047] Grade 4: Overwhelming; more than 90% of neurons are changed
or lost, with variable but frequently severe inflammatory
infiltration
[0048] Structures involved in the pathologic process most often and
with greatest severity were designated `target areas,` while those
structures discriminating between wild-type JE virus and
ChimeriVax.TM.-JE were designated `discriminator areas.` The
substantia nigra constituted the `target area` and the caudate
nucleus, globus pallidus, putamen, anterior/medial thalamic
nucleus, lateral thalamic nucleus, and spinal cord (cervical and
lumbar enlargements) constituted `discriminator areas` (Monath et
al., Curr. Drugs Infect. Dis., 1:37-50, 2001), as previously shown
for YF 17D (Levenbook et al., J. Biol. Stand. 15:305-313, 1987).
All neuropathological evaluations were done by a single,
experienced investigator who was blinded to the treatment code.
Separate scores for target area, discriminator areas, and
target+discriminator areas were determined for each monkey, and
test groups compared with respect to average scores. Other areas of
the brainstem (nuclei of the midbrain in addition to substantia
nigra; pons; medulla; and cerebellum) and the leptomeninges were
also examined. Statistical comparisons of mean neuropathological
scores (for the target area, discriminator areas, and
target+discriminator areas) were performed by Student's t test,
2-tailed. In addition to neuropathological examination, the liver,
spleen, adrenal glands, heart, and kidneys were examined for
pathologic changes by light microscopy.
Genome Stability
[0049] To ascertain the genetic stability of the YF/JE chimeric
virus, and to search for `hot spots` in the vaccine genome that are
susceptible to mutation, multiple experiments were performed in
which RNA was used to transfect cells and the progeny virus
serially passaged in vitro, with partial or complete genomic
sequencing performed at low and high passage levels. Passage series
were performed starting with the transfection step in FRhL or
Vero-PM cells. Serial passage of the virus was performed at low MOI
in cell cultures grown in T25 or T75 flasks. At selected passage
levels, duplicate samples of viral genomic RNA were extracted,
reverse-transcribed, amplified by PCR, and the prM-E region or full
genomic sequence determined.
Results
Generation of Single-Site Mutant Viruses by Empirical Passage
[0050] The chimeric YF/JESA14-14-2 (ChimeriVax.TM.-JE) virus
recovered from transfected FRhL cells (FRhL.sub.1) was passed
sequentially in fluid cultures of these cells at an MOI of
approximately 0.001. As is described below, at passage 4 we noted a
change in plaque morphology, which was subsequently shown to be
associated with a T.fwdarw.G transversion at nucleotide 1818
resulting in an amino acid change (Met.fwdarw.Lys) at position 279
of the E protein.
[0051] Plaques were characterized at each passage level and
classified into 3 categories based on their sizes measured on day 6
(large, L.about.>1.0 mm, medium, M.about.0.5-1 mm, and small,
S.about.<0.5 mm). The plaque size distribution was determined by
counting 100 plaques. FRhL.sub.3 (3.sup.rd passage) virus contained
80-94% L and 6-20% S plaques. At FRhL.sub.5 (5.sup.th passage), a
change in plaque size was detected, with the emergence of S plaques
comprising >85% of the total plaque population (FIG. 1). The
FRhL.sub.4 virus was intermediate, with 40% large and 60% small
plaques. Full genomic sequencing of the FRhL.sub.5 virus
demonstrated a single mutation at E279. The full genome consensus
sequence of the FRhL.sub.5 chimera, with careful inspection for
codon heterogeneity, confirmed that this was the only detectable
mutation present in the virus. The full genome consensus sequence
of the FRhL.sub.3 virus revealed no detectable mutations compared
to the parental YF/JESA14-14-2 chimeric virus (Arroyo et al., J.
Virol. 75:934-942, 2001) (Table 1).
[0052] Ten large, medium, and small plaques were picked from
FRhL.sub.3, .sub.-4 and .sub.-5, and amplified by passage in fluid
cultures of FRhL cells. After amplification, the supernatant fluid
was plaqued on Vero cells. Attempts to isolate the S plaque
phenotype from FRhL.sub.3 failed and all isolated L or S size
plaques produced a majority of L plaques after one round of
amplification in FRhL cells. At the next passage (FRhL.sub.4),
where 60% of plaques were of small size, it was possible to isolate
these plaques by amplification in FRhL cells. At FRhL.sub.5, the
majority of plaques (85-99%) were of small size, and amplification
of both L and S individual plaques resulted in majority of S size.
Sequencing the prM-E genes of the S and L plaque phenotypes from
FRhL.sub.3 revealed identical sequences to the parent SA14-14-2
genes used for construction of ChimeriVax.TM.-JE, whereas S plaques
isolated from either FRhL.sub.4 or FRhL.sub.5 virus revealed the
mutation (Met.fwdarw.Lys) at E279.
Animal Protocols
[0053] All studies involving mice and nonhuman primates were
conducted in accordance with the USDA Animal Welfare Act (9 C.F.R.,
Parts 1-3) as described in the Guide for Care and Use of Laboratory
Animals.
Virulence for Weaned Mice
[0054] Ten female ICR mice 4 weeks of age were inoculated IC with
approximately 3.0 log.sub.10 PFU of FRhL.sub.3, .sub.-4, or .sub.-5
virus in separate experiments; in each study 10 mice received an
equivalent dose (approximately 3.3 log.sub.10 PFU) of commercial
yellow fever vaccine (YF-VAX.RTM., Aventis Pasteur, Swiftwater
Pa.). None of the mice inoculated with chimeric viruses showed
signs of illness or died, whereas 70-100% of control mice
inoculated with YF-VAX.RTM. developed paralysis or died. In another
experiment, 8 mice were inoculated IC with FRhL.sub.5 (3.1
log.sub.10 PFU) or the YF/JE single-site E279 revertant (4.0
log.sub.10 PFU) and 9 mice received YF-VAX.RTM. (2.3 log.sub.10
PFU). None of the mice inoculated with the chimeric constructs
became ill, whereas 6/9 (67%) of mice inoculated with YF-VAX.RTM.
died.
Virulence for Suckling Mice
[0055] Two separate experiments were performed in which
YF/JESA14-14-2 chimeric viruses with and without the E279 mutation
were inoculated IC at graded doses into suckling mice (Table 2).
YF-VAX.RTM. was used as the reference control in these experiments.
LD.sub.50 and average survival times (AST) were determined for each
virus.
[0056] In the first experiment using mice 8.6 days old, FRhL.sub.5
virus containing the single site reversion (Met.fwdarw.Lys) at E279
was neurovirulent, with a log.sub.10 LD.sub.50 of 1.64 whereas the
FRhL.sub.3 virus lacking this mutation was nearly avirulent, with
only 1 of 10 mice dying in the highest dose groups (Table 2). At
the highest dose (approximately 3 log.sub.10 PFU), the AST of the
FRhL.sub.5 virus was shorter (10.3 days) than that of the
FRhL.sub.3 virus (15 days).
[0057] A second experiment was subsequently performed to verify
statistically that a single site mutation in the E gene is
detectable by neurovirulence test in suckling mice. In this
experiment outbred mice 4 days of age were inoculated IC with
graded doses of ChimeriVax.TM.-JE FRhL.sub.3 (no mutation),
ChimeriVax.TM.-JE FRhL.sub.5 (E279 Met.fwdarw.Lys), or a YF/JE
chimera in which a single mutation E279 (Met.fwdarw.Lys) was
introduced at by site-directed mutagenesis (Arroyo et al., J.
Virol. 75:934-942, 2001). The LD.sub.50 values of the two viruses
containing the E279 mutation were >10-fold lower than the
FRhL.sub.3 construct without the mutation (Table 2) indicating that
the E279 Met. Lys mutation increased the neurovirulence of the
chimeric virus. There were statistically significant differences
between the viruses in the survival distributions (FIG. 2). At the
lowest dose (.about.0.7 log.sub.10 PFU), the YF/JE chimeric viruses
were significantly less virulent than YF-VAX.RTM. (log rank
p<0.0001). The viruses with the E279 Met.fwdarw.Lys mutation had
similar survival curves that differed from the FRhL3 virus no
mutation), but the difference did not reach statistical
significance (log rank p=0.1216). However, at higher doses
(.about.1.7 and .about.2.7 log.sub.10 PFU), the survival
distributions of the E279 mutant viruses were significantly
different from FRhL.sub.3 virus.
[0058] Analysis of mortality ratio by virus dose revealed similar
slopes and parallel regression lines (FIG. 3). The FRhL.sub.5 virus
was 18.52 times more potent (virulent) than FRhL.sub.3 (95%
fiducial limits 3.65 and 124.44, p<0.0001).
Monkey Neurovirulence Test
[0059] None of the 20 monkeys inoculated with ChimeriVax.TM.-JE
FRhL.sub.3 or FRhL.sub.5 viruses developed signs of encephalitis,
whereas 4/10 monkeys inoculated with YF-VAX.RTM. developed grade 3
signs (tremors) between days 15-29, which resolved within 6 days of
onset. Mean and maximum mean clinical scores were significantly
higher in the YF-VAX.RTM. group than in the two ChimeriVax.TM.-JE
groups. There was no difference in clinical score between groups
receiving ChimeriVax.TM.-JE viruses with and without the E279
mutation (Table 3).
[0060] There were no differences in weight changes during the
experiment between treatment groups. Pathological examination
revealed no alterations of liver, spleen, kidney, heart, or adrenal
glands attributable to the viruses, and no differences between
treatment groups.
[0061] Histopathologic examination of the brain and spinal cord
revealed significantly higher lesion scores for monkeys inoculated
with YF-VAX.RTM. than for ChimeriVax.TM.-JE virus FRhL.sub.3 and
FRhL.sub.5 (Table 3). The combined target+discriminator scores
(.+-.SD) for YF-VAX.RTM. was 1.17 (.+-.0.47). The scores for the
ChimeriVax.TM.-JE FRhL.sub.3 (E279 Met) and FRhL.sub.5 (E279 Lys)
were 0.29 (.+-.0.20), (p=0.00014 vs. YF-VAX.RTM.) and 0.54
(.+-.0.28), (p=0.00248 vs. YF-VAX.RTM.), respectively.
[0062] The discriminator area score and combined
target+discriminator area score for ChimeriVax.TM.-JE FRhL.sub.5
containing the Met.fwdarw.Lys reversion at E279 were significantly
higher than the corresponding scores for ChimeriVax.TM.-JE
FRhL.sub.3 (Table 3).
[0063] The main symptom in monkeys inoculated with YF-VAX.RTM. was
tremor, which may reflect lesions of the cerebellum, thalamic
nuclei, or globus pallidus. No clear histological lesions were
found in the cerebellar cortex, N. dentatus, or other cerebellar
nuclei, whereas imflammatory lesions were present in the thalamic
nuclei and globus pallidus in all positive monkeys.
[0064] Interestingly, there was an inverse relationship between
neurovirulence and viscerotropism of the E279 revertant, as
reflected by viremia. The WHO monkey neurovirulence test includes
quantitation of viremia as a measure of viscerotropism (World
Health Organization, "Requirements for yellow fever vaccine,"
Requirements for Biological Substances No. 3, revised 1995, WHO
Tech. Rep. Ser. 872, Annex 2, Geneva: WHO, 31-68, 1998). This is
rational, based on observations that intracerebral inoculation
results in immediate seeding of extraneural tissues (Theiler, "The
Virus," In Strode (ed.), Yellow Fever, McGraw Hill, New York, N.Y.,
46-136, 1951). Nine (90%) of 10 monkeys inoculated with YF-VAX.RTM.
and 8 (80%) of 10 monkeys inoculated with ChimeriVax.TM.-JE
FRhL.sub.3 became viremic after IC inoculation. The level of
viremia tended to be higher in the YF-VAX.RTM. group than in the
ChimeriVax.TM.-JE FRhL.sub.3 group, reaching significance on Day 4.
In contrast, only 2 (20%) of the animals given FRhL.sub.5 virus
(E279 Met.fwdarw.Lys) had detectable, low-level viremias (Table 4),
and the mean viremia was significantly lower than FRhL.sub.3 virus
on days 3 and 4 (and nearly significant on day 5). Thus, the
FRhL.sub.5 revertant virus displayed increased neurovirulence, but
decreased viscerotropism compared to FRhL.sub.3 virus. Sera from
monkeys inoculated with ChimeriVax.TM.-JE FRhL.sub.3 and FRhL.sub.5
were examined for the presence of plaque size variants. Only L
plaques were observed in sera from monkeys inoculated with the
FRhL.sub.3, whereas the virus in blood of monkeys inoculated with
FRhL.sub.5 had the appropriate S plaque morphology.
Immunogenicity
[0065] All monkeys in all three groups developed homologous
neutralizing antibodies 31 days post-inoculation to yellow fever
(YF-VAX.RTM. group) or ChimeriVax.TM.-JE (ChimeriVax.TM. groups),
with the exception of 1 animal (FRhL.sub.5, RAK22F), which was not
tested due to sample loss. However, the geometric mean antibody
titer (GMT) was significantly higher in the monkeys inoculated with
FRhL.sub.3 (GMT 501) than with FRhL.sub.5 (GMT 169, p=0.0386,
t-test).
Genome Stability
[0066] Two separate transfections of ChimeriVax.TM.-JE RNA were
performed in each of two cell strains, FRhL and Vero, and progeny
viruses were passed as is shown in FIG. 4. The FRhL passage series
B resulted in appearance of the E279 reversion at FRhL.sub.4 as
described above. Interestingly, a separate passage series (A) in
FRhL cells also resulted in the appearance of a mutation
(Thr.fwdarw.Lys) in an adjacent residue at E281, and one of the
passage series in Vero cells resulted in a Val.fwdarw.Lys mutation
at E271. Other mutations selected in Vero cells were in domain III
or within the transmembrane domain. All viruses containing
mutations shown in FIG. 2 were evaluated in the adult mouse
neurovirulence test and were found to be avirulent.
II. Yellow Fever/Dengue Chimera Including Hinge Region Mutation
Summary
[0067] ChimeriVax.TM.-DEN1 virus was produced using the prME genes
of a wild type strain of dengue 1 virus [(Puo359) isolated in 1980
in Thailand] inserted into the yellow fever virus (strain 17D)
backbone (Guirakhoo et al., J. Virol. 75:7290-7304, 2001). During
production of a Pre-Master Seed virus for ChimeriVax.TM.-DEN1 in
Vero cells, a clone (clone E) containing a single nucleotide change
from A to G at position 1590, which resulted in an amino acid
substitution from K to R at position 204 on the envelope protein E,
was isolated and plaque purified. The virus exhibited attenuation
for 4-day-old suckling mice and produced a lower viremia
(viscerotropism) than its parent (non-mutant) virus when inoculated
by subcutaneous route into monkeys. Another clone (clone J-2)
without mutation was selected, plaque purified, and used to produce
a PMS virus stock at passage 7 (P7). This virus did not undergo any
mutations when passaged under laboratory conditions up to P10 in
Vero cells. However, upon one passage under cGMP conditions to
produce a Master Seed virus (P8) from PMS stock, the same mutation
at position 1590 (A to G) emerged. Similar to clone E, the P8 virus
produced larger plaques than P7 virus and was attenuated for
suckling mice. The E204 position, which is conserved in all dengue
viruses, can thus be manipulated in ChimeriVax.TM.-DEN (serotypes
1-4) viruses to achieve a balance between attenuation and
immunogenicity of the vaccine candidates for humans.
Results and Discussion
Production of Pre-Master Seeds for ChimeriVax-DEN1 Viruses
[0068] Production of plaque purified Pre-Master Seed (PMS) viruses
for DEN1 was carried out as follows. Plaque purification was
started with the virus at Passage 2 (P2) post RNA transfection. Two
PMS viruses (uncloned at P2 and cloned at P7) were produced in
Aventis Vero LS10 cells at passage 142 using a qualified cell bank
obtained from Aventis at passage 140. Cloned viruses were obtained
after 3 rounds of plaque purification and sequenced across the full
genome to assure lack of mutations. Generally, if a clone contained
an amino acid substitution, it was not used as a PMS virus
candidate. Other clones were prepared and sequenced until a clone
without mutation was identified, which was then subjected to plaque
purification and sequencing.
Sequencing
[0069] For sequencing, viral RNA was extracted from each individual
virus sample (generally 0.25 ml) using TRI-Reagent LS (Molecular
Research Center) or Trizol LS (a similar reagent from Gibco) and
dissolved in 0.20 ml of RNase-free water. The extracted RNA was
then used as a template for RT-PCR. The entire genome was amplified
in five overlapping amplicons of .about.2-3 kb in length (fragments
I through V) with the Titan One-Tube RT-PCR kit (Roche). The RT-PCR
fragments were purified using QIAquick PCR Purification kit
(Qiagen) or agarose gel-purified using QIAquick Gel Extraction kit
(Qiagen). Sequencing reactions were done using CEQ Dye Terminator
Cycle Sequencing kit (Beckman) and a collection of YF-specific
oligonucleotide primers of both positive and negative orientation
to read both strands of the amplicons. Sequencing reaction products
were purified using DyeEx Spin kit (Qiagen), and resolved with a
CEQ2000 automated sequencer (Beckman Coulter). Generated sequencing
data were aligned and analyzed with Sequencher 3.0 (GeneCodes)
software. Nucleotide heterogeneities were registered only when a
heterogeneous signal was observed in all chromatograms representing
both plus- and minus-strand sequencing reactions.
[0070] As is shown in Table 5, the uncloned P2 virus did not have
any mutations, but acquired 5 amino acid mutations (heterogeneity)
within the envelope protein E by P5. Interestingly, the only
mutation that was stable (further selected) at P15 was the 204
mutation. A repeat passage experiment starting from uncloned P2
virus (up to P15) revealed the same mutation (K to R) at E204
position being selected in Vero cells.
[0071] Different clones of ChimeriVax-DEN1 (A-J) were selected by
direct plaque to plaque purification and sequenced at various
stages to identify mutations. The most frequent mutation was the
E251 (V>F) substitution, which occurred in clones A, B, D, and G
followed by E204 (K>R), which was found in clones E and F, as
well as in uncloned viruses. The mutation at E311 (E>D) was only
found in clones C and D. Interestingly, clone J was free from
mutations up to P10. However, when a Master Seed (MS) of this virus
was produced from P7 (PMS) under cGMP manufacturing, the same
substitution at E204 reemerged (only after 1 passage). This
mutation was stable when P20 virus was sequenced (Table 5). Clones
containing the E204 mutation produced larger plaques (.about.2 mm
in diameter) than non-mutant viruses (.about.1 mm in diameter)
(Table 9). The original construct of this virus at Vero P4
(previously shown to produce a low level of viremia in monkeys)
also contained the same E204 mutation (Guirakhoo et al., J. Virol.
75:7290-7304, 2001). The role of this mutation in the biology of
the virus could not be understood previously because: a) the
original construct contained an additional mutation (nucleotide A
to G causing an amino acid change from H to R) at M39 besides the
E204 mutation; b) the neurovirulence of the original construct was
evaluated only in 3-4 week old mice, which are not sensitive enough
to reveal attenuation of ChimeriVax-DEN1 virus or any other
ChimeriVax.TM.-DEN viruses (Guirakhoo et al., J. Virol.
75:7290-7304, 2001); and c) there was no ChimeriVax.TM.-DEN1 virus
(without mutation) available for comparison to determine changes in
neurovirulence or viscerotropism phenotype of the virus.
[0072] Since chimeric viruses are attenuated for 3-4 week old mice,
we developed a more sensitive test (using suckling mice of 4-8 days
old) to test subtle differences in neurovirulence of different
clones.
Mouse Neurovirulence
[0073] The mouse neurovirulence test, using 3-4 week old mice, is
performed as a release test to ensure that neurovirulence of
chimeras does not exceed that of the virus vector (YF-VAX.RTM.)
used to construct ChimeriVax.TM. viruses. Because all chimeras
constructed so far (with or without mutations) are not virulent for
adult mice (3-4 weeks old), these animals cannot be used to
identify subtle changes in neurovirulence of chimeras associated
with single amino acid substitutions. In contrast, suckling mice of
4-10 days of age are more sensitive to minor changes in the genome
of chimeras, which are involved in virulence. In the course of
development of ChimeriVax.TM.-DEN viruses, several mutations were
observed across the genome of all 4 chimeras (Guirakhoo et al., J.
Virol. 75:7290-7304, 2001). These mutations were corrected in all
chimeras, and the reconstructed viruses (except for DEN1 chimeras)
were successfully evaluated for safety and immunogenicity in
monkeys. Due to instability of DEN1 plasmids, the reconstruction of
this chimera (without mutation) was not accomplished on time, and
could therefore not be tested in monkeys. During plaque
purification to produce a PMS for DEN1 chimera, 10 different clones
(A-J) were sequenced to identify a clone without mutations (Table
5). All but one clone (J) contained 1 or 2 mutations within the
envelope protein E. Representative clones of DEN1 chimeras were
evaluated for their neurovirulence using 4 day-old suckling mice
(Table 6). Animals were inoculated by the i.c. route with two 0.02
ml of undiluted, 1:10, or 1:100 dilutions of each chimeric DEN1
virus and observed for 21 days. The actual doses were determined by
back titration of inocula in a plaque assay. As is shown in Table
6, all clones except clone E exhibited similar neurovirulence for 4
day-old mice with average survival times (AST) significantly lower
than that of YF-VAX.RTM. (p<0.01 using JMP software, Version
4.0.2). Clone E (E204K>R) was significantly less virulent than
all other DEN1 clones (p<0.0001). Interestingly, one of the 2
mutations identified in the original DEN1 chimera was the E204
K>R substitution. This virus induced a low level of viremia
(mean peak titer 0.7 log.sub.10 PFU/ml) for 1.3 days when
inoculated into monkeys (Guirakhoo et al., J. Virol. 75:7290-7304,
2001). Clone J, which did not contain any mutations and was shown
to be significantly less virulent than YF-VAX.RTM. in 4 days old
mice, P=0.001, was selected for production of the cGMP MS
virus.
Safety and Immunogenicity (Viremia and Neutralizing Antibody
Responses) of Chimeric DEN1 Viruses in Monkeys
[0074] The effect of the E204 mutation on viscerotropism (viremia)
of the virus was assessed by inoculation of monkeys with
ChimeriVax-DEN1 viruses with (clone E, P6) or without (clone J, P7)
the E204 mutation. The original DEN1 chimera (ChimeriVax-DEN-1,
uncloned P4, 1999, Group 1) was selected as a control, because its
viremia and immunogenicity profiles had already been evaluated in
monkeys as a monovalent or a tetravalent (combined with 3 other
chimeras) vaccine (Guirakhoo et al., J. Virol. 75:7290-7304,
2001).
[0075] Groups of 4 rhesus monkeys were inoculated with 5 log.sub.10
PFU/0.5 ml of DEN1 chimeras (Table 7). Viremia was measured (by
plaque assay on Vero cells) on sera obtained from Day 2 to Day 11
post infection. All monkeys inoculated with DEN1 PMS virus (Group
3) became viremic, whereas 3/4 and 2/4 monkeys inoculated with
clone E or uncloned viruses, respectively, became viremic (Table
8). The mean peak virus titer (2.5 log.sub.10 PFU/ml) and duration
(8.5 days) of viremia in Group 3 monkeys (DEN1 PMS) was
significantly higher (p=0.024 and 0.0002 for peak virus titer and
duration, respectively) than Groups 1 and 2. Despite the lack of
viremia in some monkeys, all animals developed neutralizing
antibody titers against homologous viruses. For neutralization
assays, sera from each group of monkeys were heat inactivated and
mixed with the homologous virus (the same virus that had been used
for inoculation of animals in each group). Consistent with the
level of viremia, the neutralizing titers in monkeys immunized with
the PMS virus (without mutation) were higher than the other 2
groups (p=0.0002). The sera of Group 1 monkeys (immunized with a
DEN1 chimera with 2 mutations on the envelope proteins, prM and E)
revealed the lowest neutralizing titers (Table 9), indicating that
the M39 mutation may have further attenuated the virus (p=0.0045).
These experiments demonstrated that there might be a direct
correlation for ChimeriVax.TM.-DEN viruses between 1) the magnitude
of viremia and the level of neutralizing antibodies in monkeys, and
2) neurovirulence of chimera for mouse and viremia/immunogenicity
in monkeys (clone E was attenuated for 4 days old mice and induced
a lower level of viremia and neutralizing antibodies than the PMS
virus, which was neurovirulent for mice of similar age).
[0076] In summary, the mutation at E204 residue of
ChimeriVax.TM.-DEN1 controls the replication of the DEN1 chimera in
vertebrate hosts, as shown by viremia and neutralizing responses.
Mutation of this residue, which is conserved in all dengue
serotypes (Table 10), can thus be used in the construction of
chimeras with desired phenotypes appropriate for human dengue
vaccine.
TABLE-US-00001 TABLE 1 Comparison of the amino acid differences in
the E protein of ChimeriVax .TM.-JE FRhL.sub.3 and ChimeriVax
.TM.-JE FRhL.sub.5 virus with published sequences of JE SA14-14-2
vaccine, wild-type JE strains, parental SA14, and Nakayama virus.
ChimeriVax .TM.-JE FRhL.sub.3 and FRhL.sub.5 viruses were sequenced
across their entire genomes and the mutation at E279 was the only
difference found. Virus E107 E138 E176 E177 E227 E244 E264 E279
E315 E439 ChimeriVax .TM.-JE FRhL.sub.3 E279 Met F K V A S G H M V
R ChimeriVax .TM.-JE FRhL.sub.5 E279 Lys F K V A S G H K V R JE
SA14-14-2 PDK.sup.1 F K V T S G Q M V R JE SA14-14-2 PHK.sup.2 F K
V A S G H M V R JE SA14.sup.1,3 L E I T S G Q K A K JE
Nakayama.sup.4 L E I T P E Q K A K .sup.1Nitayaphan S. et al. 1990.
Virology 177: 541-552 .sup.2Ni H. et al. 1994. J. Gen. Virol. 75:
1505-1510; PDK = primary dog kidney .sup.3Aihara S. et al. 1991.
Virus Genes 5: 95-109; PHK = primary hamster kidney .sup.4McAda P.
et al. 1987. Virology 158: 348-360
TABLE-US-00002 TABLE 2 Neurovirulence for suckling mice of
ChimeriVax .TM.-JE viruses with and without a mutation at E279 and
YF 17D vaccine Mouse Virus, passage Intracerebral Average LD.sub.50
age and E279 amino dose Mortality Survival (Log.sub.10 Experiment
(days) acid (Log.sub.10 PFU) (%) Time (Days) PFU) 1 8.6 YF-VAX
.RTM. 1.15 10/10 (100) 8.4 0.11 0.15 5/10 (50) 10 0-0.85 1/10 (10)
14 ChimeriVax .TM.- 2.60 1/10 (10) 15 >2.6 JE, FRhL.sub.3, 1.6
1/10 (10) 13 E279 Met 0.6 0/10 (0) N/A -0.45 0/10 (0) N/A
ChimeriVax .TM.- 3.0 10/10 (100) 10.3 1.64 JE, FRhL.sub.5, 2.0 8/10
(80) 11.25 E279 Lys 1.0 2/10 (20) 14.5 0 2/10 (20) 16 2 4 YF-VAX
.RTM. 0.95 11/11 (100) 8.4 -0.3 -0.05 9/11 (82) 8.8 -1.05 2/12 (17)
10 ChimeriVax .TM.- 2.69 7/12 (58) 10.6 2.5 JE, FRhL.sub.3, 1.69
4/12 (33) 11.5 E279 Met 0.69 0/12 (0) NA ChimeriVax .TM.- 2.88
10/12 (83) 9.3 1.45 JE, FRhL.sub.5, 1.88 11/12 (92) 10.3 E279 Lys
0.88 4/12 (33) 12.2 -0.11 2/12 (17) 14 -1.11 0/12 (0) NA
YF/JE.sub.279 site- 3.55 12/12 (100) 9.4 1.15 specific 2.55 11/12
(92) 10.1 revertant, 1.55 11/12 (92) 10.2 E279 Lys 0.55 3/12 (25)
10.7 -0.44 2/12 (17) 14
TABLE-US-00003 TABLE 3 Neuropathological evaluation, monkeys
inoculated IC with ChimeriVax .TM.-JE FRhL.sub.3, FRhL.sub.5 or
yellow fever 17D (YF-VAX .RTM.) and necropsied on day 30
post-inoculation. Dose.sup.1 Clinical Individual and group mean
log.sub.10 score.sup.2 histopathological score PFU/ Maximum Target
+ 0.25 score/Mean Target Discriminator Discriminator Test virus
Monkey Sex mL daily score area.sup.3 areas.sup.4 areas YF-VAX .RTM.
RT702M M 4.05 1/0 2.00 0.51 1.26 Connaught RT758M M 4.28 1/0 0.25
0.01 0.13 Lot # 0986400 RT653M M 4.07 1/0 2.00 0.39 1.20 RT776M M
4.25 3/1 2.00 1.29 1.65 RT621M M 4.34 3/2 1.00 0.46 0.73 RAH80F F
4.14 3/1 1.50 0.71 1.10 RAL02F F 4.13 1/1 2.00 0.80 1.40 RT698F F
3.78 3/1 1.50 0.64 1.07 RAI12F F 4.11 1/1 2.00 1.45 1.73 RP942F F
4.05 1/0 2.00 0.81 1.41 Mean 4.12 1 1.63 0.71 1.17 SD 0.16 1 0.59
0.42 0.47 ChimeriVax .TM.- RT452M M 3.55 1/0 0.50 0.08 0.29 JE,
FRhL.sub.3 RR257M M 3.52 1/0 1.00 0.14 0.57 Lot# I031299A RT834M M
3.71 1/0 0.50 0.38 0.44 RT620M M 3.71 1/0 1.00 0.14 0.57 RT288M M
3.76 1/0 0.50 0.19 0.35 RAJ98F F 3.79 1/1 0.00 0.11 0.05 RAR08F F
3.52 1/0 0.00 0.13 0.07 RV481F F 3.52 1/0 0.00 0.06 0.03 RT841F F
3.71 1/0 0.50 0.05 0.28 RT392F F 3.76 1/0 0.50 0.07 0.29 Mean 3.66
0 0.45 0.14 0.29 SD 0.11 0 0.37 0.10 0.20 P-value (t Test.sup.5)
vs. YF-VAX .RTM. 0.037/0.025 0.00008 0.00191 0.00014 ChimeriVax
.TM.- RT628M M 4.20 1/0 0.50 0.57 0.54 JE, FRhL.sub.5 RT678M M 4.19
1/0 1.00 0.12 0.60 Lot # 99B01 RT581M M 4.17 1/0 1.00 0.46 0.73
RR726M M 4.32 1/0 1.00 0.66 0.83 RR725M M ND.sup.6 1/0 1.00 0.33
0.67 RAJ55F F 4.27 0/0 1.00 0.14 0.57 RT769F F 4.44 1/0 1.00 0.58
0.79 RAK22F F 4.24 1/0 0.00 0.12 0.06 RT207F F 4.49 1/1 1.00 0.22
0.61 RT490F F 4.34 1/0 0.00 0.04 0.02 Mean 4.30 0 0.75 0.32 0.54 SD
0.11 0 0.42 0.23 0.28 P-value (t Test) vs. YF-VAX .RTM. 0.024/0.025
0.00154 0.02436 0.00248 P-value (t Test) vs. ChimeriVax .TM.-JE
FRhL.sub.3 0.343/1.00 0.10942 0.03223 0.03656 .sup.1Back-titration
.sup.2Clinical score: 0 = no signs; 1 = rough coat, not eating; 2 =
high pitched voice, inactive, slow moving; 3 = tremor,
incoordination, shaky movements, limb weakness; 4 = inability to
stand, paralysis, moribund, or dead. The maximum score on any day
and the mean score over the 30-day observation period are shown.
.sup.3Substantia nigra .sup.4Corpus striatum and thalamus, right
and left side (N. caudatus, globus pallidus, putamen, N. ant./lat.
thalami, N. lat. thalami; cervical and lumbar enlargements of the
spinal cord (6 levels) .sup.5Student's t test, two-sided,
heteroscedastic, comparing YF-VAX .RTM. and ChimeriVax .TM.-JE
viruses. .sup.6Not done
TABLE-US-00004 TABLE 4 Viremia, rhesus monkeys inoculated IC with
YF-VAX .RTM. or ChimeriVax .TM.-JE FRHL.sub.3 and FRHL.sub.5
viruses (for dose inoculated, see Table 3) YF-VAX .RTM. Control
Serum Virus Titer (Log.sub.10 PFU/mL), Day Animal 1 2 3 4 5 6 7 8 9
RT702M .sup. --.sup.1 -- 1.6 3.0 -- -- -- -- -- RAH80F -- -- -- 3.3
2.5 -- -- -- -- RT758M -- -- 2.1 3.2 2.8 -- -- -- -- RAL02F -- --
-- 1.3 -- -- -- -- -- RT653M -- -- -- 2.7 -- -- -- -- -- RT698F --
1.0 2.3 3.7 2.5 -- 1.0 -- -- RT776M -- -- -- -- -- -- -- -- --
RAI12F -- -- -- 2.0 2.5 2.5 2.0 -- -- RT621M -- 1.0 2.0 3.3 2.0 --
-- -- -- RP942F -- 1.0 2.6 3.6 2.0 -- -- -- -- Mean Titer.sup.2 0.8
1.4 2.7 1.7 0.9 0.9 SD 0.1 0.8 1.0 0.9 0.6 0.4 ChimeriVax .TM.-JE
FRHL.sub.3 E279 Met Serum Virus Titer.sup.1 (Log.sub.10 PFU/mL),
Day Animal 1 2 3 4 5 6 7 8 9 RAJ98F -- -- 1.9 1.3 -- -- -- -- --
RT452M -- 1.3 2.1 1.6 -- -- -- -- -- RAR08F -- -- 1.3 2.2 2.2 1.8
-- -- -- RR257M -- -- 1.9 2.2 1.8 -- -- -- -- RV481F -- -- 2.1 1.8
1.5 -- -- -- -- RT834M -- -- 2.5 1.3 -- -- -- -- -- RT841F -- --
2.4 1.7 -- -- -- -- -- RT620M -- -- 1.6 1.0 -- -- -- -- -- RT392F
-- -- -- -- -- -- -- -- -- RT288M -- -- -- -- -- -- -- -- -- Mean
Titer 0.8 1.7 1.5 1.0 0.8 SD 0.2 0.6 0.5 0.6 0.3 P-value.sup.3
0.696 0.386 0.003 0.065 0.745 ChimeriVax .TM.-JE FRhL.sub.5 E279
Lys Serum Virus Titer.sup.1 (Log PFU/mL), Day Animal 1 2 3 4 5 6 7
8 9 RT628M -- -- -- -- -- -- -- -- -- RAJ55F -- -- -- -- -- -- --
-- -- RT678M -- -- -- -- -- -- -- -- -- RT769F -- -- -- 2.0 -- --
-- -- -- RT581M -- -- -- -- -- -- -- -- -- RAK22F -- -- -- -- -- --
1.8 -- -- RR726M -- -- -- -- -- -- -- -- -- RT207F -- -- -- -- --
-- -- -- -- RR725M -- -- -- -- -- -- -- -- -- RT490F -- -- -- -- --
-- -- -- -- Mean Titer 0.7 0.7 0.8 0.7 0.7 0.8 SD 0.0 0.0 0.4 0.0
0.0 0.4 P-value.sup.4 0.331 <0.000 0.010 0.076 1.0 1.0 .sup.1--
= No detectable viremia; in most tests neat serum was tested, the
cutoff being 1.0 log.sub.10 PFU/mL); in some cases, neat serum was
toxic to cells, and serum diluted 1:2 or 1:5 was used (cut-off 1.3
or 1.7 log.sub.10 PFU/mL). .sup.2For the purpose of calculating
mean titers and standard deviations, 0.7 was used in place of
<1.0, 1.0 was used in place of <1.3, and 1.4 was used in
place of <1.7. .sup.3Comparison with YF-VAX .RTM. by t-test,
2-tailed .sup.4Comparison with ChimeriVax .TM.JE FRhL.sub.3 by
t-test, 2-tailed
TABLE-US-00005 TABLE 5 Nucleotide and amino acid sequences of
uncloned and various clones of ChimeriVax-DEN1 viruses and their in
vitro (Vero passages) genetic stabilities. Nt. change/ AA change/
Virus Passage Gene Nt. No.sup.a heterogeneity heterogeneity AA
No.sup.b Comments Uncloned P2 -- -- -- -- -- No mutations Uncloned
P5 E 1590 A/G K/R 204 Nucleotide heterogeneity E 1730 G/T V/F 251
Nucleotide heterogeneity E 1912 G/t E/D 311 Barely detectable
mutant E 2282 C/a L/I 435 Undetectable mutants in some samples
Uncloned P15 E 1590 A to G K to R 204 Nucleotide heterogeneity NS2B
4248 G to T G to V Nucleotide heterogeneity NS4A 6888 C/T A/V NS4A
7237 A/G I/M Uncloned P15 E 1590 A to G K to R 204 Nucleotide
heterogeneity REPEAT E 1730 G/T V/F 251 Nucleotide heterogeneity
from P2 NS4A 7237 A/G I/M 263 Barely detectable mutant NS4B 7466
C/t P/S 52 Clone A P3, P7 E 1730 G to T V to F 251 Domain II j
strand, no function assigned E 2282 C to A L to I 435 Before
anchor; L and I in D2 and YF respectively. (a gap left, nt
7080-7220) Clone B P3, P7, P10 E 1730 G to T V to F 251 Clone C P3,
P6 E 1912 G to T E to D 311 Domain III, a strand, no function
assigned. Clone D P3, P6 E 1730 G to T V to F 251 Clone E P3, P6 E
1590 A to G K to R 204 Domain II, f-g loop of, no function ass.
Clone F P3 M 788 C to T -- -- E 1590 A to G K to R 204 Clone G P3 E
1730 G to T V to F 251 Clone H P3 E 1912 G to T E to D 311 Domain
III, d strand (L in D2 and D3; I E 2030 G to T V to L 351 in D4)
Clone I P3 E 1590 A to G K to R 204 Clone J P3, P6, P7, -- -- -- --
-- (J-2) P10 Cline J P8 E 1590 A to G (a/G) K to R 204 Some parent
(a) nucleotide still (J-2) (cGMP MS) E 1590 A to G K to R 204
present P10 from (cGMP MS) Clone J P10 REPEAT E 1590 A to G K to R
204 (J-2) from P7 Clone J P20 From E 1590 A to G K to R 204 (J-2)
P10 repeat NS4A 6966 G/T S/I 171 NS4A 7190 G/a V/I 246 .sup.aFrom
the beginning of the genome. .sup.bFrom the N-terminus of indicated
protein; numbering according to Rice et al., Science 229: 726-733,
1985. Clones with 204 mutations are shown in bold letters.
TABLE-US-00006 TABLE 6 Neurovirulence of different clones of
chimeric DEN1 viruses in 4-day old suckling mice. ChimeriVax- Dose
No. dead/total AST DEN1 Mutation Dilution (BT) (% dead) Days
Uncloned None Neat 5.0 11/11 (100) 9.1 (P2) 1:10 4.1 11/11 (100)
10.2 Clone B E251 Neat 5.8 10/11 (91) 9.8 (P7) V to F 1:10 5.0
11/11 (100) 10.2 Clone C E311 Neat 5.8 11/11 (100) 8.5 (P6) E to D
1:10 4.9 11/11 (100) 9.5 Clone E E204 Neat 5.9 3/11 (27) 13 (P6) K
to R 1:10 4.8 1/11 (9) 14 1:100 4.0 1/11 (9) 15 Clone J None Neat
3.6 11/11 (100) 10.8 (P3) 1:10 3.0 11/11 (100) 11.3 1:100 1.8 9/11
(82) 11.3 YF-VAX .RTM. NA 1:20 2.5 12/12 (100) 8.3
TABLE-US-00007 TABLE 7 Immunogenicity Study in Rhesus Monkeys,
ChimeriVax .TM.-DEN1 viruses, Sierra Biomedical NON-GLP Study Dose
Group* Virus Mutation (0.5 ml) 1 ChimeriVax-DEN-1 M39 and 5 logs
Uncloned, P4, 1999** E204 2 ChimeriVax-DEN-1, E204 5 logs P6, clone
E 3 ChimeriVax-DEN-1, None 5 logs PMS (P7), clone J *Four monkeys
(2M/2F) per group. **Guirakhoo et al 2001
TABLE-US-00008 TABLE 8 Viremia in monkeys immunized with 5
log.sub.10 PFU (S.C.) of different clones of ChimeriVax-DEN1
viruses Virus Viremia (log.sub.10 PFU/ml) by post-immunization day:
Monkey (Mutation) 2* 3 4 5 6 7 8 9 10 11 R18265M ChimeriVax .TM.-
--** -- -- -- -- -- -- -- -- -- R175110F DEN1, 99, P4, -- -- -- 1.7
-- -- -- -- -- -- R17572M uncloned 1.3 1.0 -- 1.0 -- -- -- -- -- --
R171114F (M39, E204) -- -- -- -- -- -- -- -- -- -- R182103M
ChimeriVax .TM.- -- -- -- -- -- -- -- -- -- -- R17098F DEN1, P6,
clone -- 1.7 -- -- -- -- -- -- -- -- R18261M E, (E204) 1.7 2.5 1.3
2.0 -- R175118F -- -- 1.0 -- -- -- -- -- -- -- R182104M ChimeriVax
.TM.- 1.0 1.9 1.7 1.7 1.8 1.7 1.0 1.0 1.7 -- R175108F DEN1, P7,
clone -- 1.7 2.8 2.2 1.0 2.0 1.7 2.0 2.2 1.7 R182111M J, PMS 2.3
3.0 3.3 2.8 1.7 1.7 -- -- -- -- R175104F (None) -- 2.4 1.3 2.0 2.3
1.7 1.7 2.2 3.0 3.1 *Monkeys were immunized on Day 1. **<1.0
log.sub.10 PFU/ml
TABLE-US-00009 TABLE 9 Viremia and neutralizing antibody titers
(50%) in monkeys immunized with 5 log.sub.10 PFU (S.C.) of
different clones of ChimeriVax-DEN1 viruses No. Plaque viremic/no.
Mean Neut. Ab size Monkey Mutation tested (%) Peak titer Duration
titer (mm) R18265M YF-DEN1, 99, 2/4 (50) 1.5 1.5 640 2-4 R175110F
P4, uncloned 640 R17572M (M39, E204) 320 R171114F 640 R182103M
YF-DEN1, 01, 3/4 (75) 1.7 2 5120 2-4 R17098F P6, clone E 2560
R18261M (E204) 2560 R175118F 5120 R182104M YF-DEN1, 01, 4/4 (100)
2.5 8.5 5120 1 R175108F P7, clone J, 10240 R182111M PMS (None)
10240 R175104F 10240
TABLE-US-00010 TABLE 10 Position of 204 residues in ChimeriVax
.TM.-DEN1-4 E proteins Amino Acid residues, E protein ChimeriVax-
200 201 202 203 204 205 206 207 208 DEN1 T M K E K S W L V DEN2 Q M
E N K A W L V DEN3 T M K N K A W M V DEN4 K M K K K T w L V
* * * * *