ATTENUATED CHIKUNGUNYA VIRUS
[0001] This application claims the benefit of United States Provisional Patent
Application No. 61/706,589, filed September 27, 2012, which is incorporated herein by
reference in its entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The sequence listing that is contained in the file named
"CLFR.P0397WO_ST25.txt", which is 99 KB (as measured in Microsoft Windows®) and
was created on September 26, 2013, is filed herewith by electronic submission and is
incorporated by reference herein.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0003] The present invention relates generally to the field of molecular biology and
virology. More particularly, it concerns Chikungunya polypeptides and viruses that are
attenuated in mammalian hosts.
2. Description of Related Art
[0004] Chikungunya virus (ChikV is a member of the Togaviridae family; genus
Alphavirus (Khan et al., 2002) and is pathogenic to humans. ChikV is an arthropod borne
virus (arbovirus) spread by the bite of an aedene mosquito. As with all alphaviruses its
genome is composed of a small ~ 11 Kb plus polarity single-stranded RNA. The genome
encodes 3 structural proteins, El, E2, and C and 4 nonstructural proteins nsPl-4. As a
member of the Togaviruses these viruses are enveloped and as arboviruses they contain a
membrane envelope derived from the insect or vertebrate host. The alphavirus genus contains
29 known species which cause encephalitis, fever, and/or arthralgia.
[0005] ChikV was first isolated from the blood of a febrile patient in Tanzania in
1953 where the virus was endemic (Pialoux et al, 2007). Outbreaks occur repeatedly in west,
central, and southern Africa and have caused several human epidemics in those areas since
that time. However, ChikV is a re-emerging pathogenic virus and is now also endemic in
south east Asia (see, e.g., the world wide web at searo.who.int/index.htm). Recently, ChikV
spread from Asia and the Indian Ocean to Italy (Rezza et al. 2007; Mavalankar et al. 2008).
Of the two strain lineages of ChikV, the African; remains enzootic by cycling between
mosquitoes and monkeys but the Asian strain is transmitted directly between mosquitoes and
humans. This cycle of transmission may have allowed the virus to become more pathogenic
as the reservoir host was eliminated (Powers et al, 2000).
[0006] In humans, ChikV causes a debilitating disease characterized by fever,
headache, nausea, vomiting, fatigue, rash, muscle pain and joint pain; the symptoms
commonly associated with Dengue virus infection (with the exception of the arthralgia).
Incubation can be 2-12 days, but most commonly 3-7 days with "silent" infections occurring
with unknown frequency (WHO, Weekly epidemiological record. 2007). ChikV can be
transmitted from mother to child (Ramful et al. 2007) and can produce chronic persisting
symptoms including crippling arthralgia, encephalitis and myocarditis (rare) (Paul et al.
201 1). ChikV epidemics from 2004-201 1 have resulted in 1.4-6.5 million reported cases, with
imported cases to 40 countries (Suhrbier et al. 2012). Aedes aegypti is the primary vector of
ChikV, but recent outbreaks, which involved mortalities, have been propagated through the
Aedes albopictus mosquito (Mavalankar et al. 2008; Dubrulle et al. 2009). Importantly, this
mosquito vector has spread to 12 European countries as well as to the Australian continent
(Johnson et al. 2008). Despite significant morbidity and mortality associated with ChikV
infections and its growing prevalence and geographic distribution there is currently no
vaccine or antiviral for ChikV approved for human use (Barrett et al. 2009).
SUMMARY OF THE INVENTION
[0007] Embodiments of the invention concern recombinant Chikungunya virus E2
polypeptides comprising amino acid deletions in the transmembrane domain. For example, a
recombinant E2 polypeptide comprising the deletion can be efficiently expressed on insect
cell membranes, but cannot be efficiently expressed in mammalian cell membranes.
Accordingly, a recombinant Chikungunya virus comprising a deleted E2 of the embodiments
efficiently replicates in insect cells, but inefficiently replicate in mammalian cells and are
therefore highly attenuated relative to mammals.
[0008] Accordingly, in a first embodiment, there is provided a recombinant
polypeptide wherein the polypeptide comprises an amino acid sequence at least 85% identical
to a wild type Chikungunya virus E2 polypeptide and comprises a deletion in the
transmembrane domain (TMD). In some aspects, the recombinant polypeptide is at least 90%
identical to a Chikungunya virus E2 polypeptide from the West African strain 37997 (SEQ
ID NO:l), India isolate RGCB699-09 (SEQ ID NO:9) or Maritius isolate BNI1446 (SEQ ID
NO: 11). In some aspects, a recombinant polypeptide is at least 91%, 92%, 93%, 94%, 95% or
96% identical to SEQ ID NO:l, 9 or 11. In preferred aspects a deletion in TMD according to
the embodiments is a deletion of 8-1 1 amino acids in the TMD (which corresponds to amino
acid positions 365-390 of SEQ ID NO: 1). For example, the deletion can be a deletion of 8, 9,
10 or 11 amino acids in the TMD.
[0009] In certain specific aspects, a recombinant polypeptide of the embodiments
comprises a deletion of 9 amino acids in the TMD. For example, the polypeptide can
comprise a deletion of the amino acids corresponding to amino acid positions 372-380, 374-
382 or 373-381 of SEQ ID NO:l. Examples of such polypeptide include, without limitation,
polypeptides comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7 or a sequence at least 90% identical to the foregoing sequences. In a further aspect, a
recombinant polypeptide of the embodiments comprises a deletion of 10 amino acids in the
TMD. For example, the polypeptide can comprise a deletion of the amino acids
corresponding to amino acid positions 372-381, 374-383 or 373-382 of SEQ ID NO:l.
Examples of such a polypeptide include, without limitation, polypeptides comprising the
amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 or a sequence at
least 90% identical to the foregoing sequences.
[0010] In a further embodiment there is provided a polynucleotide molecule encoding
a recombinant Chikungunya virus E2 polypeptide of the embodiments. For example, the
polynucleotide can comprise a sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
or 97% identical to a Chikungunya virus E2 coding sequence from the West African strain
37997 (SEQ ID NO:2), India isolate RGCB699-09 (SEQ ID NO: 10) or Maritius isolate
BNI1446 (SEQ ID NO: 12). Thus, in some specific aspects, a polynucleotide of the
embodiments comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% to SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO: 16; SEQ ID
NO: 18; or SEQ ID NO:20 (e.g., a sequence 100% identical to any of the foregoing
sequences). A polynucleotide of the embodiments can be a DNA or RNA sequence, such as a
Chikungunya virus E2 genomic RNA.
[0011] In still a further embodiment there is provided a host cell comprising a
polypeptide or a polynucleotide of the embodiments. For example, the host cell can be a
eukaryotic or prokaryotic cell. In certain aspects, the host cell is an insect cell, such as a
Spodopterafrugiperda cell. Thus, in some aspects, a culture of insect cells (e.g., SF9 cells) is
provided wherein the cells comprise a polypeptide and/or polynucleotide of the embodiments.
[0012] In still yet a further embodiment there is provided a recombinant virus particle
comprising a polypeptide or polynucleotide of the embodiments. For example, in certain
aspects, the viral genome comprises a polynucleotide sequence of the embodiments. In some
aspects, a viral particle of the embodiments can be defined as a live attenuated Chikungunya
virus. In further aspects, a recombinant virus comprises at least one additional attenuating
mutation. For example, the additional attenuating mutation can be a deletion, insertion or
substitution of one or more nucleotides in the viral genome. In certain aspects, the
recombinant virus is adapted for growth insect cell, such as a virus that have been passaged
10 or more times in an insect cell line. In still yet further aspects a recombinant virus of the
embodiments is inactivated or partially inactivated, for example by treatment with a chemical
(e.g., formalin), with heat or with radiation.
[0013] As outlined above, in some aspects, a recombinant virus according to the
embodiments can comprise one ore more additional attenuating mutations. For example, in
some aspects, a Chikungunya virus coding sequence can comprise an internal ribosomal entry
site of a encephalomyelocarditis virus substituted for the sequence encoding the 5' UTR of
the viral subgenomic RNA (see, e.g., U.S. Pat. Publn. No. 201 10052634, incorporated herein
by reference).
[0014] In yet a further embodiment there is provided a method of producing a
recombinant virus of the embodiments comprising (a) infecting a host cell with a
recombinant virus and (b) collecting progeny virus from the host cell. In further aspects, a
method of the embodiments can comprise, expressing viral genome (e.g., a genome
comprising a polynucleotide of the embodiments) in a host cell and collecting virus particles
produced by the host cell. In certain aspects the host cell is an insect cell, such an SF9 cell.
[0015] In still further embodiments there is provided an immunogenic composition
comprising a recombinant polypeptide, polynucleotide or virus particle of the embodiments
in a pharmaceutically acceptable carrier. In preferred aspects, an immunogenic composition
comprises a recombinant Chikungunya virus of the embodiments (e.g., a live attenuated
Chikungunya virus). In further aspects, an immunogenic composition further comprises
additional components such as an adjuvant, an immunomodulator, a preservative or a
stabilizer. Thus, in some aspects, a composition is provided for use in preventing the
symptoms of a Chikungunya virus infection, the composition comprising a recombinant virus
particle of the embodiments in a pharmaceutically acceptable carrier.
[0016] In yet still a further embodiment there is provided a method of producing an
immune response in a subject comprising administering an immunogenic composition of
embodiments to the subject. For example, a method of the embodiments can be further
defined as a method for preventing symptoms (e.g., fever, rash or virus-associated arthritis)
of a Chikungunya virus infection in a subject. In still further aspects, a method can be defined
as a method for reducing the probability of a Chikungunya virus infection in a subject. In
certain aspects, a subject is a subject who is at risk of acquiring a Chikungunya virus
infection, such as a subject who lives in an endemic area or who lives in or has visited a
region known to have circulating Chikungunya virus. In further aspects, a subject is a subject
that is at risk for having severe symptoms from Chikungunya virus infection such as a subject
who is immunosuppressed, elderly or who has arthritis. In preferred aspects, the subject is a
human subject.
[0017] In further aspects, an immunogenic composition of the embodiments can be
administered to a subject orally, intravenously, intramuscularly, intraperitoneally,
intradermally or subcutaneously. For example, in some aspects, the composition is
administered to a subject by an injection, e.g., an intramuscular or subcutaneous injection. In
some cases, the composition is administered multiple times, such as 2, 3, 4 or 5 times. In
certain cases, each administration is separated by a period of days, weeks, months or years.
[0018] Embodiments discussed in the context of methods and/or compositions of the
invention may be employed with respect to any other method or composition described
herein. Thus, an embodiment pertaining to one method or composition may be applied to
other methods and compositions of the invention as well.
[0019] As used herein the terms "encode" or "encoding" with reference to a nucleic
acid are used to make the invention readily understandable by the skilled artisan; however,
these terms may be used interchangeably with "comprise" or "comprising" respectively.
[0020] As used herein the specification, "a" or "an" may mean one or more. As used
herein in the claim(s), when used in conjunction with the word "comprising", the words "a"
or "an" may mean one or more than one.
[0021] The use of the term "or" in the claims is used to mean "and/or" unless
explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive,
although the disclosure supports a definition that refers to only alternatives and "and/or." As
used herein "another" may mean at least a second or more.
[0022] Throughout this application, the term "about" is used to indicate that a value
includes the inherent variation of error for the device, the method being employed to
determine the value, or the variation that exists among the study subjects.
[0023] Other objects, features and advantages of the present invention will become
apparent from the following detailed description. It should be understood, however, that the
detailed description and the specific examples, while indicating preferred embodiments of the
invention, are given by way of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The following drawings form part of the present specification and are included
to further demonstrate certain aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in combination with the detailed
description of specific embodiments presented herein.
[0025] FIG. 1: Pre-challenge viremia by plaque assay in the designated tissues at 1,
2, 3, and 7 days after injection with 103 pfu of ChikV, TM17-1, TM17-2, or TM17-3. The
values of the mutant virus compared to the WT viremias were analyzed by students' t test and
are noted where significant differences were found. In (A) the viremia detected in mouse sera
is shown. Analysis of the titers shows no significant difference between the mutants and WT
until day 2 for TM17-2, p<0.05, and day 3 pO.001 for TM17-1. (B) Foot and ankle tissue
titers differ from WT as follows: day 1 p<0.001 for TM17-1, p<0.01 TM-2, and on day 2;
TM17-1, 2 and 3 were titers were significantly lower (p<0.05 respectively). One day 3 virus
had been cleared from the TM17-2/3 infected mice. However, WT and TM17-1 had not been
cleared from the foot/ankle at day 7. (C) The titers from quadriceps are shown. All the mutant
viruses were cleared by day 3. No viremia was detected in mice injected with mock samples.
Limit of detection of the plaque assay is 80 pfu.
[0026] FIG. 2: Neutralizing antibody titers present in mouse sera 7, 10, and 2 1 days
after injection (pre-challenge) with wild type or attenuated mutant ChikV37997. Titers shown
represent the geometric means of sera from 3 different mice per group per day. Because of
the variability of the data and the small group size, no significant differences could be
established for the levels of NAb on these 3 days.
[0027] FIG. 3: Total anti-ChikV IgG concentration (mg/mL) present in mouse serum
1 days post vaccination. The amount of total WT IgG was found to be statistically higher
than IgG from TM17-1, 2, and 3 (p<0.001 for Naive, p<0.01 for TM17-1 and 3, and p<0.05
for TM17-2) while there was no significant difference found among the respective mutant
pairs.
[0028] FIG. 4: Neutralizing antibody titers present in mouse sera 7 days after
challenge with wild type ChikV(SL15649) or mock (diluent, complete MEM). Titers shown
represent the geometric means of sera from 3 different mice per group per day.
[0029] FIG. 5: Total anti-ChikV IgG concentrations (mg/mL) present in mouse
serum 7 days after challenging vaccinated mice with ChikV(SL15649) or media as
determined by ELISA. Error bars represent the standard deviation.
[0030] FIG. 6: Graph shows the growth of ChikV TM16 (TM16-1, TM16-2 and
TM-16-3) mutants in C710 insect cells versus mammalian BHK cells. In each case viral titer
is show at 24 hour and 48 hour time points (left and right bars respectively).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. The Present Invention
[0031] ChikV is a re-emerging human pathogen that has now established itself in
south east Asia as well as Africa and has recently spread to Europe (Rezza et al. 2007;
Mavalankar et al. 2008). In humans, ChikV causes a debilitating disease characterized by
fever, headache, nausea, vomiting, fatigue, rash, muscle pain and joint pain. Human ChikV
epidemics from 2004-2011 have resulted in 1.4-6.5 million reported cases, including a
number of deaths. However, despite significant morbidity and mortality associated with
ChikV infection and its growing prevalence and geographic distribution there is currently no
vaccine or antiviral for ChikV approved for human use (Barrett et al. 2009). Thus, ChikV
antigens and attenuated viruses are desperately needed for development of vaccines.
[0032] Embodiments of the invention address this need by providing deleted ChikV
E2 polypeptides that render the virus highly attenuated in mammalian hosts. In particular, the
studies detailed below demonstrate that ChikV expressing mutant E2 glycoproteins
comprising a deletion of 9 or 10 amino acids in the transmembrane domain (e.g., the TM17
mutants) are highly attenuated and can serve as the basis for viral vaccine compositions.
Importantly, large deletions, such as those studied, do not revert in vitro or in vivo (Smith et
al. 2012). Moreover, though highly attenuated in mammalian cells, the viruses can be grown
to near wild type titers in insect cells, thereby allowing for efficient production of vaccine
strains. When injected into test animals the attenuated viruses were found to be safe, in that
they did not persist in the blood or joints of the infected animals. The ChikV TM17-2 mutant,
for example, did not produce any swelling at the site of injection, produced little if any
inflammation in the foot/ankle or quad and did not persist in any tissue tested pre-challenge.
Upon challenge of animals exposed to the mutant viruses with WT ChikV the animals were
found to be protected from infection. In particular, ChikV TM17-2 provided significant
protection against infection even as compared to TM 17-1 and WT ChikV. Assay of the
serum, foot/ankle and quad post challenge did not detect any virus for mice infected with
TM17-2. Considering that infection of humans with an arbovirus confers lifelong immunity,
the ChikV TM17-2 protected better even than infection with WTChikV which allowed a
transient infection post challenge.
[0033] The mutant viruses described here provide ideal vaccine candidates. First, they
are high attenuated as demonstrated by their reduced replication efficiency in mammalian
cells and the lack of persistence and symptoms of infection upon introduction into test
animals. Second because of the large deletions that are used, the chance of reversion to wild
type has been minimized. Most importantly, the viruses produce a robust and protective
immune response. In fact certain mutant viruses such as TM17-2 produced an immune
response that provides even greater protection that infection of animals with wild type virus.
Together these studies have identified highly attenuated, non-reactogenic, and efficacious
strains of ChikV which can (and should) be further developed for use in human vaccines.
II. Reference to the Sequence Listing
[0034] The following sequences are provided in the sequence listing and may be used
in accordance with certain aspects of the embodiments.
SEQ ID NO:l - amino acid sequence for WT Chikungunya virus E2 polypeptide West
African strain 37997 (Genbank # EU224270, incorporated herein by
reference)
SEQ ID NO:2 - polynucleotide sequence encoding SEQ ID NO:l
SEQ ID NO:3 - amino acid sequence for Chikungunya virus E2 polypeptide "TM17-1"
SEQ ID NO:4 - polynucleotide sequence encoding SEQ ID NO:3
SEQ ID NO:5 - amino acid sequence for Chikungunya virus E2 polypeptide "TM17-2"
SEQ ID NO:6 - polynucleotide sequence encoding SEQ ID NO:5
SEQ ID NO:7 - amino acid sequence for Chikungunya virus E2 polypeptide "TM17-3"
SEQ ID NO:8 - polynucleotide sequence encoding SEQ ID NO:7
SEQ ID NO:9 - amino acid sequence for WT Chikungunya virus E2 polypeptide India isolate
RGCB699-09 (Genbank # GU562827, incorporated herein by reference)
SEQ ID NO: 10 - polynucleotide sequence encoding SEQ ID NO:9
SEQ ID NO: 11 - amino acid sequence for WT Chikungunya virus E2 polypeptide Maritius
isolate BNI1446 (Genbank # GU434106, incorporated herein by reference)
SEQ ID NO: 12 - polynucleotide sequence encoding SEQ ID NO: 11
SEQ ID NO: 13 - TMD of Sindbis virus E2
SEQ ID NO: 14 - TMD of WT Chikungunya virus E2
SEQ ID NO: 15 - amino acid sequence for Chikungunya virus E2 polypeptide "TM16-1"
SEQ ID NO: 16 - polynucleotide sequence encoding SEQ ID NO: 15
SEQ ID NO: 17 - amino acid sequence for Chikungunya virus E2 polypeptide "TM16-2"
SEQ ID NO: 18 - polynucleotide sequence encoding SEQ ID NO: 17
SEQ ID NO: 19 - amino acid sequence for Chikungunya virus E2 polypeptide "TM16-3"
SEQ ID NO:20 - polynucleotide sequence encoding SEQ ID NO: 19
SEQ ID NO:21 - Genomic polynucleotide sequence for WT Chikungunya virus, West
African strain 37997
SEQ ID NO:22 - Amino acid sequence for the non-structural polyprotein of WT
Chikungunya virus, West African strain 37997
SEQ ID NO:23 - Genomic polynucleotide sequence for the structural polyprotein of WT
Chikungunya virus, West African strain 37997
SEQ ID NO:24-25 - Synthetic oligonucleotide primers
III. Recombinant Polypeptide and Polynucleotides
[0035] The recombinant polypeptides and viruses of certain aspects of the
embodiments are based on deletion mutations in the transmembrane domains of membrane
glycoproteins of ChikV, in particular the ChikV EZ TMD. Like other viruses, the E2
membrane glycoprotein has a hydrophobic membrane-spanning domain which anchors the
protein in the membrane bilayer (Rice et ah, 1982). The membrane-spanning domain needs to
be long enough to reach from one side of the bilayer to the other in order to hold or anchor
the proteins in the membrane. Unlike mammalian cell membranes, the membranes of insect
cells contain no cholesterol (Clayton 1964; Mitsuhashi et ah, 1983). Because insects have no
cholesterol in their membranes, the insect-generated viral membrane will be thinner in cross
section than the viral membranes generated from mammals. Consequently, the membranespanning
domains of proteins integrated into insect membranes do not need to be as long as
those integrated into the membranes of mammals. Accordingly, as demonstrated for the first
time here ChikV E2 polypeptides with a 8-1 1 amino acid deletion in their TMD result in
viruses tat can replicate efficiently in insect cells but show reduced replication in mammalian
cells that comprise thicker membranes. Further methods of modifying the a glycoprotein
trans membrane domain are provided for instance in U.S. Patent 6,306,401; 6,589,533;
7,128,915 and 7,335,363, each incorporated herein by reference.
[0036] In certain embodiments recombinant viruses or polypeptides according to the
current embodiments may comprise two or more host range mutations or additionally
comprise other mutations such as attenuating mutations, mutations to increase
immunogenicity or viral stability or any mutations that may be used for vaccine production
and that are current known in the art.
[0037] In additional aspects, recombinant polynucleotide, polypeptides or viruses of
the embodiments can comprise additional deletions, substitutions or insertions (or amino
acids or nucleic acids). For example, sequences from other ChikV strains can be incorporated
into the recombinant molecules of the embodiments. Thus, in some aspects amino acid or
nucleic acid changes can be made in molecules by substituting the position for a
corresponding position from another strain of virus. Similarly, in the case of amino acid
substitution, changes can be made with amino acids having a similar hydrophilicity. The
importance of the hydropathic amino acid index in conferring interactive biologic function on
a protein is generally understood in the art (Kyte & Doolittle, 1982). It is accepted that the
relative hydropathic character of the amino acid contributes to the secondary structure of the
resultant protein, which in turn defines the interaction of the protein with other molecules, for
example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. As detailed
in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino
acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine
(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1);
alanine ( 0.5); histidine -0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine ( 2.3); phenylalanine (-2.5); tryptophan (-3.4). These values can be
used as a guide and thus substitution of amino acids whose hydrophilicity values are within
±2 are preferred, those that are within ± 1 are particularly preferred, and those within ±0.5 are
even more particularly preferred. Thus, any of the E2 polypeptides described herein may be
modified by the substitution of an amino acid, for different, but homologous amino acid with
a similar hydrophilicity value. Amino acids with hydrophilicities within +/- 1.0, or +/- 0.5
points are considered homologous.
IV. Viral Vaccines
[0038] Certain aspects of the present invention are drawn to a method of producing an
immunogenic composition or viral vaccine from genetically engineered membrane-enveloped
viruses, such as Chikungunya virus, for vaccination of mammals, comprising the steps of
introducing the engineered virus into insect cells and allowing the virus to replicate in the
insect cells to produce a viral vaccine.
[0039] Certain aspects of the embodiments concern host-range mutant viruses. It is
contemplated in certain aspects of the invention that one, two, three, four or more of these
types of mutations can be combined, for example, to formulate a tetravalent vaccine.
Furthermore, certain aspects of the present invention provide a method of producing a viral
vaccine against a disease spread by a wild mosquito population to a mammal, comprising the
steps of genetically engineering a mutation of one or more amino acids in a ChikV E2 protein
such as the TMD to produce an engineered virus, wherein the transmembrane protein is able
to span the membrane envelope when the virus replicates in mosquito cells, but is unable to
efficiently span the membrane envelope when the virus replicates in mammalian cells, and
wherein the virus remains capable of replicating in mosquito cells; introducing the engineered
virus into a wild mosquito population; and allowing the virus to replicate in cells of the wild
mosquito population to produce a population of mosquitoes which excludes the wild-type
pathogenic virus and harbors the vaccine strain of the virus such that a mosquito bite delivers
the vaccine to a mammal that is bitten.
[0040] In addition, certain aspects of the present invention provide a method of
vaccinating an individual in need of such treatment, comprising the steps of introducing the
viral vaccine of the present invention into the individual and allowing the vaccine to produce
viral proteins for immune surveillance and to stimulate the immune system for antibody
production in the individual.
A. Vaccine preparations
[0041] In any case, a vaccine component (e.g., an antigenic peptide, polypeptide,
nucleic acid encoding a proteinaceous composition, or virus particle) may be isolated and/or
purified from the chemical synthesis reagents, cell, or cellular components. A vaccine
component may be cultured in a population of cells, such as a cell line. Any suitable cell
population or cell line may be used. For example, a vaccine component (e.g., a polypeptide,
a nucleic acid encoding a polypeptide, or a virus particle) may be cultured in insect cells.
Suitable insect cells include, but are not limited to, C6/36 cells, Sf9 cells, other Sf series cells,
drosophila SI cells, other drosophila cell lines, or TN368 cells. It is anticipated that any
cultured insect cells may be used to grow the vaccine components or viruses disclosed herein.
[0042] The C6/36 cell line (derived from Aedes albopictus) is made up of mosquito
cells and is frequently used to study arboviruses. C6/36 cells can be transfected with a
vaccine component, such as a polypeptide or a nucleic acid encoding a polypeptide. The
production of viruses can be visualized and monitored using a focus assay.
[0043] The Sf9 cell line (derived from Spodoptera frugiperda) is commonly used to
express recombinant proteins and can be infected by viruses, including arboviruses. For
example, Sf9 cells can be infected by viruses including recombinant baculovirus and St.
Louis encephalitis, Yellow fever, DEN-1, DEN-2, Gumbo limbo, Eastern equine
encephalomyelitis, herpes simplex virus type 1, and vesicular stromatitis viruses (Zhang et
al, 1994). Yellow fever, DEN-1, and DEN-2 viruses can replicate in Sf9 cells (Zhang et al,
1994) such that Sf9 cells can be used to culture and produce such viruses. Likewise, Sf9 cells
can be used use for production of the recombinant ChikV of the embodiments.
[0044] In a method of producing a vaccine component, purification is accomplished
by any appropriate technique that is described herein or well known to those of skill in the art
(e.g., Sambrook et al., 1987). Although preferred for use in certain embodiments, there is no
general requirement that an antigenic composition of the present invention or other vaccine
component always be provided in their most purified state. Indeed, it is contemplated that a
less substantially purified vaccine component, which is nonetheless enriched in the desired
compound, relative to the natural state, will have utility in certain embodiments, such as, for
example, total recovery of protein product, or in maintaining the activity of an expressed
protein. However, it is contemplated that inactive products also have utility in certain
embodiments, such as, e.g., in determining antigenicity via antibody generation.
[0045] Certain aspects of the present invention also provide purified, and in preferred
embodiments, substantially purified vaccines or vaccine components. The term "purified
vaccine component" as used herein, is intended to refer to at least one vaccine component
(e.g., a proteinaceous composition, isolatable from cells), wherein the component is purified
to any degree relative to its naturally obtainable state, e.g., relative to its purity within a
cellular extract or reagents of chemical synthesis. In certain aspects wherein the vaccine
component is a proteinaceous composition, a purified vaccine component also refers to a
wild-type or mutant protein, polypeptide, or peptide free from the environment in which it
naturally occurs.
[0046] Where the term "substantially purified" is used, this will refer to a
composition in which the specific compound (e.g., a protein, polypeptide, or peptide) forms
the major component of the composition, such as constituting about 50% of the compounds
in the composition or more. In preferred embodiments, a substantially purified vaccine
component will constitute more than about 60%, about 70%, about 80%, about 90%, about
95%, about 99% or even more of the compounds in the composition.
[0047] In certain embodiments, a vaccine component may be purified to
homogeneity. As applied to the present invention, "purified to homogeneity," means that the
vaccine component has a level of purity where the compound is substantially free from other
chemicals, biomolecules or cells. For example, a purified peptide, polypeptide or protein will
often be sufficiently free of other protein components so that degradative sequencing may be
performed successfully. Various methods for quantifying the degree of purification of a
vaccine component will be known to those of skill in the art in light of the present disclosure.
These include, for example, determining the specific protein activity of a fraction (e.g.,
antigenicity), or assessing the number of polypeptides within a fraction by gel
electrophoresis.
[0048] It is contemplated that an antigenic composition of the invention may be
combined with one or more additional components to form a more effective vaccine. Nonlimiting
examples of additional components include, for example, one or more additional
antigens, immunomodulators or adjuvants to stimulate an immune response to an antigenic
composition of the present invention and/or the additional component(s). For example, it is
contemplated that immunomodulators can be included in the vaccine to augment a cell or a
patient's (e.g., an animal's) response. Immunomodulators can be included as purified
proteins, nucleic acids encoding immunomodulators, and/or cells that express
immunomodulators in the vaccine composition.
[0049] Immunization protocols have used adjuvants to stimulate responses for many
years, and as such adjuvants are well known to one of ordinary skill in the art. Some
adjuvants affect the way in which antigens are presented. For example, the immune response
is increased when protein antigens are precipitated by alum. Emulsification of antigens also
prolongs the duration of antigen presentation.
[0050] 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.
[0051] An immunologic composition of the present invention may be mixed with one
or more additional components (e.g., excipients, salts, etc.) that are pharmaceutically
acceptable and compatible with at least one active ingredient (e.g., antigen). Suitable
excipients are, for example, water, saline, dextrose, glycerol, ethanol and combinations
thereof.
[0052] An immunologic composition of the present invention may be formulated into
the vaccine as a neutral or salt form. A pharmaceutically acceptable salt, includes the acid
addition salts (formed with the free amino groups of the peptide) and those that are formed
with inorganic acids such as, for example, hydrochloric or phosphoric acid, or such organic
acids as acetic, oxalic, tartaric, mandelic, and the like. A salt formed with a free carboxyl
group also may be derived from an inorganic base such as, for example, sodium, potassium,
ammonium, calcium, or ferric hydroxide, and such organic bases as isopropylamine,
trimethylamine, 2 ethylamino ethanol, histidine, procaine, and combinations thereof.
[0053] In addition, if desired, an immunologic composition may comprise minor
amounts of one or more auxiliary substances such as for example wetting or emulsifying
agents, pH buffering agents, etc. that enhance the effectiveness of the antigenic composition
or vaccine.
B. Vaccine administration
[0054] Viruses of the embodiments 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. Examples of patients who can be treated using the ChikV-related vaccines
and methods of the invention include (i) children in areas in which ChikV is endemic, such as
Asia, and Africa, (ii) foreign travelers, (iii) military personnel, and (iv) patients in areas of a
ChikV epidemic. Moreover, inhabitants of regions where the disease has been observed to be
expanding (e.g., Southern Europe), or regions where it may be observed to expand in the
future (e.g., regions infested with Aedes aegypti or Aedes albopictus), can be treated
according to the invention.
[0055] Formulation of 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, 18th Ed., 1990). 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. Preferably, virus can be
prepared or administered in FDA-approved insect cells.
[0056] The immunogenic compositions of the embodiments can be administered
using methods that are well known in the art, and appropriate amounts of the vaccines
administered can 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 and
107 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. Further, the immunogenic compositions of the embodiments 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.
[0057] The manner of administration of an immunogenic compositions of the
embodiments may be varied widely. Any of the conventional methods for administration of a
vaccine are applicable. For example, a vaccine may be conventionally administered
intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially,
intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally,
intravaginally, intratumorally, intramuscularly, intraperitoneally, subcutaneously,
intravesicularlly, mucosally, intrapericardially, orally, rectally, nasally, topically, in eye
drops, locally, using aerosol, injection, infusion, continuous infusion, localized perfusion
bathing target cells directly, via a catheter, via a lavage, in creams, in lipid compositions (e.g.,
liposomes), or by other methods or any combination of the forgoing as would be known to
one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th
Ed., 1990, incorporated herein by reference).
[0058] A vaccination schedule and dosages may be varied on a patient-by-patient
basis, taking into account, for example, factors such as the weight and age of the patient, the
type of disease being treated, the severity of the disease condition, previous or concurrent
therapeutic interventions, the manner of administration and the like, which can be readily
determined by one of ordinary skill in the art.
[0059] An immunogenic compositions of the embodiments is administered in a
manner compatible with the dosage formulation, and in such amount as will be
therapeutically effective and immunogenic. For example, the intramuscular route may be
preferred in the case of toxins with short half lives in vivo. The quantity to be administered
depends on the subject to be treated, including, e.g., the capacity of the individual's immune
system to synthesize antibodies, and the degree of protection desired. The dosage of the
vaccine will depend on the route of administration and will vary according to the size of the
host. Precise amounts of an active ingredient required to be administered depend on the
judgment of the practitioner. In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound. In other embodiments, an
active compound may comprise between about 2% to about 75% of the weight of the unit, or
between about 25% to about 60%, for example, and any range derivable therein. However, a
suitable dosage range may be, for example, of the order of several hundred micrograms active
ingredient per vaccination. In other non-limiting examples, a dose may also comprise from
about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10
microgram/kg/body weight, about 50 microgram/kg/body weight, about 100
microgram/kg/body weight, about 200 microgram/kg/body weight, about 350
microgram/kg/body weight, about 500 microgram/kg/body weight, about 1
milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body
weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200
milligram/kg/body weight, about 350 milligram/kg/body weight, about 500
milligram/kg/body weight, to about 1000 mg/kg/body weight or more per vaccination, and
any range derivable therein. In non-limiting examples of a derivable range from the numbers
listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5
microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered,
based on the numbers described above. A suitable regime for initial administration and
booster administrations (e.g., inoculations) are also variable, but are typified by an initial
administration followed by subsequent inoculation(s) or other administration(s).
[0060] In many instances, it will be desirable to have multiple administrations of the
vaccine, usually not exceeding six vaccinations, more usually not exceeding four
vaccinations and preferably one or more, usually at least about three vaccinations. The
vaccinations will normally be at from two to twelve week intervals, more usually from three
to five week intervals. Periodic boosters at intervals of 1.5 years, usually three years, will be
desirable to maintain protective levels of the antibodies.
[0061] The course of the immunization may be followed by assays for antibodies for
the supernatant antigens. The assays may be performed by labeling with conventional labels,
such as radionuclides, enzymes, fluorescents, and the like. These techniques are well known
and may be found in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384
and 3,949,064, as illustrative of these types of assays. Other immune assays can be
performed—and assays of protection from challenge with the ChikV—can be performed
following immunization.
[0062] Certain aspects of the present invention include a method of enhancing the
immune response in a subject comprising the steps of contacting one or more lymphocytes
with a ChikV immunogenic composition, wherein the antigen comprises as part of its
sequence a nucleic acid or amino acid sequence encoding mutant E2 protein, according to the
invention, or an immunologically functional equivalent thereof. In certain embodiments the
one or more lymphocytes is comprised in an animal, such as a human. In other embodiments,
the lymphocyte(s) may be isolated from an animal or from a tissue (e.g., blood) of the animal.
In certain preferred embodiments, the lymphocyte(s) are peripheral blood lymphocyte(s). In
certain embodiments, the one or more lymphocytes comprise a T-lymphocyte or a Blymphocyte.
In a particularly preferred facet, the T-lymphocyte is a cytotoxic T-lymphocyte.
[0063] The enhanced immune response may be an active or a passive immune
response. Alternatively, the response may be part of an adoptive immunotherapy approach in
which lymphocyte(s) are obtained from an animal (e.g., a patient), then pulsed with a
composition comprising an antigenic composition. In a preferred embodiment, the
lymphocyte(s) may be administered to the same or different animal (e.g., same or different
donors).
C. Pharmaceutical compositions
[0064] It is contemplated that pharmaceutical compositions may be prepared using
the novel mutated viruses of certain aspects of the present invention. In such a case, the
pharmaceutical composition comprises the novel virus and a pharmaceutically acceptable
carrier. A person having ordinary skill in this art readily would be able to determine, without
undue experimentation, the appropriate dosages and routes of administration of this viral
vaccination compound. When used in vivo for therapy, the vaccine of certain aspects of the
present invention is administered to the patient or an animal in therapeutically effective
amounts, i.e., amounts that immunize the individual being treated from the disease associated
with the particular virus. It may be administered parenterally, preferably intravenously or
subcutaneously, but other routes of administration could be used as appropriate. The amount
of vaccine administered may be in the range of about 10 to about 106 pfu/kg of subject
weight. The schedule will be continued to optimize effectiveness while balancing negative
effects of treatment (see Remington's Pharmaceutical Science, 18th Ed., (1990); Klaassen In:
Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed. (1990); which
are incorporated herein by reference). For parenteral administration, the vaccine may be
formulated in a unit dosage injectable form (solution, suspension, emulsion) in association
with a pharmaceutically acceptable parenteral vehicle. Such vehicles are preferably non-toxic
and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose
solution, and 5% human serum albumin.
V. Examples
[0065] The following examples are included to demonstrate preferred embodiments
of the invention. It should be appreciated by those of skill in the art that the techniques
disclosed in the examples which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be considered to constitute
preferred modes for its practice. However, those of skill in the art should, in light of the
present disclosure, appreciate that many changes can be made in the specific embodiments
which are disclosed and still obtain a like or similar result without departing from the spirit
and scope of the invention.
Example 1 - Materials and Methods of the Studies
Biosafety
[0066] All studies involving viable ChikV were performed in certified BSL-3
laboratories in biosafety cabinets using biosafety protocols approved by the Institutional
Biosafety Committee of North Carolina State University. Animal husbandry and mouse
experiments were performed in accordance with all University of North Carolina at Chapel
Hill Institutional Animal Care and Use Committee guidelines.
Construction of ChikV TM deletion mutants.
[0067] A full-length cDNA clone of Chikungunya, West African strain 37997, in the
pSinRep5 vector (Genbank # EU224270, incorporated herein by reference (SEQ ID NO:21))
was obtained (Tsetsarkin et al. 2006). Deletions in the E2 TMD of ChikV were produced by
PCR based site-directed mutagenesis, using Pfu Turbo® DNA polymerase AD (Stratagene,
La Jolla, CA). Primers designed were to create sets of 9 amino acid (aa) deletions within
ChikV E2 so that the TMD size was 17 aa in length (TM17-1, 2 and 3) (Tablel). Reactions
were run with and without DMSO (4% final concentration) in 1.5 x buffer. PCR cycles were:
95°C, 2 min, X 25 cycles of 95°C for 15 sec, 45 sec annealing (TA = Primer Tm -5°C for
each set of primers), 68°C for 24 min. Extension, 28 min at 68°C; samples were then held at
4°C. Following mutagenesis, the PCR products were digested with Dpn I (New England
Biolabs) and transformed into SURE®2 Supercompetent E. coli cells (Stratagene) as per
manufacturer's instructions with a few alterations. Following heat shock and recovery on ice,
RT NZY+ broth (Teknova, Hollister, CA) was added and incubated at 30°C for 2 hours.
After plating on LB agar containing 50 mg/mL carbenicillin (Teknova), incubation was 30°C
for 32-48 hours. A colony PCR screen was used to identify mutations. Growth of all ChikV
clones in SURE®2 cells was in LB containing 50 mg/mL carbenicillin at 28 to 30°C for
approximately 24 to 48 hours. ChikV plasmid DNA was recovered using the Wizard® Plus
Minipreps (Promega, Madison, WI). All ChikV deletion mutant clones were confirmed by
sequence analysis (Eurofins MWG Operon, Huntsville, AL). Purified DNA produced fulllength
ChikV RNAs were transcribed in vitro with SP6 RNA polymerase and transfected into
C7-10 cells for stock virus production,
Cell and Virus Culture
[0068] BHK and C7-10 mosquito cell lines were maintained as previously described
(Hernandez et al. 2010) in minimal essential medium (MEM-E) containing Earl's salts,
supplemented with 10% fetal bovine serum (FBS), 5% tryptose phosphate broth, and 5 mM
L-glutamine. C7-10 cells were transfected by electroporation with WT ChikV and ChikV
TM17 series mutant RNAs. Supernatants were harvested 2 days post transfection and stored
at -80°C with 10% glycerol added. Infections in sub-confluent monolayers of BHK and C7-
10 were performed using virus from these transfections. Virus harvested from transfections
and infections in vitro was titrated by plaque assay in C7-10 cells to test for a host-range
phenotype. Assays were stained 48 hours post inoculation with IXMEM-E completed media
containing 0.06% neutral red and 1% agarose. Spodoptera frugiperda (Sf9) cells were
cultured at 28°C in Grace's medium (Gibco) completed with 10% FBS. Suspension cultures
were seeded at a density of 3xl0 5 cells per mL, and allowed to grow to a density of 2xl0 6
cells/mL. 24 hours prior to infection, adherent flasks were seeded with cells from suspension
cultures and incubated at 28°C. Subconfluent adherent Sf9 cells were infected with a
multiplicity of infection (MOI) of > 1 plaque forming units (pfu)/cell of ChikV or ChikV
TM17-2, for 1 hr. with rocking and inoculum was removed and replaced with Grace's
medium completed with 10% FBS or uncompleted Grace's medium. Supernatants were
harvested after 19 hr. of incubation at 28°C. Virus was titered via plaque assay in C7-10 cells
as described above. Similar studies were also completed for the ChikV TM16 series of the
mutants.
Mouse Studies
[0069] Previous studies have described ChikV disease in C57BL/6J mice (Couderc et
al. 2008; Morrison et al. 201 1) which were also chosen for the testing of the ChikV vaccine
strains. C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and
were injected at 14 days of age in the left hind footpad (Kamala 2007). Mice were weighed
every day and no mortality occurred from ChikV infection. Swelling and inflammation were
measured laterally and longitudinally along the foot below the ankle. Fourteen day old mice
were infected via subcutaneous injection into the left foot pad with ~10 pfu of WT ChikV,
TM17-1, TM17-2, and TM17-3, ChikV in IOm of completed MEM with 10% glycerol.
Animals, including those from a naive group of mice injected with media only, were
sacrificed 1, 2, 3, 7, 10, and 2 1 days post injection (dpi) to evaluate viremia, persistence in
the tissues, neutralizing antibody titer (NAb), IgG production, and tissue disease.
Observations were made 1-10 days post vaccination to evaluate physical stress, swelling, or
disability to the mouse foot pad due to virus infection. One of the first markers for pathology
is swelling at the site of infection. WT virus produced severe swelling, TM17-3
reactogenicity was only slight whileTM17-l and TM17-2 had no measurable swelling.
Because swelling is linked to other pathology only the mutants which did not produce
swelling at the site of infection were challenged, eliminating TM17-3. Animals from which
tissues were prepared to evaluate disease were perfused with paraformaldehyde, imbedded in
paraffin and processed for H and E staining on 7, 10 and 2 1 dpi. Based on results from the
initial evaluation of the vaccine candidates; naive mice and mice injected with TM17-1, and
TM17-2 were used to test for protection from infection with a more pathogenic strain of
ChikV. Twenty eight dpi the majority of mice from each group were challenged via
subcutaneous injection in the foot pad with 103 pfu WT ChikV(SL15649) (Morrison et al.
2011), while 3 mice from each vaccine group were injected with media as a control test for
any residual response from the original vaccination. Mice were sacrificed 1, 2, 3, and 7 days
post challenge to again evaluate viremia, tissue disease, and NAb.
Viremiafrom Mice
[0070] Due to the selective nature of the ChikV strain for growth in mosquitoes
(Tsetsarkin et al. 2006; Delatte et al. 2010; Vazeille et al. 2007), and the attenuation of the
vaccine strains in mammalian cells, vaccine titers and viremias from mice were quantified by
plaque assay in C7-10 as described (Hernandez et al. 2010). Viremias resulting from the
challenge virus ChikV SL15649 were quantified by plaque assay on BHK due to the selective
nature of this virus for mammalian cells (personal observation and this study). The limit of
detection for these assays was <40 pfu per gram of tissue (pfu/g), and the results expressed
are the arithmetic means of titers obtained from 3 mice per group per day, shown in FIG. 1.
Persistence of Infection in Tissues
[0071] ChikV is known to persist in the joints of the host, producing chronic
arthralgia. To determine if mutant virus persisted in the vaccinated animals, tissues and sera
from infected and naive mice 10 and 1 days post vaccination were homogenized and RNA
extracted using Trizol® LS reagent and the Purelink® RNA kit (Life Technologies Inc.
Grand Island, NY) and suspended in water. Extracted RNAs were then analyzed via RT-PCR
(reverse transcription-polymerase chain reaction) using the following primer pairs; Sense
primer: CHIKV 10007F (5'-CAG TGA TCC CGA ACA CGG TG-3'; SEQ ID NO:24) Antisense
primer: CHIKV 10260R (5'-CCA CAT AAA TGG GTA GAC TCC-3'; SEQ ID
NO:25) which recognize the ChikV strain (sequences courtesy of Kristen Long, UNC Chapel
Hill). The plasmid icCHIKV SL15649 was used as a positive control, and extracted RNA was
used as a negative control. RT-PCR had a sensitivity of detection of ~10 pfu.
Plaque Reduction Neutralization Test
[0072] Neutralizing antibody (NAb) titers were determined by plaque reduction
neutralization test (PRNT) in BHK cells (Smith et al. 2012). Mice sera were heat inactivated
at 56°C for 20 minutes prior to being serially diluted in duplicate 1 to 2, starting with a 1 to
20 dilution. After diluting the sera, approximately 20 pfu of WT ChikV were added to each
dilution, allowed to incubate at RT for 15 minutes, and then plated on BHK and allowed to
produce plaques for 2 days at 37°C. NAb titers (PRNT50) were determined based upon the
highest serial dilutions where 50% of the pfu added were observed, and results are expressed
as the geometric mean of titers from the 3 mice per group per day.
Anti-ChikV IgG ELISA
[0073] 96-well Poly-D-Lysine pretreated ELISA plates (Becton Dickinson, Bedford,
MA) were coated with >100 ng of purified WT ChikV per well at 37°C for 1 hour, blocking
with PBS-D buffer with 0.2% Tween-20, and 10% FBS at 4°C overnight. A standard of
serially diluted Anti-ChikV IgGl (#3583, ViroStat Inc, Portland, ME) was added to the plate,
as well as 1:100 dilutions of heat inactivated mouse sera in duplicate. Serum samples
obtained 2 1 dpi and 7 days post challenge were added for 1.5 hours at RT and removed. A
1:2000 dilution of Anti-mouse IgG horseradish peroxidase conjugated (Sigma-Aldrich
#A8924). Ab was then added to the plate for another 1.5 hours at RT. ELISAs were
developed using TMB substrate (Promega) for 30 minutes in the dark at RT, stopped with 1%
SDS, read using a Tecan Rainbow® 96-well plate reader at an absorbance wavelength of 405
nm and reference wavelength of 0. IgG concentrations are given in mg/mL as calculated from
the standard curve of control antibody, and results shown are the arithmetic mean of
concentrations obtained from 6 mice per group.
Reactogenicity
[0074] The first part of the study evaluated inflammation and swelling of the foot and
ankle at the site of injection of each of the viruses injected, compared to a control group.
Inflammation was monitored for 10 dpi. Severity grades were assigned as minimal, mild,
moderate or marked. WT ChikV infected mice displayed mild to moderate inflammation
beginning 2 dpi (data not shown) while ChikV TM17-3 exhibited inflammation in the
minimal category and was eliminated from the study at the challenge phase.
Histopathology
[0075] Mice were sacrificed and perfused by intracardial injection with 4%
paraformaldehyde, pH 7.3 on the days indicated. Hind limb tissues were embedded in
paraffin and 5mih sections were prepared (Morrison et al. 2011). Hemotoxylin and Eosin (H
and E) stain was used to determine the extent of inflammation of the tissue and tissue disease.
Sections were evaluated for fasciitis in the foot/ankle and quadriceps (quad) as in (Morrison
et al. 201 1).
Example 2 - Study Results and Discussion
Host Range mutant design
[0076] The 26 amino acid sequence defining the ChikV TMD was determined by
comparison to the SIN TMD and the junction with the endodomain (Hernandez et al. 2000;
Rice et al. 1982; Ahlquist et al. 1985; Strauss et al. 1994; Hernandez et al. 2005). Because of
the specific geometry of the helical TMD and the differences in the amino acid sequence in
ChikV; it was not clear which amino acid deletions might result in desired HR phenotype. A
series of 3 TM17 mutants were made, deleting the sequences shown in Table 1A. TM17-3
represented the most central deletion whereas two other mutants, ChikV TM17-1 and TM17-
2 were designed to shift the deleted sequence toward the amino and carboxyl termini,
respectively. Likewise, a series of TM16 mutants were made, deleting the sequences shown
in Table IB. Virus titers of the ChikV mutants were determined after growth in both BHK
and C7-10 cells. All ChikV TM 17 mutants had titers in the range of 106 from BHK and 10
from C7-10 cells (Table 1A). Similarly, ChikV TM 16 mutants had titers in the range of 106
from BHK and 10 -108 from C7-10 cells (Table 1C and FIG. 6).
[0077] Table 1A: Transmembrane domain sequences of SIN (SEQ ID NO: 13)
compared to ChikV37997 (SEQ ID NO: 14) are shown. Three transmembrane deletions (each
deletion of 9 amino acids) of ChikV were produced in vitro and studied. The underlined
portions of sequence represent the segments of the TMD which were deleted. Titers shown
demonstrate the host range phenotype existing in each of these deletion mutants. TM17
designates the predicted number of amino acids remaining in the TMD in these mutants.
*HR indicates heat resistant strain.
[0078] Table IB: Transmembrane domain sequences of SIN (SEQ ID NO: 13)
compared to ChikV37997 (SEQ ID NO: 14) are shown. Three additional transmembrane
deletions (each deletion of 10 amino acids) of ChikV were produced. The underlined portions
of sequence represent the segments of the TMD which were deleted.
*HR indicates heat resistant strain.
[0079] Table 1C: ChikV TM16 mutant viruses shown in Table IB were grown in
C7-10 mosquito cells or mammalian BHK cells and titers (in PFU) were assed at 24 and 48
hours.
Cell line; ChikV: l i m
mutant
C710 TM16-1 4.02E+08 7.25E+07
C710 TM16-2 2.53E+08 3.83E+07
C710 TM16-3 4.66E+08 4.94E+08
BHK TM16-1 3.56E+06 1.44E+07
BHK TM16-2 2.56E+06 1.50E+06
BHK TM16-3 4.57E+06 1.48E+06
Safety and Immunogenicity
[0080] Chikungunya is a virus which causes arthritis and will also establish persistent
infection in the joints (Suhrbier et al. 2012). For this reason, serum as well as tissues
surrounding the ankle were examined. The WT La Reunion strain was used to construct the
mutant viruses. The virus titers may seem high but this virus is mosquito adapted (Tsetsarkin
et al. 2007; Vanlandingham et al. 2006) and was titered on mosquito C7-10 cells. BHK cells,
which were not found to be good indicator cells for this ChikV strain, gave much lower titers
and did not reflect the actual viremic levels. The first part of the study evaluated
inflammation and swelling of the foot/ankle at the site of injection of each of the viruses
injected, compared to a control group. Swelling at the site of injection is indicative of primary
reactogenicity and is a good predictor of further tissue disease (Morrison et al. 201 1). Both
TM17-1 and TM17-2 did not produce any inflammatory response at the site of injection and
proceeded to the challenge portion of the study.
Virus viremia post injection.
[0081] Viremia from all virus infected mice was determined from sera and tissue
samples on 1, 2, 3 and 7 dpi and are shown in FIG. 1A. All mice were injected with 10
pfu/ IOmE of virus each of the viruses and grew to a level of 107 pfu/mL within 24 hours pi.
Serum virus titers were not found to be significantly different from WT until day 2 for TM17-
2 (p<0.05), and day 3 for TM17-1 (pO.001). ChikV TM 17-1 and 2 had not cleared all virus
from the serum on day 7 (102 pfu/mL). The infection profile changes when the foot/ankles are
examined (FIG. IB). Foot and ankle tissue titers differ from WT as follows; day 1 titers are
significantly different from WT (pO.001 for TM17-1, p<0.01 for TM17-2) for both TM17-1
and 2 and on day 2 only TM17-1 differs (p<0.05). Both TM17-1 and 2 are significantly lower
than WT on day 3 (p<0.05 for both). By day 7 both TM17-2 and 3 are cleared from the
foot/ankle. ChikV titers from the quadriceps of the 3 mutants tested did not vary from WT
titers on days 1 and 2 (FIG. 1C). However, for TM17-1, 2, and 3 virus was not detected on
day 3 while WT virus infected animals still expressed 104 pfu/g. Wild type virus is detected
by RT-PCR in the serum, foot/ankle, and quad 2 1 days after infection indicating a persistent
infection of the affected tissues and is discussed further below.
Viruspersistence infoot/ankle and quad.
[0082] Virus persistence in the mice is defined by the presence of virus past 7 dpi.
Persistence was evaluated by RT-PCR at days 10 and 2 1 post injection and is shown in Table
2. The presence of a PCR product was scored as positive or negative for each of three mice.
On day 10 the ChikV infected mice tested positive for 1 mouse in the serum, 3 in the
foot/ankle and 1 mouse in the quad. By day 2 1 this same profile was seen for a second group
of 3 ChikV mice. Of the mice infected with the mutant viruses TM17-1 tested positive ( 1
mouse) in the foot/ankle on day 21, TM17-2 tested positive ( 1 mouse) from the serum on day
21, and TM17-3 tested positive (2 mice) from the serum on both days 10 and 2 1 post
infection. The limit of detection of this assay was 10 pfu.
[0083] Table 2: Evaluation of persistence of ChikV RNA 10 and 2 1 days after
injection of mice (pre-challenge) with wild type ChikV, attenuated mutants CHIKV TM-171-
3, or mock (diluent) by RT-PCR. Tissues were either positive or negative for the presence of
viral RNA and the number of positive symbols represents the number of mice per each
sample group that tested positive (n=3). The limit of detection for this assay was equivalent to
10 pfu.
Vaccine efficacy
[0084] To ascertain the level of vaccine efficacy animals were sacrificed 1, 2, and 3
days post challenge (28 dpi) to determine viremia and pathology. Challenge was injection
with 103 pfu of WT ChikV SL15649, into mice infected with TM17-1, TM17-2, WT ChikV
or no vaccine (naive). Shown in Table 3 are the viremia values measured for the indicated
tissue on 3 consecutive days post challenge. It was of interest that ChikV was not more
protective against challenge giving a titer of 8.5 x 10 pfu/g from the foot/ankle and 5.1x102
pfu/g from the quad on day 1. WT ChickV infected mice continued to be infected with
challenge virus in the quad on day 2 (1.3x104 pfu/g) which was cleared by day 3 post
challenge. As is presented in Table 3, TM17-1 had a titer of 1.3 x 104 pfu/mL in the serum on
day 1 post challenge (day 29). This is challenge virus since all pre-challenge viremia was
cleared for this mutant by day 2 1(refer to Table 2). ChikV TM17-1 also had a titer of 6.4 x
102 pfu/mL virus in the quad on day 2 post challenge. No further viremia was detected for
this mutant from any tissue on day 3 post challenge in any mouse. ChikV TM17-2 had no
detectable viremia in any of the tissues sampled on the 3 days post challenge. The challenge
virus, ChikV SL15649 gave serum titers of 4.7 x 10 and 8.6 x 105 pfu/mL on days 1 and 2
respectively, but was cleared by day 3. ChikV SL15649 was also found to have viremia in the
foot/ankle an all 3 days; 4.3 x 10 , 7.3 x 103 and 9.8 x 102 pfu/g respectively for each day.
ChikV SL15649 titers were also measured for all three days when the quad was analyzed.
These values are 1.7 x 105, 4.0 x 10 5, and 2.1 x 103 pfu/g respectively on each of the 3 days
post challenge. These data collectively demonstrate that ChikV infection targets the joints
and surrounding musculature and that vaccination with TM17-2 protected all tissues assayed
from WT virus challenge beginning day 1 of the viremic period.
[0085] Table 3: The titers of the viremia detected (in pfu/g) by plaque assay 1, 2, and
3 days after challenging mice with 103 pfu of WT ChikV SL15649. Challenge was 28 days
after injection with TM17-1, TM17-2, ChikV37997, or no vaccine (naive). ChikV TM-3 was
not challenged due to the detection of mild reactogenicity at the injection site. P values of the
titers compared to the naive virus control are given with ns designating; not statistically
significant.
Serum Foot/Ankle Quadricep
Vaccine
Day 1 Day 2 Day 3 Day l Day 2 Day 3 Day l Day 2 Day 3
1.3xl04 ND* ND ND ND ND ND 6.4xl0 2 ND
TM17-1 p<0.05 ns ns p<0.05 p<0.001 p<0.05 p<0.01 p<0.01 ns
ND ND ND ND ND ND ND ND
TM17-2 p<0.001 p<0.001 ns p<0.05 p<0.001 p<0.05 ND p<0.001 p<0.01 ns
ND ND ND 8.5xl0 3 ND ND 5.1xl02 1.3xl04 ND
ChikV37997 p<0.001 p<0.01 ns p<0.001 p<0.001 p<0.05 p<0.001 p<0.01 ns
4.7xl0 7 8.6xl0 5 ND 4.3xl0 3 7.3xl0 3 9.8xl0 2 1.7xl05 4.0xl0 5 2.1xl03
Naive
ND below detection limit of the assay, 80 pfu/mL.
[0086] Efficacy was shown further by measuring the amount of Nab on day 7 post
challenge (FIG. 4). NAb titers generated by the WT virus ChikV post inoculation were high ~
1000 PRNT50 on day 7, which was expected. PRNT50 titers remained high for WT ChikV
and were not found to be comparable to titers of the ChikV HR mutants because of the
variability of the data and the small sample size, thus all TM17 mutants appear to have
similar neutralization to WT 7, 10 and 1 dpi. On day 7 post challenge. ChikV TM17-1 gave
a PRNT50 titer of 4000 while TM17-2 was 2000, compared to the mock control and the
amount of NAb produced by ChikV SL15649. These values are all essentially equivalent to
the WT values.
Total ChikVIgG concentration
[0087] It was important to determine the total ChikV specific IgG post infection. To
determine the total concentration of ChikV-specific IgG elicited by vaccination, an enzymelinked
immunosorbant assay (ELISA) was performed 2 1 dpi. As shown in FIG. 3, WT LAV
infected animals were found to have more than 1.5 mg/mL of IgG present. Animals
vaccinated with TM17-1, TM17-2, and TM17-3 had significantly lower titers of IgG, overall,
compared to WT vaccinated animals. There was no significant difference in IgG titers
between the vaccine candidates. All 3 TM17 mutants were found to elicit ~ 4 times less total
ChikV specific Ab TM17-1, (p<0.01), TM17-2 (p<0.05), TM17-3 (p<0.01) and
mock,(p<0.001) while there was no significant difference found between the respective
mutant pairs. (WT ChikV). Thus while virus neutralization for each of these viruses was not
found to significantly differ, the total amount of IgG of each of the HR mutants was
significantly lower than that of the WT inoculation indicating a favorable low ratio of nonneutralizing
to neutralizing antibody. Surprisingly, the levels of total IgG did not change for
TM17-1 or TM17-2 whether they were challenged or remained unchallenged. Because this
was an unexpected finding no additional mice were planned to evaluate cellular immune
response.
Histopathology
[0088] It was important to determine if any tissue pathology presented as a result of
vaccination with TM17-1 or TM17-2. To determine this, sections of mouse foot/ankle joints
were taken at 7 days postvaccination, H&E stained, and scored blindly. Pathology was
assessed by scoring slides from each animal based on muscle inflammation, muscle necrosis,
tendonitis, synovitis, and perivasculitis. For scoring pathology, the following scale was used:
0, 0 to 2%; 1, 2 to 20%; 2, 20 to 40%; 3, 40 to 60%; 4, 60 to 80%; and 5, 80 to 100%. For
scoring of synovium and perivascular inflammation, the following scale was used: 0, no
change; 1, minimal; 2, mild (inflammatory infiltrate); 3, moderate; 4, severe (destruction of
synovial membrane). Scores for individual animals postvaccination are shown in Table 4.
Infection of mice with WT ChikV produced severe muscle inflammation and necrosis with
apparent destruction of the synovial membrane. In contrast, animals vaccinated with TM17-1
displayed mild muscle inflammation with no other signs of pathology. Importantly, animals
vaccinated with TM17-2 displayed no signs of any pathology at 7 days postvaccination.
These results confirm the primary reactogenicity studies, in which no swelling was seen in
animals vaccinated with TM17-2.
[0089] Table 3: Pathology scoring assigned to slides for individual animals for
foot/ankle sections taken 7 days after vaccination.
Score
Group/ Muscle Muscle
mouse Inflammation Necrosis" Tendonitis" Synovitis Perivasculitis
WT/1 5 5 1 3 3
WT/2 5 5 1 4 2
WT/3 5 5 1 4 2
TM17-1/1 1 0 0 0 0
TM17-1/2 0 0 0 0 0
TM17-1/3 1 0 0 0 0
TM17-2/1 0 0 0 0 0
TM17-2/2 0 0 0 0 0
TM17-2/3 0 0 0 0 0
aScale: 0,0 to 2%; 1, 2 to 20%; 2, 20 to 40%; 3, 40 to 60%; 4, 60 to 80%; 5, 80 to
100%.
bScale: 0, no change; 1, minimal; 2, mild (inflammatory infiltrate); 3, moderate; 4,
severe (destruction of synovial membrane).
[0090] To determine if vaccination with TM17-1 or TM17-2 protected animals from
developing ChikV-associated pathology during challenge, foot/ankle sections of mice were
taken at 7 days postchallenge, H&E stained, and scored for pathology as described above
(see, Table 5). Naive mice challenged with ChikV SL15649 displayed moderate muscle
inflammation and necrosis. Mice vaccinated with TM17-1 displayed minimal muscle
inflammation following challenge with ChikV SL15649, with no other pathology apparent.
Most importantly, samples taken from mice vaccinated with TM17-2 prior to challenge with
ChikV SL15649 had no detectable pathology and appeared similar to samples taken from
naive mice challenged with medium alone. Taken together, these data suggest that TM17-2
is not only nonreactogenic, it is also sufficient to protect mice from pathology associated with
ChikV infection. This suggests that TM17-2 is a ChikV vaccine strain that warrants further
investigation and development as a live-attenuated vaccine strain (LAV).
[0091] Table 5 : Pathology scoring assigned to slides for individual animals for foot/ankle sections taken 7 days postchallenge.
Score
Muscle Muscle
Group/mouse (challenge) Inflammation 1 Necrosis Tendonitis Synovitis Perivasculitis'"
Naive/ 1 (mock) 0 0 0 0 0
Na 've/2 (mock) 0 0 0 0 0
Na'ive/3 (mock) 0 0 0 0 0
Naive/ 1 (challenged) 3 1 0 1 0
Na'ive/2 (challenged) 4 3 0 2 0
Na'ive/3 (challenged) 3 2 0 3 0
TM17-1/1 (mock) 0 0 0 0 0
TM17-1/2 (mock) 0 0 0 0 0
TM17-1/3 (mock) 0 0 0 0 0
TM17-1/1 (challenged) 0 0 0 0 0
TM17-1/2 (challenged) 2 0 0 0 0
TM17-1/3 (challenged) 0 0 0 0 0
TM17-2/1 (mock) 0 0 0 0 0
TM17-2/2 (mock) 0 0 0 0 0
TM17-2/3 (mock) 0 0 0 0 0
TM17-2/1 (challenged) 0 0 0 0 0
TM17-2/2 (challenged) 0 0 0 0 0
TM17-2/3 (challenged) 0 0 0 0 0
Challenge, challenged with ChikV SL15649; mock, mock challenged.
Scale: 0,0 to 2%; 1, 2 to 20%; 2, 20 to 40%; 3, 40 to 60%; 4, 60 to 80%; 5, 80 to 100%.
Scale: 0, no change; 1, minimal; 2, mild (inflammatory infiltrate); 3, moderate; 4, severe (destruction of synovial membrane).
{00090134} - 32 -
Discussion
[0092] This study of ChikV HR mutants TM17-1, 2, and 3 can provide the basis for
viral vaccine compositions. The large deletions, such as those studied, do not revert in vitro
or in vivo (Smith et al. 2012). One mutant, ChikV TM17-2 did not produce any swelling at
the site of injection, produced little if any inflammation in the foot/ankle or quad and did not
persist in any tissue tested pre-challenge. Of the 2 HR mutants that were challenged, ChikV
TM17-2 also protected against infection compared to TM 17-1 and WTChikV. Assay of the
serum, foot/ankle and quad on days 1-3 post challenge did not detect any virus for mice
infected with TM17-2, while TM17-1 and LAR both allowed growth of challenge virus.
Considering that infection of humans with an arbovirus confers lifelong immunity, ChikV
TM17-2 protected better than infection with WTChikV which allowed a transient infection
post challenge. Upon inspection of the histology, TM17-2 did not display any evidence of
inflammation or tissue disease day 7 post challenge. These results suggest that ChikV TM17-
2 is an attenuated, non-reactogenic, efficacious vaccine strain which should be further
developed for use in humans.
[0093] Interpretation of the data suggests that the protection conferred by
ChikVTM17-2 is not solely antibody-dependent. While antibodies are believed to be the
primary method of protection against ChikV infection (Couderc et al. 2009), cell mediated
immunity has been shown to be sufficient for protection against alphavirus disease in the
absence of strong antibody response (Linn et al. 1998; Paessler et al. 2007). Although the
studies here do not point directly to a specific mechanism for protection by this particular
mutant there is one notable point to consider. All the ChikV TM17 mutants deleted the same
number of amino acids (9) and the only distinction between these mutants is the position of
the deletion with respect to the amino and carboxyl terminus of the TMD.
[0094] Upon an initial inspection of the post inoculation titers of WTChikV and
TM17-1, 2 and 3, it may seem that the virus titers for WTChik and the mutants are high;
however it should be considered that the mutants are mosquito adapted to the A. albopictus
cell lines C7-10 and C6/36. These viruses were not found to plaque well on BHK and all
assays were performed on C7-10 cells. The HR phenotype of these mutants has been
proposed in previous studies as a marker of attenuation and now has additional support from
studies in monkeys for DV2 and ChikV.
* * *
[0095] All of the methods disclosed and claimed herein can be made and executed
without undue experimentation in light of the present disclosure. While the compositions and
methods of this invention have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be applied to the methods and in the
steps or in the sequence of steps of the method described herein without departing from the
concept, spirit and scope of the invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be substituted for the
agents described herein while the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those skilled in the art are deemed to be within the
spirit, scope and concept of the invention as defined by the appended claims.
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WE CLAIMS:-
1. A recombinant polypeptide wherein the polypeptide comprises an amino acid
sequence at least 90% identical to SEQ ID NO:l and comprises a deletion of 8-1 1 amino
acids in the transmembrane domain (TMD) corresponding to amino acid positions 365-390 of
SEQ ID NO: 1.
2. The polypeptide of claim 1, wherein the polypeptide is at least 91%, 92%, 93%, 94%,
95% or 96% identical to SEQ ID NO: 1.
3. The polypeptide of claim 1, wherein the polypeptide comprises a deletion of 9 amino
acids in the TMD.
4. The polypeptide of claim 3, wherein the polypeptide comprises a deletion of the
amino acids corresponding to amino acid positions 372-380 of SEQ ID NO:l.
5. The polypeptide of claim 3, wherein the polypeptide comprises a deletion of the
amino acids corresponding to amino acid positions 374-382 of SEQ ID NO:l.
6. The polypeptide of claim 3, wherein the polypeptide comprises a deletion of the
amino acids corresponding to amino acid positions 373-381 of SEQ ID NO:l.
7. The polypeptide of claim 1, wherein the polypeptide comprises SEQ ID NO:3; SEQ
ID NO:5 or SEQ ID NO:7.
8. The polypeptide of claim 1, wherein the polypeptide comprises a deletion of 10 amino
acids in the TMD.
9. The polypeptide of claim 8, wherein the polypeptide comprises a deletion of the
amino acids corresponding to amino acid positions 372-381 of SEQ ID NO:l.
10. The polypeptide of claim 8, wherein the polypeptide comprises a deletion of the
amino acids corresponding to amino acid positions 374-383 of SEQ ID NO:l.
11. The polypeptide of claim 8, wherein the polypeptide comprises a deletion of the
amino acids corresponding to amino acid positions 373-382 of SEQ ID NO:l.
12. The polypeptide of claim 1, wherein the polypeptide comprises SEQ ID NO: 15; SEQ
ID NO: 17 or SEQ ID NO: 19.
13. A polynucleotide molecule encoding a polypeptide of anyone of claims 1-12.
14. The polynucleotide of claim 13, comprising a sequence at least 90% identical to SEQ
ID NO:2.
15. The polynucleotide of claim 14, comprising a sequence of SEQ ID NO:4; SEQ ID
NO:6; SEQ ID O:8; SEQ ID NO: 16; SEQ ID NO: 18; or SEQ ID NO:20.
16. A host cell comprising the polynucleotide of claim 13.
17. The cell of claim 16, wherein the cell is an insect cell.
18. The cell of claim 17, wherein the cell is a SF9 cell.
19. A recombinant virus particle comprising a polypeptide of anyone of claims 1-12 or
the polynucleotide of claim 13.
20. The recombinant virus of claim 19, further defined as a live attenuated Chikungunya
virus.
21. The recombinant virus of claim 20, further comprising a genome encoding at least
additional attenuating mutation.
22. The recombinant virus of claim 20, wherein the virus is adapted for growth insect
cells.
23. An immunogenic composition comprising a recombinant virus of claim 20 in a
pharmaceutically acceptable carrier.
24. The immunogenic composition of claim 23, further comprising an adjuvant, a
preservative or a stabilizer.
25. A composition for use in preventing the symptoms of a Chikungunya virus infection,
said composition comprising a recombinant virus of claim 20 in a pharmaceutically
acceptable carrier.
26. The composition of claim 25, formulated for administration by injection.
27. The composition of claim 26, formulated for intramuscular or subcutaneous injection.
28. The composition of claim 25, further defined as a vaccine.
29. The use of a recombinant virus in accordance with claim 20 in the preparation of a
medicament.
30. The use of claim 28, wherein the medicament is for use in preventing the symptoms
of a Chikungunya virus infection.
31. The use of claim 28, wherein the medicament is formulated for administration by
injection.
32. The use of claim 28, wherein the medicament is formulated for intramuscular or
subcutaneous injection.