Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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FELINE LEUKEMIA VIRUS VACCINE
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. 119(e) of provisional
applications U.S. Serial No. 62/582,050 filed November 6, 2017, U.S. Serial
No.
62/596,508 filed December 8, 2017, and U.S. Serial No. 62/599,401 filed
December
15, 2017, the content of all of which are hereby incorporated by reference in
their
entireties.
FIELD OF THE INVENTION
The present invention relates to new vaccines for feline leukemia virus.
Methods of making and using the vaccine alone or in combinations with other
protective agents are also provided.
BACKGROUND
Feline leukemia virus (FeLV) is a retrovirus that infects domestic cats,
resulting in significant morbidity and mortality worldwide. Though
predominantly
transmitted through saliva, FeLV also has been reported to spread through
contact
with body fluids [Pacitti et al., Vet Rec 118:381-384 (1986)
doi:10.1136/vr.118.14.381; Levy et al., J Feline Med Surg 10:300-316 (2008)
doi:10.1016/j.jfms.2008.03.002]. The clinical signs in cats observed during
FeLV
infections include: cytoproliferative disorders (lymphoid or myeloid tumors),
cytosuppressive disorders (infectious diseases associated with
immunosuppression,
anemia, myelosuppression), inflammatory disorders, neurological disorders,
abortions, and enteritis [Hoover et al., J Am Vet Med Assoc 199:1287-1297
(1991);
Levy and Crawford, Textbook of Veterinary Internal Medicine, 6th ed (Ettinger
SJ,
Feldman EC., eds.) WB Saunders, Philadelphia, PA. (2005)]. The prevalence of
antigenemia may vary from 1-5% in healthy cats to 15-30% in afflicted cats
[Hosie et
al. Veterinary Records, 128: 293-297 (1989); Braley, Feline Practice 22: 25-29
(1994); Malik et al., Australian Veterinary Journal 75:323-327 (1997); Arjona
et al.,
Journal of Clinical Microbiology 38:3448-3449 (2000)]. FeLV frequently
establishes
a lasting infection with a concomitant persistent viremia, often leading to
the death
of the host cat.
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The RNA genome of FeLV encodes only three genes: (i) an ENV gene, which
encodes the envelope glycoprotein, (ii) a GAG gene, which encodes the major
structural components of the virus, and (iii) a POL gene, which encodes the
RNA
polymerase [Thomsen et al., Journal of General Virology 73:1819-1824 (1992)].
The FeLV envelope (ENV) gene encodes a gp85 precursor protein which is
proteolytically processed by one or more cellular enzymes to yield the major
envelope glycoprotein gp70 and the associated transmembrane protein p15E
[DeNoronha, et al., Virology 85:617-621 (1978); Nunberg et al., PNAS 81:3675-
3679 (1983)]. The transmembrane protein p15E contains a sequence conserved
among gammaretroviruses with immunosuppressive properties [Mathes et al.,
Nature 274:687-689 (1978)]. Recently, The European Medicines Agency's
Committee for Medicinal Products for Veterinary Use (CVMP) has adopted a
positive opinion for a vaccine comprising a recombinant p45 FeLV-envelope
antigen
derived from the gp70 surface glycoprotein of the FeLV subgroup A that is
expressed in Escherichia coli as active substance. The FeLV envelope
glycoprotein
is the target of FeLV-specific cytotoxic T cell responses, as well as
neutralizing
antibodies and accordingly, one of the major immunogens of FeLV [Flynn et al.,
J.
Virol. 76(5): 2306-2315 (2002)].
A variety of factors, such as the immune status of the host, the age of the
host, the infecting FeLV strain, the viral load, as well as the route of the
exposure of
the FeLV can all affect the ultimate outcome of that exposure. At one time
veterinarians and researchers classified FeLV infections in relation to the
relative
persistence of the concomitant antigenemia, which in the more fortunate cases
were
a transient antigenemia and/or the elimination of infection. Assays for
antigenemia
include p27 enzyme-linked immunosorbent assay (ELISA), virus isolation, and
immunofluorescence assays [Hoover et al., J Am Vet Med Assoc 199:1287-1297
(1991); Rojko and Kociba, J Am Vet Med Assoc 199:1305-1310 (1991)]. Such tests
remain helpful in clinical applications, as well as for determining whether
the clinical
disease is a result of an actively circulating virus.
Four vaccines for FeLV are currently available in the United States, including
two whole-virus adjuvanted killed vaccines; a dual-adjuvanted, multiple-
antigen
vaccine; and a nonadjuvanted, canarypox virus-vectored vaccine. Notably,
different
vaccines have been shown to have various degrees of efficacy [Sparkes, J Small
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Anim Pract 38:187-194 (1997) doi:10.1111/j.1748-5827.1997.tb03339.x.]. Earlier
studies demonstrated the efficacy of whole-virus adjuvanted killed vaccines
after
challenge, including testing vaccinated and unvaccinated cats for viral RNA,
proviral
DNA, FeLV antibodies, and the p27 antigen [Hines et al., J Am Vet Med
Assoc 199:1428-1430 (1991); Pedersen, J Vet Intern Med 7:34-39 (1993)
doi:10.1111/j.1939-1676.1993.tb03166.x.]; Torres et al., Vet Immunol
lmmunopathol 134:122-131(2010) doi:10.1016/j.vetimm.2009.10.017]. There are
limited data evaluating the efficacy of the nonadjuvanted recombinant FeLV
vaccine
available for use [Stuke et al., Vaccine 32:2599-2603 (2014)
doi:10.1016/j.vaccine.2014.03.016].
More recently, the efficacy of two commercially available feline leukemia
vaccines, one an inactivated whole-virus vaccine and the other a live
canarypox
virus-vectored vaccine have been compared following a challenge with virulent
feline leukemia virus [Patel et al., Clinical and Vaccine Immunology 22(7):798-
805
(2015)]. In this study the whole-virus adjuvanted killed vaccine was again
found to
provide superior protection against FeLV infection. However, the use of whole-
virus
killed adjuvanted FeLV vaccines has been implicated as one factor that leads
to the
development of feline injection-site sarcomas [Kass et al., J. AM Vet Med
Assoc 203
(3): 396-405 (1993)]. Although subsequent studies have been unable to
establish a
direct link between killed adjuvanted vaccines and feline injection-site
sarcomas, a
perception remains that nonadjuvanted vaccines are safer. Indeed, the American
Association of Feline Practitioners Feline Vaccination Guidelines suggest the
use of
nonadjuvanted FeLV vaccines to lower the risk of feline injection-site
sarcomas and
to reduce local inflammation [AAFP Feline Advisory Panel, 15: 785-808 (2013)].
A number of vector strategies have been employed through the years for
vaccines in an effort to protect against certain pathogens. One such vector
strategy
includes the use of alphavirus-derived replicon RNA particles (RP) [Vander
Veen, et
al. Anim Health Res Rev. 13(1):1-9. (2012) doi: 10.1017/S1466252312000011;
Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727 (2010)] which have been
developed
from several different alphaviruses, including Venezuelan equine encephalitis
virus
(VEE) [Pushko et al., Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek
et
al., Journal of Virology 67:6439-6446 (1993)], and Semliki Forest virus (SFV)
[Liljestrom and Garoff, Biotechnology (NY) 9:1356-1361 (1991)]. RP vaccines
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deliver propagation-defective alphavirus RNA replicons into host cells and
result in
the expression of the desired antigenic transgene(s) in vivo [Pushko et al.,
Virology
239(2):389-401 (1997)]. RPs have an attractive safety and efficacy profile
when
compared to some traditional vaccine formulations [Vander Veen, et al. Anim
Health
Res Rev. 13(1):1-9. (2012) ]. The RP platform has been used to encode
pathogenic
antigens and is the basis for several USDA-licensed vaccines for swine and
poultry.
Unfortunately heretofore, pet owners were forced to choose between
(i) nonadjuvanted FeLV vaccines that were believed to be safer, but found to
be
significantly less efficacious than killed, adjuvanted vaccines [see, Stuke et
al.,
Vaccine 32: 2599-2603 (2014); Patel et al., Clin Vaccine Immunol 22 (7):798-
808
(2015)] and (ii) the adjuvanted FeLV vaccines, which though more efficacious,
are
perceived by some to lead to injection-site sarcomas. Accordingly, there
remains a
present need for an improved, safe nonadjuvanted FeLV vaccine, which while not
inducing feline injection-site sarcomas still protects vaccinates from the
debilitating
disease state caused by FeLV infection as efficaciously as its inactivated
whole-
virus adjuvanted vaccine counterpart.
The citation of any reference herein should not be construed as an admission
that such reference is available as "prior art" to the instant application.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides vectors that encode one or more
feline leukemia virus (FeLV) antigens. Such vectors can be used in immunogenic
compositions comprising these vectors. The immunogenic compositions of the
present invention may be used in vaccines. In one aspect of the present
invention,
a vaccine protects the vaccinated subject (e.g., mammal) against FeLV. In a
particular embodiment of this type, the vaccinated subject is a feline. In a
more
particular embodiment, the vaccinated subject is a domestic cat. The present
invention further provides combination vaccines for eliciting protective
immunity
against FeLV and other diseases, e.g., other feline infectious diseases.
Methods of
making and using the immunogenic compositions and vaccines of the present
invention are also provided.
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In specific embodiments, the vector is an alphavirus RNA replicon particle
that encodes one or more antigens that originate from a feline pathogen. In
particular embodiments, the feline pathogen is FeLV. In more particular
embodiments, the alphavirus RNA replicon particles encode a FeLV glycoprotein
5 (gp85). In related embodiments, the alphavirus RNA replicon particles
encode an
antigenic fragment of gp85. In a particular embodiment of this type, the
antigenic
fragment of gp85 is the FeLV glycoprotein gp70. In certain related
embodiments,
the antigenic fragment of gp85 is the FeLV glycoprotein gp45. In still more
particular embodiments, the alphavirus RNA replicon particle is a Venezuelan
Equine Encephalitis (VEE) alphavirus RNA replicon particle. In yet more
specific
embodiments the VEE alphavirus RNA replicon particle is a TC-83 VEE alphavirus
RNA replicon particle. In other embodiments, the alphavirus RNA replicon
particle
is a Sindbis (SIN) alphavirus RNA replicon particle. In still other
embodiments, the
alphavirus RNA replicon particle is a Semliki Forest virus (SFV) alphavirus
RNA
replicon particle. In an alternative embodiment a naked DNA vector comprises a
nucleic acid construct that encodes one or more antigens that originate from a
feline
pathogen. In particular embodiments of this type, the naked DNA vectors
comprise
a nucleic acid construct that encodes a FeLV gp85, or antigenic fragment
thereof.
In certain embodiments an alphavirus RNA replicon particle of the present
invention encodes one or more FeLV antigens or antigenic fragments thereof. In
particular embodiments of this type, the alphavirus RNA replicon particles
encode
two to four FeLV antigens or antigenic fragments thereof. In other
embodiments,
immunogenic compositions comprise alphavirus RNA replicon particles that
encode
one or more FeLV antigens or antigenic fragments thereof. In related
embodiments,
the immunogenic compositions comprise alphavirus RNA replicon particles
encodes
two to four FeLV antigens or antigenic fragments thereof. In particular
embodiments
of this type, the alphavirus RNA replicon particles encode an FeLV
glycoprotein
(gp85) or an antigenic fragment thereof. In more particular embodiments of
this
type, the antigenic fragment of gp85 is the FeLV glycoprotein gp70. In certain
related embodiments, the antigenic fragment of gp85 is the FeLV glycoprotein
gp45.
In more particular embodiments, the immunogenic composition comprises
alphavirus RNA replicon particles that are Venezuelan Equine Encephalitis
(VEE)
alphavirus RNA replicon particles. In yet more specific embodiments the VEE
alphavirus RNA replicon particles are TC-83 VEE alphavirus RNA replicon
particles.
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In still other embodiments, the immunogenic composition comprises two or
more sets of alphavirus RNA replicon particles. In particular embodiments of
this
type, one set of alphavirus RNA replicon particles encodes a particular
antigen,
whereas the other set of alphavirus RNA replicon particles encodes a second
antigen. In a specific embodiment of this type the first set of alphavirus RNA
replicon particles encode the FeLV antigen or an antigenic fragment thereof,
and the
second set of alphavirus RNA replicon particles encode a feline calicivirus
(FCV)
antigen or an antigenic fragment thereof. In certain embodiments of this type,
the
FCV antigen originates from a virulent systemic feline calicivirus (VS-FCV)
isolate.
In other embodiments the FCV antigen originates from a classical (F9-like)
feline
calicivirus isolate. In yet other embodiments, the second set of alphavirus
RNA
replicon particles encode two FCV antigens, one of which originates from a
virulent
systemic feline calicivirus isolate, whereas the other originates from a
classical (F9-
like) feline calicivirus isolate.
In yet other embodiments, the immunogenic composition comprises one set
of alphavirus RNA replicon particles that encode a first antigen, another set
of
alphavirus RNA replicon particles that encode a second antigen, and a third
set of
alphavirus RNA replicon particles that encode a third antigen. In a particular
embodiment of this type, the first set of alphavirus RNA replicon particles
encode
the FeLV antigen or an antigenic fragment thereof, the second set of
alphavirus
RNA replicon particles encode a feline calicivirus (FCV) antigen which
originates
from a virulent systemic feline calicivirus or an antigenic fragment thereof,
and the
third set of alphavirus RNA replicon particles encode a feline calicivirus
(FCV)
antigen which originates from a classical (F9-like) feline calicivirus or an
antigenic
fragment thereof.
Accordingly, in particular embodiments in which the immunogenic
compositions comprise multiple sets (e.g., 2-10) of alphavirus RNA replicon
particles, in which the first set of alphavirus RNA replicon particles encode
the FeLV
antigen or an antigenic fragment thereof, the one or more other sets of
alphavirus
RNA replicon particles can encode one or more non-FeLV antigens. In certain
embodiments of this type, the non-FeLV antigen is a protein antigen that
originates
from feline herpesvirus (FHV). In other embodiments, the non-FeLV antigen is a
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protein antigen that originates from feline calicivirus (FCV). In yet other
embodiments, the non-FeLV antigen is a protein antigen that originates from
feline
pneumovirus (FPN). In still other embodiments, the non-FeLV antigen is a
protein
antigen that originates from feline parvovirus (FPV). In yet other
embodiments, the
non-FeLV antigen is a protein antigen that originates from feline infectious
peritonitis
virus (FIPV). In still other embodiments, the non-FeLV antigen is a protein
antigen
that originates from feline immunodeficiency virus. In still other
embodiments, the
non-FeLV antigen is a protein antigen that originates from borna disease virus
(BDV). In yet other embodiments, the non-FeLV antigen is a protein antigen
that
originates from feline influenza virus. In still other embodiments, the non-
FeLV
antigen is a protein antigen that originates from feline panleukopenia virus
(FPLV).
In yet other embodiments the non-FeLV antigen is a protein antigen that
originates
from feline coronavirus (FCoV). In still other embodiments the non-FeLV
antigen is
a protein antigen that originates from feline rhinotracheitis virus (FVR). In
yet other
embodiments the non-FeLV antigen is a protein antigen that originates from
Chlamydophila felis.
The present invention also includes all of the nucleic acid constructs of the
present invention including synthetic messenger RNA, RNA repl icons, as well
as all
of the alphavirus RNA replicon particles of the present invention, the naked
DNA
vectors, and the immunogenic compositions and/or vaccines that comprise the
nucleic acid constructs (e.g., synthetic messenger RNA, RNA replicons), the
alphavirus RNA replicon particles, and/or the naked DNA vectors of the present
invention.
In particular embodiments, a nucleic acid construct of the present invention
encodes one or more FeLV antigens or antigenic fragments thereof. In related
embodiments of this type, the nucleic acid construct encodes two to four FeLV
antigens or antigenic fragments thereof. In other embodiments, alphavirus RNA
replicon particles comprise a nucleic acid construct that encodes one or more
FeLV
antigens or antigenic fragments thereof. In particular embodiments, alphavirus
RNA
replicon particles comprise a nucleic acid construct that encodes two to four
FeLV
antigens or antigenic fragments thereof.
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In still other embodiments, the immunogenic compositions comprise
alphavirus RNA replicon particles that comprise a nucleic acid construct that
encodes two to four FeLV antigens or antigenic fragments thereof. In
particular
embodiments of this type, the alphavirus RNA replicon particles comprise a
nucleic
acid construct encoding an FeLV glycoprotein (gp85) or an antigenic fragment
thereof. In a particular embodiment of this type, the antigenic fragment of
gp85 is
the FeLV glycoprotein gp70. In other related embodiments, the antigenic
fragment
of gp85 is the FeLV glycoprotein gp45. In more particular embodiments, the
immunogenic composition comprises alphavirus RNA replicon particles that are
Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particles. In yet
more specific embodiments the VEE alphavirus RNA replicon particles are TC-83
VEE alphavirus RNA replicon particles.
In yet other embodiments, the immunogenic composition comprises two or
more sets of alphavirus RNA replicon particles. In particular embodiments of
this
type, one set of alphavirus RNA replicon particles comprises a first nucleic
acid
construct, whereas the other set of alphavirus RNA replicon particles comprise
a
second nucleic acid construct. In a specific embodiment of this type the first
nucleic
acid construct encodes the FeLV antigen or an antigenic fragment thereof, and
the
second nucleic acid construct encodes a feline calicivirus (FCV) antigen or an
antigenic fragment thereof. In certain embodiments of this type, the FCV
antigen
originates from a virulent systemic feline calicivirus (VS-FCV) isolate. In
other
embodiments the FCV antigen originates from a classical (F9-like) feline
calicivirus
isolate. In yet other embodiments, the second nucleic acid construct encodes
two
FCV antigens, one of which originates from a virulent systemic feline
calicivirus
isolate, whereas the other originates from a classical (F9-like) feline
calicivirus
isolate.
In still other embodiments, the immunogenic composition comprises one set
of alphavirus RNA replicon particles that comprise a first nucleic acid
construct,
another set of alphavirus RNA replicon particles that comprise a second
nucleic acid
construct, and a third set of alphavirus RNA replicon particles that comprise
a third
nucleic acid construct. In a particular embodiment of this type, the first
nucleic acid
construct encodes the FeLV antigen or an antigenic fragment thereof, the
second
nucleic acid construct encodes a feline calicivirus (FCV) antigen which
originates
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from a virulent systemic feline calicivirus or an antigenic fragment thereof,
and the
third nucleic acid construct encodes a feline calicivirus (FCV) antigen which
originates from a classical (F9-like) feline calicivirus or an antigenic
fragment
thereof.
In yet other embodiments, the immunogenic composition comprises one set
of alphavirus RNA replicon particles that comprise a first nucleic acid
construct,
another set of alphavirus RNA replicon particles that comprise a second
nucleic acid
construct, a third set of alphavirus RNA replicon particles that comprise a
third
nucleic acid construct, and a fourth set of alphavirus RNA replicon particles
that
comprise a fourth nucleic acid construct. In yet other embodiments, the
immunogenic composition comprises a set of alphavirus RNA replicon particles
that
comprise a first nucleic acid construct, another set of alphavirus RNA
replicon
particles that comprise a second nucleic acid construct, a third set of
alphavirus
RNA replicon particles that comprise a third nucleic acid construct, a fourth
set of
alphavirus RNA replicon particles that comprise a fourth nucleic acid
construct ,and
a fifth set of alphavirus RNA replicon particles that comprise a fifth nucleic
acid
construct. In such embodiments, the nucleotide sequences of the first nucleic
acid
construct, the second nucleic acid construct, third nucleic acid construct,
the fourth
nucleic acid construct, and the fifth nucleic acid construct are all
different.
Accordingly, an immunogenic composition of the present invention can
contain alphavirus RNA replicon particles that comprise a nucleic acid
construct that
encodes at least one non-FeLV antigen for eliciting protective immunity to a
non-
FeLV pathogen. In particular embodiments of this type, the non-FeLV antigen is
a
protein antigen that originates from feline herpesvirus (FHV). In other
embodiments,
the non-FeLV antigen is a protein antigen that originates from feline
calicivirus
(FCV). In yet other embodiments, the non-FeLV antigen is a protein antigen
that
originates from feline pneumovirus (FPN). In still other embodiments, the non-
FeLV
antigen is a protein antigen that originates from feline parvovirus (FPV). In
yet other
embodiments, the non-FeLV antigen is a protein antigen that originates from
feline
infectious peritonitis virus (FIPV). In still other embodiments, the non-FeLV
antigen
is a protein antigen that originates from feline immunodeficiency virus. In
still other
embodiments, the non-FeLV antigen is a protein antigen that originates from
borna
disease virus (BDV). In yet other embodiments, the non-FeLV antigen is a
protein
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antigen that originates from feline influenza virus. In still other
embodiments, the
non-FeLV antigen is a protein antigen that originates from feline
panleukopenia
virus (FPLV). In yet other embodiments the non-FeLV antigen is a protein
antigen
that originates from feline coronavirus (FCoV). In still other embodiments the
non-
5 FeLV antigen is a protein antigen that originates from feline
rhinotracheitis virus
(FVR). In still other embodiments the non-FeLV antigen is a protein antigen
that
originates from Chlamydophila felis.
The present invention further provides combination immunogenic compositions
10 and/or vaccines (multivalent vaccines) that include alphavirus RNA
replicon particles
that encode an antigen or antigenic fragment thereof originating from FeLV
together
with one or more modified live (e.g., attenuated) or killed feline pathogens.
In
particular embodiments, the immunogenic compositions comprise a modified live
or
killed Chlamydophila felis combined with alphavirus RNA replicon particles
that
encode an antigen or antigenic fragment thereof originating from FeLV. In
other
embodiments, the immunogenic compositions comprise a modified live or killed
feline rhinotracheitis Virus (FVR) combined with alphavirus RNA replicon
particles
that encode an antigen or antigenic fragment thereof originating from FeLV. In
still
other embodiments, the immunogenic compositions comprise a modified live or
killed feline calicivirus (FCV) combined with alphavirus RNA replicon
particles that
encode an antigen or antigenic fragment thereof originating from FeLV. In yet
other
embodiments, the immunogenic compositions comprise a modified live or killed
feline panleukopenia virus (FPL) combined with alphavirus RNA replicon
particles
that encode an antigen or antigenic fragment thereof originating from FeLV. In
still
other embodiments, the immunogenic compositions comprise a modified live or
killed Chlamydophila felis, a modified live or killed FVR, a modified live or
killed
FCV, a modified live or killed FPL, and alphavirus RNA replicon particles that
encode an antigen or antigenic fragment thereof originating from FeLV. In
particular
embodiments of this type, the feline antigen of the FeLV is the FeLV viral
glycoprotein (gp85). In certain embodiments, vaccines comprise an
immunologically effective amount of one or more of these immunogenic
compositions.
In particular embodiments the FeLV antigen is the FeLV glycoprotein (gp85).
In specific embodiments of this type, the FeLV glycoprotein gp85 comprises an
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amino acid sequence comprising 95% identity or more with the amino acid
sequence of SEQ ID NO: 2. In more specific embodiments of this type, the FeLV
glycoprotein (gp85) comprises the amino acid sequence of SEQ ID NO: 2. In even
more specific embodiments of this type the FeLV glycoprotein (gp85) is encoded
by
the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 10. In related
embodiments, the FeLV glycoprotein gp70 comprises an amino acid sequence
comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 4.
In
more specific embodiments of this type, the FeLV glycoprotein (gp85) comprises
the
amino acid sequence of SEQ ID NO: 4. In even more specific embodiments of this
type the FeLV glycoprotein (gp70) is encoded by the nucleotide sequence of SEQ
ID NO: 3 or SEQ ID NO: 11.
The present invention further comprises vaccines and multivalent vaccines
comprising the immunogenic compositions of the present invention. In
particular
embodiments, the vaccines are nonadjuvanted vaccine. In certain embodiments,
the
vaccine aids in the prevention of disease due to FeLV. In related embodiments,
antibodies are induced in a feline subject when the feline is immunized with
the
vaccine.
The present invention also provides methods of immunizing a feline against a
feline pathogen, e.g., FeLV, comprising administering to the feline an
immunologically
effective amount of a vaccine or multivalent of the present invention. In
particular
embodiments the vaccine is administered via intramuscular injection. In
alternative
embodiments the vaccine is administered via subcutaneous injection. In other
embodiments the vaccine is administered via intravenous injection. In still
other
embodiments the vaccine is administered via intradermal injection. In yet
other
embodiments the vaccine is administered via oral administration. In still
other
embodiments the vaccine is administered via intranasal administration. In
specific
embodiments, the feline is a domestic cat.
The vaccines and multivalent vaccines of the present invention can be
administered as a primer vaccine and/or as a booster vaccine. In specific
embodiments, a vaccine of the present invention is administered as a one shot
vaccine
(one dose), without requiring subsequent administrations. In certain
embodiments, in
the case of the administration of both a primer vaccine and a booster vaccine,
the
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primer vaccine and the booster vaccine can be administered by the identical
route. In
certain embodiments of this type, the primer vaccine and the booster vaccine
are both
administered by subcutaneous injection. In alternative embodiments, in the
case of the
administration of both a primer vaccine and a booster vaccine, the
administration of the
primer vaccine can be performed by one route and the booster vaccine by
another
route. In certain embodiments of this type, the primer vaccine can be
administered by
subcutaneous injection and the booster vaccine can be administered orally.
The invention further provides for a method of immunizing a feline against
FeLV comprising injecting the feline with an immunologically effective amount
of the
above described inventive vaccines. In particular embodiments the vaccines can
include from about 1 x 104 to about 1 x 1019 RPs or higher, for example. In
more
particular embodiments the vaccines can include from about 1 x 105 to about 1
x 109
RPs. In even more particular embodiments the vaccines can include from about 1
x
106 to about 1 x 108 RPs. In particular embodiments the feline is a domestic
cat.
In particular embodiments the vaccines of the present invention are
administered in 0.05 mL to 3 mL doses. In more particular embodiments the dose
administered is 0.1 mL to 2 mLs. In still more particular embodiments the dose
administered is 0.2 mL to 1.5 mLs. In even more particular embodiments the
dose
administered is 0.3 to 1.0 mLs. In still more particular embodiments the dose
administered is 0.4 mL to 0.8 mLs. In yet more particular embodiments the dose
administered is 0.5 mL to 1.5 mLs.
These and other aspects of the present invention will be better appreciated
by reference to the following Detailed Description.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an improved, safe nonadjuvanted FeLV
vaccine. In one aspect, the vaccines of the present invention do not induce
feline
injection-site sarcomas, yet still provide protection to the vaccinates from
the
debilitating disease state caused by FeLV infection as efficaciously as an
inactivated
whole-virus adjuvanted vaccine.
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Accordingly, the vaccine compositions of the present invention include an
immunologically effective amount of a vector encoding an antigen from one or
more
strains of feline leukemia virus that aids in eliciting protective immunity in
the
recipient vaccinated animal. Furthermore, the present invention provides new
immunologic compositions to improve the reliability of vaccination to aid in
the
reduction of antigenemia in a feline infected by FeLV and to thereby yield a
transient
antigenemia and/or lead to the elimination of the infection. In a particular
aspect of
the present invention, the vaccines comprise an alphavirus RNA replicon
particle
(RP) encoding the FeLV viral glycoprotein (gp85). In more specific
embodiments,
the vaccines comprise alphavirus RNA replicon particles (RPs) that comprise
the
capsid protein and glycoproteins of Venezuelan Equine Encephalitis Virus (VEE)
and encode the FeLV viral glycoprotein (gp85) and/or an antigenic fragment
thereof
(e.g., gp70 or gp45). In even more specific embodiments, the vaccines comprise
alphavirus RNA replicon particles (RPs) that comprise the capsid protein and
glycoproteins of the avirulent TC-83 strain of VEE and encode the FeLV viral
glycoprotein (gp85) and/or an antigenic fragment thereof (e.g., gp70 or gp45).
In another aspect of the present invention, the vaccines comprise naked DNA
vectors that encode the FeLV viral glycoprotein (gp85) and/or an antigenic
fragment
thereof (e.g., gp70 or gp45). The vaccines of the present invention can be
administered to a feline in the absence of an adjuvant and still effectively
aid in the
protection of the vaccinated feline against FeLV.
In order to more fully appreciate the invention, the following definitions are
provided.
The use of singular terms for convenience in description is in no way
intended to be so limiting. Thus, for example, reference to a composition
comprising "a polypeptide" includes reference to one or more of such
polypeptides.
In addition, reference to an "alphavirus RNA replicon particle" includes
reference to
a plurality of such alphavirus RNA replicon particles, unless otherwise
indicated.
As used herein the term "approximately" is used interchangeably with the
term "about" and signifies that a value is within fifty percent of the
indicated value
i.e., a composition containing "approximately" 1 x 108 alphavirus RNA replicon
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14
particles per milliliter contains from 0.5 x 108 to 1.5 x 108 alphavirus RNA
replicon
particles per milliliter.
As used herein, the term "feline" refers to any member of the Felidae family.
Domestic cats, pure-bred and/or mongrel companion cats, and wild or feral cats
are
all felines.
As used herein, the term "replicon" refers to a modified RNA viral genome
that lacks one or more elements (e.g., coding sequences for structural
proteins) that
if they were present, would enable the successful propagation of the parental
virus
in cell cultures or animal hosts. In suitable cellular contexts, the replicon
will amplify
itself and may produce one or more sub-genomic RNA species.
As used herein, the term "alphavirus RNA replicon particle", abbreviated
"RP", is an alphavirus-derived RNA replicon packaged in structural proteins,
e.g.,
the capsid and glycoproteins, which also are derived from an alphavirus, e.g.,
as
described by Pushko et al., [Virology 239(2):389-401 (1997)]. An RP cannot
propagate in cell cultures or animal hosts (without a helper plasmid or
analogous
component), because the replicon does not encode the alphavirus structural
components (e.g., capsid and glycoproteins).
The term "non-FeLV", is used to modify terms such as pathogen, and/or
antigen (or immunogen) to signify that the respective pathogen, and/or antigen
(or
immunogen) is neither an FeLV pathogen nor a FeLV antigen (or immunogen) and
that a non-FeLV protein antigen (or immunogen) does not originate from an
FeLV.
The terms "originate from", "originates from" and "originating from" are used
interchangeably with respect to a given protein antigen and the pathogen or
strain of
that pathogen that naturally encodes it, and as used herein signify that the
unmodified and/or truncated amino acid sequence of that given protein antigen
is
encoded by that pathogen or strain of that pathogen. The coding sequence
within a
nucleic acid construct of the present invention for a protein antigen
originating from
a pathogen may have been genetically manipulated so as to result in a
modification
and/or truncation of the amino acid sequence of the expressed protein antigen
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relative to the corresponding sequence of that protein antigen in the pathogen
or
strain of pathogen (including naturally attenuated strains) it originates
from.
As used herein, the terms "protecting", or "providing protection to", or
5 "eliciting protective immunity to", "aids in prevention of disease", and
"aids in the
protection" do not require complete protection from any indication of
infection. For
example, "aids in the protection" can mean that the protection is sufficient
such that,
after challenge, symptoms of the underlying infection are at least reduced,
and/or
that one or more of the underlying cellular, physiological, or biochemical
causes or
10 mechanisms causing the symptoms are reduced and/or eliminated. It is
understood
that "reduced," as used in this context, means relative to the state of the
infection,
including the molecular state of the infection, not just the physiological
state of the
infection.
15 As used herein, a "vaccine" is a composition that is suitable for
application to
an animal, e.g., feline (including, in certain embodiments, humans, while in
other
embodiments being specifically not for humans) comprising one or more antigens
typically combined with a pharmaceutically acceptable carrier such as a liquid
containing water, which upon administration to the animal induces an immune
response strong enough to minimally aid in the protection from a disease
arising
from an infection with a wild-type micro-organism, i.e., strong enough for
aiding in
the prevention of the disease, and/or preventing, ameliorating or curing the
disease.
As used herein, a multivalent vaccine is a vaccine that comprises two or
more different antigens. In a particular embodiment of this type, the
multivalent
vaccine stimulates the immune system of the recipient against two or more
different
pathogens.
The terms "adjuvant" and "immune stimulant" are used interchangeably
herein, and are defined as one or more substances that cause stimulation of
the
immune system. In this context, an adjuvant is used to enhance an immune
response to one or more vaccine antigens/isolates. Accordingly, "adjuvants"
are
agents that nonspecifically increase an immune response to a particular
antigen,
thus reducing the quantity of antigen necessary in any given vaccine, and/or
the
frequency of injection necessary in order to generate an adequate immune
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response to the antigen of interest. In this context, an adjuvant is used to
enhance
an immune response to one or more vaccine antigens/isolates. The American
Association of Feline Practitioners Feline Vaccination Guidelines suggest the
use of
nonadjuvanted FeLV vaccines [AAFP Feline Advisory Panel, 15: 785-808 (2013)].
As used herein, a "nonadjuvanted vaccine" is a vaccine or a multivalent
vaccine that does not contain an adjuvant.
As used herein, the term "pharmaceutically acceptable" is used adjectivally to
mean that the modified noun is appropriate for use in a pharmaceutical
product.
When it is used, for example, to describe an excipient in a pharmaceutical
vaccine,
it characterizes the excipient as being compatible with the other ingredients
of the
composition and not disadvantageously deleterious to the intended recipient
animal,
e.g., feline.
Parenteral administration" includes subcutaneous injections, submucosal
injections, intravenous injections, intramuscular injections, intradermal
injections,
and infusion.
As used herein the term "antigenic fragment" in regard to a particular protein
(e.g., a protein antigen) is a fragment of that protein that is antigenic,
i.e., capable of
specifically interacting with an antigen recognition molecule of the immune
system,
such as an immunoglobulin (antibody) or T cell antigen receptor. For example,
an
antigenic fragment of an FeLV viral glycoprotein (gp85) is a fragment of the
gp85
protein that is antigenic. Preferably, an antigenic fragment of the present
invention
is immunodominant for antibody and/or T cell receptor recognition. In
particular
embodiments, an antigenic fragment with respect to a given protein antigen is
a
fragment of that protein that retains at least 25% of the antigenicity of the
full length
protein. In preferred embodiments an antigenic fragment retains at least 50%
of the
antigen icity of the full length protein. In more preferred embodiments, an
antigenic
fragment retains at least 75% of the antigen icity of the full length protein.
Antigenic
fragments can be as small as 20 amino acids or at the other extreme, be large
fragments that are missing as little as a single amino acid from the full-
length
protein. In particular embodiments the antigenic fragment comprises 25 to 150
amino acid residues. In other embodiments, the antigenic fragment comprises 50
to
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17
250 amino acid residues. The gp45 glycoprotein and the gp70 glycoprotein are
antigenic fragments of the gp85 glycoprotein.
As used herein one amino acid sequence is 100% "identical" or has 100%
"identity" to a second amino acid sequence when the amino acid residues of
both
sequences are identical. Accordingly, an amino acid sequence is 50%
"identical" to
a second amino acid sequence when 50% of the amino acid residues of the two
amino acid sequences are identical. The sequence comparison is performed over
a
contiguous block of amino acid residues comprised by a given protein, e.g., a
protein, or a portion of the polypeptide being compared. In a particular
embodiment,
selected deletions or insertions that could otherwise alter the correspondence
between the two amino acid sequences are taken into account.
As used herein, nucleotide and amino acid sequence percent identity can be
determined using C, MacVector (MacVector, Inc. Cary, NC 27519), Vector NTI
(Informax, Inc. MD), Oxford Molecular Group PLC (1996) and the Clustal W
algorithm with the alignment default parameters, and default parameters for
identity.
These commercially available programs can also be used to determine sequence
similarity using the same or analogous default parameters. Alternatively, an
Advanced Blast search under the default filter conditions can be used, e.g.,
using
the GCG (Genetics Computer Group, Program Manual for the GCG Package,
Version 7, Madison, Wisconsin) pileup program using the default parameters.
As used herein, the term "inactivated" microorganism is used interchangeably
with the term "killed" microorganism. For the purposes of this invention, an
"inactivated" microorganism is an organism which is capable of eliciting an
immune
response in an animal, but is not capable of infecting the animal. An antigen
of the
present invention (e.g., an inactivated feline panleukopenia virus) may be
inactivated by an agent selected from the group consisting of binary ethyl
eneimine,
formalin, beta-propiolactone, thimerosal, or heat. In a particular embodiment,
inactivated feline calicivirus isolates combined with an RP of the present
invention
are inactivated by binary ethyleneimine.
The alphavirus RNA replicon particles of the present invention may be
lyophilized and rehydrated with a sterile water diluent. On the other hand,
when the
alphavirus RNA replicon particles are stored separately, but intend to be
mixed with
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18
other vaccine components prior to administration, the alphavirus RNA replicon
particles can be stored in the stabilizing solution of those components, e.g.,
a high
sucrose solution.
A vaccine of the present invention can be readily administered by any
standard route including intravenous, intramuscular, subcutaneous, oral,
intranasal,
intradermal, and/or intraperitoneal vaccination. The skilled artisan will
appreciate
that the vaccine composition is preferably formulated appropriately for each
type of
recipient animal and route of administration.
Thus, the present invention also provides methods of immunizing a feline
against FeLV and/or other feline pathogens. One such method comprises
injecting
a feline with an immunologically effective amount of a vaccine of the present
invention, so that the feline produces appropriate FeLV antibodies.
Multivalent Vaccines:
The present invention also provides multivalent vaccines. For example, the
coding sequence of a protein antigen or antigenic fragment thereof, or
combination
of such coding sequences of protein antigens useful in a feline vaccine can be
added to an alphavirus RNA replicon particle (RP) or combined in the same RP
as
one that encodes a feline antigen of the FeLV [e.g., the FeLV viral
glycoprotein
(gp85)] in the vaccine. Accordingly, such multivalent vaccines are included in
the
present invention.
Examples of pathogens that one or more of such protein antigens can
originate from include feline rhinotracheitis Virus (FVR), feline calicivirus
(FCV),
feline panleukopenia Virus (FPL) feline herpesvirus (FHV), other FeLV strains,
feline parvovirus (FPV), feline infectious peritonitis virus (FIPV), feline
immunodeficiency virus, borna disease virus (BDV), rabies virus, feline
influenza
virus, canine influenza virus, avian influenza, canine pneumovirus, feline
pneumovirus, Chlamydophila felis (FKA Chlamydia psittaci), Bordetella
bronchiseptica, and Bartonella spp. (e.g., B. henselae). In particular
embodiments,
a coding sequence for a capsid protein or analogous protein from one or more
of
these feline or canine pathogens can be inserted into the same RP as the FeLV
antigen. Alternatively, or in combination therewith, a coding sequence for a
capsid
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19
protein or analogous protein from one or more of these feline or canine
pathogens
can be inserted into one or more other RPs, which can be combined with the RP
that encodes the FeLV antigen in a vaccine.
In addition, an alphavirus RNA replicon particle(RP) that encodes a feline
antigen of the FeLV [e.g., the FeLV viral glycoprotein (gp85)] can be added
together
with one or more other live, attenuated virus isolates such as a live
attenuated other
FCV strain, a live attenuated feline herpesvirus and/or a live attenuated
feline
parvovirus and/or a live, attenuated feline leukemia virus, and/or a live,
attenuated
feline infectious peritonitis virus and/or a live, attenuated feline
immunodeficiency
virus and/or a live, attenuated borna disease virus and/or a live, attenuated
rabies
virus, and/or a live, attenuated feline influenza virus and/or a live,
attenuated canine
influenza virus, and/or a live, attenuated avian influenza, and/or a live,
attenuated
canine pneumovirus, and/or a live, attenuated feline pneumovirus. In addition,
a
live, attenuated Chlamydophila felis, and/or a live, attenuated Bordetella
bronchiseptica and/or a live, attenuated Bartonella spp. (e.g., B. henselae)
can also
be included in such multivalent vaccines.
Furthermore, an alphavirus RNA replicon particle (RP) that encodes a feline
antigen of the FeLV [e.g., the FeLV viral glycoprotein (gp85)] can be added
together
with one or more other killed virus isolates such as a killed FCV strain,
and/or a
killed feline herpesvirus and/or a killed feline parvovirus and/or a killed
feline
leukemia virus, and/or a killed feline infectious peritonitis virus and/or a
killed feline
immunodeficiency virus and/or a killed borna disease virus and/or a killed
rabies
virus, and/or a killed feline influenza virus and/or a killed canine influenza
virus,
and/or a killed avian influenza virus, and/or a killed canine pneumovirus,
and/or a
killed feline pneumovirus. In addition, bacterins of Chlamydophila felis,
and/or
Bordetella bronchiseptica and/or Bartonella spp. (e.g., B. henselae) can also
be
included in such multivalent vaccines.
It is also to be understood that this invention is not limited to the
particular
configurations, process steps, and materials disclosed herein as such
configurations, process steps, and materials may vary somewhat.
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It is also to be understood that the terminology employed herein is used for
the purpose of describing particular embodiments only and is not intended to
be
limiting, since the scope of the present invention will be limited only by the
appended claims and equivalents thereof.
5
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21
SEQUENCE TABLE
SEQ ID NO: Description Type
1 FeLV viral glycoprotein (gp85) nucleic acid
DNA
2 FeLV viral glycoprotein (gp85) amino acid
3 FeLV viral glycoprotein (gp70) nucleic acid
DNA
4 FeLV viral glycoprotein (gp70) amino acid
Feline Calicivirus (VS-FCV) nucleic acid
DNA
6 Feline Cal icivirus (VS-FCV) amino acid
7 Feline Cal icivirus (F9-like) nucleic acid
DNA
8 Feline Cal icivirus (F9-like) amino acid
9 GGCGCGCCGCACC nucleic acid
FeLV viral glycoprotein (gp85) nucleic acid
RNA
11 FeLV viral glycoprotein (gp70) nucleic acid
RNA
12 Feline Calicivirus (VS-FCV) nucleic acid
RNA
13 Feline Calicivirus (F9-like) nucleic acid
RNA
TTAATTAA nucleic acid
SEQUENCES
Feline Leukemia Virus envelope glycoprotein (gp85) SEQ ID NO: 1
5 atggagtcaccaacacaccctaaaccttctaaagacaaaaccctctcgtggaatctcgccttccttgt
gggcatcctgttcacaatcgacatcggcatggccaacccttcgccgcatcagatctacaatgtgacat
gggtcattactaatgtgcagacaaacacccaggcaaatgctacttctatgcttggtactctgactgat
gcttatccaaccctgcacgtcgacctttgcgatctcgtcggtgacacatgggagcccatcgtgctgaa
tccaactaatgtcaaacatggtgccaggtattcttctagcaaatacgggtgtaagaccactgatcgga
10 agaaacagcaacaaacctacccattctacgtgtgcccgggtcacgcaccgtccctgggtccgaaggga
acacattgtgggggagcccaagacggtttttgcgctgcttggggttgtgaaacaaccggagaagcctg
gtggaagcctacctcatcttgggactacattactgtgaaaagaggctctagccaggataacagctgcg
aaggaaagtgtaatcccctggtgcttcaattcacccagaaaggccggcaggcatcatgggatggaccg
aaaatgtggggacttagactctatcgcaccggatacgaccccatcgctctgtttactgtgtcacgcca
agtctccaccattactccgccacaggccatggggccgaatctggtcctccccgatcagaagccaccct
cacggcaaagtcaaaccggctcaaaagtggccacccaacggccccagacaaatgagtccgcacctagg
tcagtggcacctacaacaatgggtccaaagcggatcggaaccggagacaggctcattaacctcgtgca
agggacttatctggcccttaacgctactgaccccaacaagaccaaggattgctggctctgccttgtga
gcagacctccttactatgaggggatcgccattctcggaaactactcaaatcagaccaacccccctccg
tcgtgtctgagcaccccccagcacaagcttactatttcagaagtcagtggacagggaatgtgcatcgg
aaccgtgccaaagactcatcaagccctttgcaacaaaactcaacaagggcacactggagctcattatc
tcgccgcacctaacgggacctactgggcttgcaatactggattgaccccgtgtatctctatggccgtg
ctgaattggacttccgacttctgcgtgcttattgagctttggcctagagtgacataccatcagcctga
gtacgtctatacccatttcgccaaggcagtcagattccggcgggagcctatctccctgactgtggcct
tgatgctcggtggactgacagtgggaggaattgcagctggagtcggaactggaaccaaggccctgctc
gaaactgctcagttccggcagctgcagatggccatgcacactgacatccaggctctggaggaatcaat
ttcagcccttgagaaaagcttgacctcgctgtctgaagtggtcctccaaaacaggcgcggtttggaca
tcctgttccttcaagagggtggtctgtgcgccgctctcaaggaggaatgctgtttctacgctgaccat
-2-23333.5.5-2.5-2333-ebobbqb-eppooqbbbpopppoqoTepaeb000Te33333-2-2-eppoTeb
qoaeqqoqb-eqoaeppoopbbbTepobbpoqooboobaeqq-eooppoTeqbbpobb000qbqb
bopoqq-eqqq3boTeq333-ebTeTebbpaeqboopTeqopb3bqq-ebbbbqbTeb-e-eqoaebb 09
aebbbqqaqoaEyepaelye-ebbbp-ebp000paqqbpobqqqqbbq33333-epobTepp-ebbb-eb
qbqob-eq-epaebbpoqb-eqb-ebbb-eb-epp-e-eqbpopoTeTeqopbbbTeoqoaq33-2333.5-2-2
bbqbbqq3b-e-eb-ebboopoopb-ebqbTebbbbTeoboobqbqqqqbbbTeb-epopobbbb-ebb
qbqq-eopoppbbfreppoobbbbqqbaq33333.5Teaebb0000bqoqbaeqqqq3333-2Teop
.5-20-2-20.5POPPPPPPP.EY2T25-20-2qOPPPPqbTebbT2T2-2-2-20q00q00q0Pqqb0PObbbb
gg
opopp-e-eqbTepoopp000pp-eqooqb-eq-eqoae-ebbbqoaeopb-ebbbqb-eqoaebqbTeqq.
aebqqbTeopq333-eq333-eqoobTebooppqq33-2-ebb-eqqbqpqaq33-233.53-2-eqob-epo
33-23-2-eqopppopqbTepooppTepqbbbqqae-eqbTepTeTeTeppoopopooqb-eqooTep
:ON GI CGS (0Ld6) uplaidodA16 adolanue snm epe)Inei auued
Og
Pbnnoobbon-eb0000pbaenbpob
ppnruebpobpon-enbpobponaennonn-ebn000bbpoonbbnbbonon-e-ebpaebbpponbnnnbpo
bnbbnobbpoppononnpnbnboaebbonnononnonn-ebnonnobnoonponobnopoonbbbnpon
pponnonon-ebnonaeoaennnbbnp000fyebppoppnnnbbnobbfrebonnbbnpbbbpobponono
pbonnbnoppobpobb-ebpowebnnoboppbbbobnobpppobbnpoppn-ebobobnbbnobbboop St
npoaebnobaenonnnbnobnp-ebb-ebbppononoboobobnbnonbbnbbfrefreponnoonnbnoon
Paebbnnnbbobobbpaepppoonoonbbnfrepbnonbnobonoaebnnobp-eppb-ebnn000bponn
rreponp-ebb-ebbnonobbpoonpaebnaeopobnpoobbnpfreobnobpobboonnbponobnoweb
onobn000bbppooppbbnoppbbonfrebbnobpobnnp-ebb-ebbbnbpaebnaebbnbbonobn-ebn
noobbnbnaebn000non-enoofyebbbobboonn-ebponbpobbppoobonnnpooaen-enonbaenb ot
pbnoobponpoopnpaebnfreb-enoobbnnnob-ebnn-ennobnbobnonnaeboonnaebbnnp-ebno
bnboobbnpnonon-enbnb000aebnn-ebbnaen-epobnnobbbnaenoaebbboppnoopoboobon
orrennponob-ebbnaeopobbfrepopponowepoppobnnn000bpponponaefreppoobnboopp
bborreobnbnp-ebbbpaebbnbponfrepbponnn-enaennobppopob-2333333-23.Eyebnonbnbon
boon333333-epoopbpon-epponaenowebbononnpoobon-ebbbfrebnpnaennoonoopbpob g
pbnbnnoobnonobbnobnn-ebbppoopfrepopp000aebnaenobae-enn000bbnon-ennaebbbp
pobnbonoop-ennponobbpaeb-ebbooppbbon-ebbob-eppoonbbbnppoppaenoopobbnbpon
bfrenoopoboonfrebnp-epaebp0000bbopp000poobbnfreppponobbooppponfreppobbaeo
n000poofrepbpon-eb0000noonbbnon-epboobbbbnpoobbpopooboonaennpoopoononbp
poobaeonbnbnaennnbnonobonp0000pbaen-ebboopobon-enonaeb-ennaebbbbnbnp-epp 0
boaebbnpbbbnponpobbpobboobb-eppbp000ponn-eponnobnbbn0000np-enbnfreppbbpp
bobnobpoppn-ebbpoofyenonobbpb-ewebnbnaennpaenaebbbnnonponoopnoobp-ebbnb
bnoofrepb-ebbooppowebnbnnbbbbnnobnobobnnnnnbbaebpp000fyebbbbbnbnnpopop
pbbfrepboonbbbn000nboopobaeonbbb000bnbnbaenonnpooaenoopppoppobpowebp
pbborrebnopoopfrepnbnbbbaen-eppob-enonnonn-enbbpoobnbbnpoppponbnp-enoppoon
SZ
ppbnobnbonp000fyebbbnpopaebnbbonbononpbobnnnoopbonbaeobn000ppoon-ennob
ruebnaebnonaenbbnnobnpnonnaenobn-eppobbp000popppaebpobnbnp-enaennponbbb
npaebnbnppaenon-ebponpoboobonn000ppoobbnpobbonpaebon-epoponnbnoonpobbb
nbnnoonnoobononp-ebbnbonon000pp-epaebp-e-enonnoopp-en000popoppooponfrebbnp
0 1, :ON CI 1 (:) S (g8d6) uplaidodA16 adolanue snm eparei auued OZ
*d-gadaxoxiooxoayi YIVOAAS al(MIA,10.AMIN'I I 3d 9,17I
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S.NIGOSSSIAI I AGMS S Id IMMVESLLE39MVV3,19(10V993HISIdS'ISdVH9d 3AX,Id OI
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IVNVOINIZANI IAMIANA I OHd S d NVTAIS I G I IZI I SNI,IV'INMS '-=(MIS d d HI d
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z :ON Q 1 (:) S (g8d6) uplaidodA16 adolanue snm eparei auued
Pbqq3obb3Teb0000pb3eqbpob g
ppqq-ebpobpoTeqb-eobpoqopqqoqq-ebq000bbpooqbbqbboqoqp-ebpaebb-epoqbqqqb-23
bqbbqobbpoppoqoqq-eqbqboaebboqqoqoqqoqq-ebqoqqobqooTeoqobqopooqbbbTeaq
ppoqqaqoTebqoqopoopqqqbbTe333.5-2.5-2-23-2-eqqqbbqobbb-ebaqqbbTebbbpobpoqoqo
pbaqqbqoppobpobb-ebpowebqqobae-ebbbobqob-eppobbTeoppTebobobqbbqobbboop
ZZ
1760080/810M1/13cl
9t9980/610Z OM
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1760080/810M1/13cl
9t9980/610Z OM
LZ-VO-OZOZ LZVO800 VD
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1760080/810M1/13cl
9t9980/610Z OM
LZ-VO-OZOZ LZVO800 VD
09
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1760080/810M1/13cl
9t9980/610Z OM
LZ-VO-OZOZ LZVO800 VD
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26
The following examples serve to provide further appreciation of the invention
but are
not meant in any way to restrict the effective scope of the invention.
EXAMPLES
EXAMPLE 1
INCORPORATION OF THE CODING SEQUENCES FOR FELV GP85
INTO THE ALPHAVIRUS RNA REPLICON PARTICLES
INTRODUCTION
RNA viruses have been used as vector-vehicles for introducing vaccine
antigens, which have been genetically engineered into their genomes. However,
their use to date has been limited primarily to incorporating viral antigens
into the
RNA virus and then introducing the virus into a recipient host. The result is
the
induction of protective antibodies against the incorporated viral antigens.
Alphavirus
RNA replicon particles have been used to encode pathogenic antigens. Such
alphavirus replicon platforms have been developed from several different
alphaviruses, including Venezuelan equine encephalitis virus (VEE) [Pushko et
al.,
Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek et al., Journal of
Virology
67:6439-6446 (1993) the contents of which are hereby incorporated herein in
their
entireties], and Semliki Forest virus (SFV) [Liljestrom and Garoff,
Biotechnology
(NY) 9:1356-1361 (1991), the contents of which are hereby incorporated herein
in
their entireties]. Moreover, alphavirus RNA replicon particles are the basis
for
several USDA-licensed vaccines for swine and poultry. These include: Porcine
Epidemic Diarrhea Vaccine, RNA Particle (Product Code 19U5.P1 ), Swine
Influenza Vaccine, RNA (Product Code 19A5.D0), Avian Influenza Vaccine, RNA
(Product Code 1905.D0), and Prescription Product, RNA Particle (Product Code
9PP0.00).
ALPHAVIRUS RNA REPLICON PARTICLE CONSTRUCTION
An amino acid sequence for FeLV gp85 were used to generate codon-
optimized (feline codon usage) nucleotide sequences in silico. Optimized
sequences were prepared as synthetic DNA by a commercial vendor (ATUM,
Newark, CA). Accordingly, a synthetic gene was designed based on the amino
acid
sequence of gp85. The construct (gp85_wt) was wild-type amino acid sequence
CA 03080427 2020-04-27
WO 2019/086646
PCT/EP2018/080094
27
[SEQ ID NO: 2], codon-optimized for feline, with flanking sequence appropriate
for
cloning into the alphavirus replicon plasmid.
The VEE replicon vectors designed to express FeLV gp85 were constructed
as previously described [see, U.S. 9,441,247 B2; the contents of which are
hereby
incorporated herein by reference in their entireties], with the following
modifications.
The TC-83-derived replicon vector "pVEK" [disclosed and described in
U.S. 9,441,247 B2] was digested with restriction enzymes Ascl and Pad. A DNA
plasmid containing the codon-optimized open reading frame nucleotide sequence
of
the FeLV gp85 genes, with 5' flanking sequence (5'-GGCGCGCCGCACC-3') [SEQ
ID NO: 9] and 3' flanking sequence (5'-TTAATTAA-3'), was similarly digested
with
restriction enzymes Ascl and Pad. The synthetic gene cassette was then ligated
into the digested pVEK vector, and the resulting clone was re-named "pVHV-
FeLV
gp85". The "pVHV" vector nomenclature was chosen to refer to pVEK-derived
replicon vectors containing transgene cassettes cloned via the Ascl and Pad l
sites
in the multiple cloning site of pVEK.
Production of TC-83 RNA replicon particles (RP) was conducted according to
methods previously described [U.S. 9,441,247 B2 and U.S. 8,460,913 B2; the
contents of which are hereby incorporated herein by reference]. Briefly, pVHV
replicon vector DNA and helper DNA plasmids were linearized with Notl
restriction
enzyme prior to in vitro transcription using MegaScript T7 RNA polymerase and
cap
analog (Promega, Madison, WI). Importantly, the helper RNAs used in the
production lack the VEE subgenomic promoter sequence, as previously described
[Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727 (2010)]. Purified RNA for the
replicon and helper components were combined and mixed with a suspension of
Vero cells, electroporated in 4 mm cuvettes, and returned to OptiPro SFM cell
culture media (Thermo Fisher, Waltham MA). Following overnight incubation,
alphavirus RNA replicon particles were purified from the cells and media by
passing
the suspension through a ZetaPlus BioCap depth filter (3M, Maplewood, MN),
washing with phosphate buffered saline containing 5% sucrose (w/v), and
finally
eluting the retained RP with 400 mM NaCI buffer. Eluted RP were formulated to
a
final 5% sucrose (w/v), passed through a 0.22 micron membrane filter, and
dispensed into aliquots for storage. Titer of functional RP was determined by
immunofluorescence assay on infected Vero cell monolayers.
CA 03080427 2020-04-27
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28
EXAMPLE 2
COMPARATIVE EFFICACY AND SAFETY OF FELV VACCINES IN CATS
A vaccine comprising an alphavirus RNA replicon particle (RP) comprising
the capsid protein and glycoproteins of the avirulent TC-83 strain of
Venezuelan
Equine Encephalitis Virus (VEE) and encoding the FeLV viral glycoprotein
(gp85),
was formulated in 5% sucrose. The liquid vaccine was frozen for storage before
use. This vaccine was compared with a commercially available vaccine
comprising
a recombinant canary pox encoding FeLV, as shown in Table 1 below. Five groups
of eight feline subjects were vaccinated either with a single dose at 8-9
weeks, or in
a prime/boost regimen of 8-9 weeks of age and then 21 days later. The doses
for
each experimental vaccinate group is provided in Table 1 below.
TABLE 1
VACCINATION PROTOCOL
Vaccinate No. of Vaccine RP/dose
Vaccination
Group Animals Days
1 8 RP-FeLV 4.35x 108 0,21
2 8 RP-FeLV 3.55x 107 0,21
3 8 RP-FeLV 1.5x 108 21
(one shot)
4 8 PureVax # Does not apply 0,21
5 8 Placebo none 0, 21
#A vaccine containing a recombinant canary pox encoding FeLV sold by Merial
All cats were subcutaneously vaccinated with 1.0 mL of their respective
vaccine regimen. Cats were 8-9 weeks of age at the time of the initial
vaccination
(including cats in Group 3). The cats of Group 4 were vaccinated at the times
provided with the quantity of vaccine as directed on the label of the
commercial
vaccine. Following the vaccination the cats were observed for adverse
reactions to
the vaccines by observing the general health daily, as well as palpating the
site of
injection for the two days following each vaccination and twice per week for
two
weeks following each vaccination. No adverse reactions were observed for any
of
the vaccines.
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29
All cats were challenged with a virulent culture of FeLV four weeks after the
booster vaccination (four weeks after the one-shot vaccination for the Group 3
cats).
The cats were challenged on four separate days over one week (study days 49,
52,
54 and 56) by administering 1.0 mL of challenge virus by the oronasal route
(0.3 mL
in each nostril and 0.4 mL orally). Three weeks after challenge serum samples
were collected each week through ten weeks post-challenge. Serum samples were
tested by ELISA for the presence of FeLV p27 antigen. An animal is considered
infected with FeLV if it is persistently antigenemic. Antigenemia is defined
as a
positive p27 ELISA result for three consecutive weeks or on five or more
occasions
during the eight week testing period. An FeLV vaccine must protect 75% of the
cats
vaccinated with the test product for USDA licensure. In addition, in order for
the
challenge to be regarded as valid, 80% of the control cats must be
persistently
antigenemic [see, Shipley et al., JAVMA, Vol. 199, No. 10, (Nov 15, 1991)].
The
results of the challenge are summarized in the Table 2 below.
TABLE 2
VACCINATION AND CHALLENGE
Treatment Vaccine RP dose %Cats %Cats
Group Antigenemic Protected
RP-FeLV 4.35x 108
1 0% 100%
RP-FeLV 3.55 x 107
2 0% 100%
RP-FeLV 1.5 x 108
3 130/0 870/0
(one shot)
PureVax # Does not apply
4 43% 57%
Placebo Does not apply
5 88% 12%
A vaccine containing a recombinant canary pox encoding FeLV sold by Merial
* All other groups received a two-dose regimen, see, Table 1 above.
As Table 2 demonstrates, the RP-FeLV vaccines protected 100% of the cats
when administered in a two-dose regimen (i.e., primary and booster
vaccination) at
both doses tested. Moreover, the RP-FeLV vaccine protected 87% of the cats
when
administered as a single dose. In direct contrast, the commercially available
vaccine only protected 57% of the cats, even with a two-dose regimen. In
addition,
the challenge is regarded as valid because greater than 80% of the control
cats
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were persistently antigenemic [see, Table 2]. Finally, all of the RP-FeLV
vaccine
formulations were found safe in cats.
EXAMPLE 3
5
DETERMINATION OF THE DOSE DEPENDENCE OF AN RP-FELV VACCINE
BY VACCINATION AND CHALLENGE.
The RP-FeLV vaccine of Example 2 was formulated in a vaccine formulation
10 that included enzymatically hydrolyzed casein (NZ-amine), gelatin,
and sucrose.
The vaccine was then lyophilized. Four groups of ten cats each were vaccinated
as
summarized in Table 3 below:
TABLE 3
15 VACCINATION PROTOCOL
Treatment No. of Vaccine RP/dose Vaccination
Group Animals Days
1 10 RP-FeLV 1.1 x 106 0,21
2 10 RP-FeLV 2.1 x 106 0, 21
3 10 RP-FeLV 6.5x 107 0,21
4 10 Non-vaccinated None NA
Controls
All cats were vaccinated with 1.0 mL of respective test product,
subcutaneously. The cats were 8-9 weeks of age at the time of initial
vaccination.
Following the vaccination the cats were observed for adverse reactions to the
20 vaccines by observing their general daily health, as well as palpating
the site of
injection for the two days following each vaccination and twice per week for
the two
weeks following each vaccination. No adverse reactions to any of the vaccines
were observed.
25
All of the cats were challenged with a virulent culture of FeLV three weeks
after the booster vaccination. Cats were challenged on four separate days over
one
week (study days 42, 45, 47 and 49) by administering 1.0 mL of challenge virus
by
the oronasal route (0.3 mL in each nostril and 0.4 mL orally). Three weeks
after
challenge serum samples were collected each week through twelve weeks post-
30 challenge. Serum samples were tested by ELISA for the presence of FeLV
p27
antigen. An animal is considered infected with FeLV if it is found to be
persistently
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antigenemic. Antigenemia is defined as a positive p27 ELISA result for three
consecutive weeks, or on five or more occasions during the eight-week testing
period. For USDA licensure an FeLV vaccine must protect 75% of the cats
vaccinated with the test product. For the challenge to be considered valid,
80% of
the control cats must be persistently antigenemic [Shipley et al., JAVMA, Vol.
199,
No. 10, Nov 15, 1991]. The results of the challenge are summarized in the
Table 4
below:
TABLE 4
DOSE DEPENDENCE OF RP-FELV
Treatment Vaccine RP/dose %Cats %Cats
Group Antigenemic Protected
1 RP-FeLV 1.1 x 105 10% 90%
2 RP-FeLV 2.1 x 106 0% 100%
3 RP-FeLV 6.5x 107 0% 100%
4 Non-vaccinated None 90% 10%
Controls
In this study of short term immunity, the minimum protective dose of the
RP-FeLV vaccine for 100% protection of the cats was between about 1.0 x 105 to
about 2.0 x 106 RPs, when administered in a two dose (primary and booster
vaccination) regimen. The challenge was valid because at least 80% of the
control
cats were persistently antigenemic. All RP-FeLV vaccine formulations tested
were
safe in cats.
The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the invention
in
addition to those described herein will become apparent to those skilled in
the art
from the foregoing description. Such modifications are intended to fall within
the
scope of the appended claims.
It is further to be understood that all base sizes or amino acid sizes, and
all
molecular weight or molecular mass values, given for nucleic acids or
polypeptides
are approximate, and are provided for description.
