Needle-free administration of FeLV vaccines
United States Patent: 7,582,302
Issued: September 1, 2009
Inventors: Tseggai; Tesfai
(Athens, GA), Pardo; Maria Camila (Athens, GA), Leard; Alton Timothy
Assignee: Merial Limited
Appl. No.: 11/146,299
Filed: June 6, 2005
Executive MBA in Pharmaceutical Management, U. Colorado
The invention provides a novel method of
vaccination of an animal of the felidae family against feline leukemia.
The FeLV recombinant vaccine based on viral vector with the aid of a
liquid jet needle-free injector can result in distribution of the vaccine
essentially in the dermis and the hypodermis of the animal.
Description of the
SUMMARY OF THE INVENTION
The objective of the present invention is to provide a new method of
vaccination of an animal of the felidae family, which is efficient, easier
and less expensive to use, and which leads to increased safety.
This objective is met by administering a FeLV recombinant vaccine based on
viral vector with the aid of a liquid jet needle-free injector, ensuring
distribution of the vaccine essentially in the dermis and the hypodermis
of the animal.
A first object of the present invention is a vaccination method against
FeLV, which may comprise the step of administration essentially in the
dermis and the hypodermis of an animal of the felidae family an efficient
amount of a FeLV recombinant vaccine based on a viral vector using a
liquid jet needle-free injector, which administration elicits a safe and
protective immune response against FeLV.
Another object is a vaccination kit or set, which may comprise such a
liquid jet needle-free injector and at least one vaccine vial containing a
FeLV recombinant vaccine based on a viral vector, operatively assembled to
perform the administration of the vaccine essentially in the dermis and
the hypodermis of an animal of the felidae family and to elicit a safe and
protective immune response against FeLV.
Another object of the invention is the use of a recombinant viral vector
which may encode and express at least one FeLV immunogen and of an
acceptable vehicle or diluent, for the preparation of a liquid vaccine
designed to be administered essentially in the dermis and the hypodermis
of animals of the felidae family using a liquid jet needle-free injector,
and resulting in eliciting a safe and protective immune response against
It is noted that in this disclosure and particularly in the claims and/or
paragraphs, terms such as "comprises", "comprised", "comprising" and the
like can have the meaning attributed to it in U.S. Patent law; e.g., they
can mean "includes", "included", "including", and the like; and that terms
such as "consisting essentially of" and "consists essentially of" have the
meaning ascribed to them in U.S. Patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the prior
art or that affect a basic or novel characteristic of the invention.
These and other embodiments are disclosed or are obvious from and
encompassed by, the following Detailed Description.
The present invention concerns a vaccination method against FeLV,
comprising the step of administration essentially in the dermis and the
hypodermis of an animal of the felidae family an efficient amount of a
FeLV recombinant vaccine based on a viral vector using a liquid jet
needle-free injector, which administration elicits a safe and protective
immune response against FeLV.
"Essentially" means what some vaccines may also be found in the epidermis
or in the muscles.
A protective immune response is characterized by a significant reduction
of the antigenemia after challenge or by significant neutralizing antibody
titers. A safe immune response is characterized by the limitation of the
side effects linked to the vaccine administration, notably by a
significant reduction or by the absence of local injection site reaction
and by a significant reduction or by the absence of symptoms, like
anorexia and depression following vaccine administration.
An animal of the felidae family encompasses cats, this including new born,
kitten, male, female, pregnant female.
The vaccine comprises a recombinant viral vector and an acceptable vehicle
or diluent. The recombinant viral vector includes notably herpesvirus,
adenovirus and poxvirus such as fowlpox (U.S. Pat. No. 5,174,993; U.S.
Pat. No. 5,505,941 and U.S. Pat. No. 5,766,599) or canarypox (U.S. Pat.
No. 5,756,103)). The vehicle or diluent includes but is not limited to
sterile water, physiological saline, glucose, buffer and the like. The
vehicle or diluent may also include polyols, glucides or pH buffering
agents. The vehicle or diluent may, for example, also comprise amino
acids, peptides, antioxidants, bactericide, bacteriostatic compounds.
The recombinant viral vector encodes and expresses at least one FeLV
immunogen, notably FeLV env gene or FeLV env and gag/pol genes. A complete
sequence of the FeLV genome can be found in Chen et al., J Virol. 1998
Sep. 72(9):7048-56, which coupled with routine experimentation enables one
of ordinary skill in the art may ascertain the sequence of any FeLV
In a preferred aspect of this embodiment, the method of the invention is
performed with a recombinant canarypox virus expressing FeLV env or FeLV
env and gag/pol genes (e.g. vCP97 construct, see example 53 in U.S. Pat.
As an alternative aspect of the invention, the method of the invention is
performed with a recombinant fowlpox virus expressing FeLV env or FeLV env
and gag/pol genes.
The invention further encompasses at least one FeLV immunogen contained in
a vector molecule or an expression vector and operably linked to a
promoter element and optionally to an enhancer.
In an advantageous embodiment, the promoter is the promoter of the
cytomegalovirus (CMV) immediate early gene. In another advantageous
embodiment, the promoter and/or enhancer elements are oxygen-inducible.
Examples of oxygen-inducible promoters and/or enhancers that can be used
in the methods of the present invention include, but are not limited to,
early growth response-1 (Egr1) promoter (see, e.g., Park et al., J Clin
Invest. August 2002; 110(3):403-1), hypoxia-inducible factor (HIF)
inducible enhancers (see e.g., Cuevas et al., Cancer Res. Oct. 15,
2003;63(20):6877-84) and Mn-superoxide dismutase (Mn-SOD) promoters (see,
e.g., Gao et al., Gene. Oct. 17, 1996;176(1-2):269-70).
In another embodiment, the enhancers and/or promoters include various cell
or tissue specific promoters (e.g., muscle, endothelial cell, liver,
somatic cell or stem cell), various viral promoters and enhancers and
various FeLV immunogen sequences isogenically specific for each animal
species. Examples of muscle-specific promoters and enhancers have been
described are known to one of skill in the art (see, e.g., Li et al., Gene
Ther. December 1999;6(12):2005-11; Li et al., Nat Biotechnol. March
1999;17(3):241-5 and Loirat et al., Virology. Jul. 20, 1999;260(1):74-83;
the disclosures of which are incorporated by reference in their
Promoters and enhancers that may be employed in the present invention
include, but are not limited to LTR or the Rous sarcoma virus, TK of
HSV-1, early or late promoter of SV40, adenovirus major late (MLP),
phosphoglycerate kinase, metallothionein, .alpha.-1 antitrypsin, albumin,
collagenese, elastase I, .beta.-actin, .beta.-globin, .gamma.-globin,
.alpha.-fetoprotein, muscle creatin kinase.
A "vector" refers to a recombinant DNA or RNA plasmid or virus that
comprises a heterologous polynucleotide to be delivered to a target cell,
either in vitro or in vivo. The heterologous polynucleotide may comprise a
sequence of interest for purposes of therapy, and may optionally be in the
form of an expression cassette. As used herein, a vector need not be
capable of replication in the ultimate target cell or subject. The term
includes cloning vectors also included are viral vectors.
The term "recombinant" means a polynucleotide semisynthetic, or synthetic
origin which either does not occur in nature or is linked to another
polynucleotide in an arrangement not found in nature.
"Heterologous" means derived from a genetically distinct entity from the
rest of the entity to which it is being compared. For example, a
polynucleotide, may be placed by genetic engineering techniques into a
plasmid or vector derived from a different source, and is a heterologous
polynucleotide. A promoter removed from its native coding sequence and
operatively linked to a coding sequence other than the native sequence is
a heterologous promoter.
The polynucleotides of the invention may comprise additional sequences,
such as additional encoding sequences within the same transcription unit,
controlling elements such as promoters, ribosome binding sites,
polyadenylation sites, additional transcription units under control of the
same or a different promoter, sequences that permit cloning, expression,
homologous recombination, and transformation of a host cell, and any such
construct as may be desirable to provide embodiments of this invention.
The present invention encompasses a vector expressing a FeLV immunogen or
variants or analogues or fragments. Elements for the expression of a FeLV
immunogen are advantageously present in an inventive vector. In minimum
manner, this comprises, consists essentially of, or consists of an
initiation codon (ATG), a stop codon and a promoter, and optionally also a
polyadenylation sequence for certain vectors such as plasmid and certain
viral vectors, e.g., viral vectors other than poxviruses. When the
polynucleotide encodes a polyprotein fragment, e.g. a FeLV immunogen,
advantageously, in the vector, an ATG is placed at 5' of the reading frame
and a stop codon is placed at 3'. Other elements for controlling
expression may be present, such as enhancer sequences, stabilizing
sequences, such as intron and signal sequences permitting the secretion of
Methods for making and/or administering a vector or recombinants or
plasmid for expression of gene products of genes of the invention either
in vivo or in vitro can be any desired method, e.g., a method which is by
or analogous to the methods disclosed in, or disclosed in documents cited
in: U.S. Pat. Nos. 4,603,112; 4,769,330; 4,394,448; 4,722,848; 4,745,051;
4,769,331; 4,945,050; 5,494,807; 5,514,375; 5,744,140; 5,744,141;
5,756,103; 5,762,938; 5,766,599; 5,990,091; 5,174,993; 5,505,941;
5,338,683; 5,494,807; 5,591,639; 5,589,466; 5,677,178; 5,591,439;
5,552,143; 5,580,859; 6,130,066; 6,004,777; 6,130,066; 6,497,883;
6,464,984; 6,451,770; 6,391,314; 6,387,376; 6,376,473; 6,368,603;
6,348,196; 6,306,400; 6,228,846; 6,221,362; 6,217,883; 6,207,166;
6,207,165; 6,159,477; 6,153,199; 6,090,393; 6,074,649; 6,045,803;
6,033,670; 6,485,729; 6,103,526; 6,224,882; 6,312,682; 6,348,450 and 6;
312,683; U.S. patent application Ser. No. 920,197, filed Oct. 16, 1986; WO
90/01543; WO 91/11525; WO 94/16716; WO 96/39491; WO 98/33510; EP 265785;
EP 0 370 573; Andreansky et al., Proc. Natl. Acad. Sci. USA
1996;93:11313-11318; Ballay et al., EMBO J. 1993;4:3861-65; Felgner et
al., J. Biol. Chem. 1994;269:2550-2561; Frolov et al., Proc. Natl. Acad.
Sci. USA 1996;93:11371-11377; Graham, Tibtech 1990;8:85-87; Grunhaus et
al., Sem. Virol. 1992;3:237-52; Ju et al., Diabetologia 1998;41:736-739;
Kitson et al., J. Virol. 1991;65:3068-3075; McClements et al., Proc. Natl.
Acad. Sci. USA 1996;93:11414-11420; Moss, Proc. Natl. Acad. Sci. USA
1996;93:11341-11348; Paoletti, Proc. Natl. Acad. Sci. USA
1996;93:11349-11353; Pennock et al., Mol. Cell. Biol. 1984;4:399406;
Richardson (Ed), Methods in Molecular Biology 1995;39, "Baculovirus
Expression Protocols," Humana Press Inc.; Smith et al. (1983) Mol. Cell.
Biol. 1983;3:2156-2165; Robertson et al., Proc. Natl. Acad. Sci. USA
1996;93:11334-11340; Robinson et al., Sem. Immunol. 1997;9:271; and
Roizman, Proc. Natl. Acad. Sci. USA 1996;93:11307-11312. Thus, the vector
in the invention can be any suitable recombinant virus or virus vector,
such as a poxvirus (e.g., vaccinia virus, avipox virus, canarypox virus,
fowlpox virus, raccoonpox virus, swinepox virus, etc.), adenovirus (e.g.,
human adenovirus, canine adenovirus), herpesvirus (e.g. canine herpesvirus),
baculovirus, retrovirus, etc. (as in documents incorporated herein by
reference); or the vector can be a plasmid. The herein cited and
incorporated herein by reference documents, in addition to providing
examples of vectors useful in the practice of the invention, can also
provide sources for non-FeLV immunogens, e.g., non-FeLV immunogens, non-FeLV
immunogens peptides or fragments thereof, cytokines, etc. to be expressed
by vector or vectors in, or included in, the compositions of the
The present invention also relates to preparations comprising vectors,
such as expression vectors, e.g., therapeutic compositions. The
preparations can comprise, consist essentially of, or consist of one or
more vectors, e.g., expression vectors, such as in vivo expression
vectors, comprising, consisting essentially or consisting of (and
advantageously expressing) one or more of FeLV immunogens. Advantageously,
the vector contains and expresses a polynucleotide that includes, consists
essentially of, or consists of a coding region encoding one or more FeLV
immunogens a pharmaceutically or veterinarily acceptable carrier,
excipient or vehicle. Thus, according to an embodiment of the invention,
the other vector or vectors in the preparation comprises, consists
essentially of or consists of a polynucleotide that encodes, and under
appropriate circumstances the vector expresses one or more other proteins
of a FeLv immunogen or a fragment thereof.
According to another embodiment, the vector or vectors in the preparation
comprise, or consist essentially of, or consist of polynucleotide(s)
encoding one or more proteins or fragment(s) thereof of a FeLV immunogen,
the vector or vectors have expression of the polynucleotide(s). The
inventive preparation advantageously comprises, consists essentially of,
or consists of, at least two vectors comprising, consisting essentially
of, or consisting of, and advantageously also expressing, advantageously
in vivo under appropriate conditions or suitable conditions or in a
suitable host cell, polynucleotides from different FeLV isolates encoding
the same proteins and/or for different proteins, but advantageously for
the same proteins. Preparations containing one or more vectors containing,
consisting essentially of or consisting of polynucleotides encoding, and
advantageously expressing, advantageously in vivo, FeLV peptide, fusion
protein or an epitope thereof.
According to one embodiment of the invention, the expression vector is a
viral vector, in particular an in vivo expression vector. In an
advantageous embodiment, the expression vector is an adenovirus vector.
Advantageously, the adenovirus is a human Ad5 vector, an E1-deleted and/
or an E3-deleted adenovirus.
In one particular embodiment the viral vector is a poxvirus, e.g. a
vaccinia virus or an attenuated vaccinia virus, (for instance, MVA, a
modified Ankara strain obtained after more than 570 passages of the Ankara
vaccine strain on chicken embryo fibroblasts; see Stickl & Hochstein-Mintzel,
Munch. Med. Wschr., 1971, 113, 1149-1153; Sutter et al., Proc. Natl. Acad.
Sci. U.S.A., 1992, 89, 10847-10851; available as ATCC VR-1508; or NYVAC,
see U.S. Pat. No. 5,494,807, for instance, Examples 1 to 6 and et seq of
U.S. Patent No. 5,494,807 which discuss the construction of NYVAC, as well
as variations of NYVAC with additional ORFs deleted from the Copenhagen
strain vaccinia virus genome, as well as the insertion of heterologous
coding nucleic acid molecules into sites of this recombinant, and also,
the use of matched promoters; see also WO96/40241), an avipox virus or an
attenuated avipox virus (e.g., canarypox, fowlpox, dovepox, pigeonpox,
quailpox, ALVAC or TROVAC; see, e.g., U.S. Pat. Nos. 5,505,941,
5,494,807), swinepox, raccoonpox, camelpox, or myxomatosis virus.
According to another embodiment of the invention, the poxvirus vector is a
canarypox virus or a fowlpox virus vector, advantageously an attenuated
canarypox virus or fowipox virus. In this regard, is made to the canarypox
available from the ATCC under access number VR-111. Attenuated canarypox
viruses are described in U.S. Pat. No. 5,756,103 (ALVAC) and WO01/05934.
Numerous fowipox virus vaccination strains are also available, e.g. the
DIFTOSEC CT strain marketed by MERIAL and the NOBILIS VARIOLE vaccine
marketed by INTERVET; and, reference is also made to U.S. Pat. No.
5,766,599 which pertains to the atenuated fowlpox strain TROVAC.
For information on the method to generate recombinants thereof and how to
administer recombinants thereof, the skilled artisan can refer documents
cited herein and to WO90/12882, e.g., as to vaccinia virus mention is made
of U.S. Pat. Nos. 4,769,330, 4,722,848, 4,603,112, 5,110,587, 5,494,807,
and 5,762,938 inter alia; as to fowipox, mention is made of U.S. Pat. Nos.
5,174,993, 5,505,941 and U.S. Pat. No. 5,766,599 inter alia; as to
canarypox mentionis made of U.S. Pat. No. 5,756,103 inter alia; as to
swinepox mention is made of U.S. Pat. No. 5,382,425 inter alia; and, as to
raccoonpox, mention is made of WO00/03030 inter alia.
When the expression vector is a vaccinia virus, insertion site or sites
for the polynucleotide or polynucleotides to be expressed are
advantageously at the thymidine kinase (TK) gene or insertion site, the
hemagglutinin (HA) gene or insertion site, the region encoding the
inclusion body of the A type (ATI); see also documents cited herein,
especially those pertaining to vaccinia virus. In the case of canarypox,
advantageously the insertion site or sites are ORF(s) C3, C5 and/or C6;
see also documents cited herein, especially those pertaining to canarypox
virus. In the case of fowipox, advantageously the insertion site or sites
are ORFs F7 and/or F8; see also documents cited herein, especially those
pertaining to fowlpox virus. The insertion site or sites for MVA virus
area advantageously as in various publications, including Carroll M. W. et
al., Vaccine, 1997, 15 (4), 387-394; Stittelaar K. J. et al., J. Virol.,
2000, 74 (9), 4236-4243; Sutter G. et al., 1994, Vaccine, 12 (11),
1032-1040; and, in this regard it is also noted that the complete MVA
genome is described in Antoine G., Virology, 1998, 244, 365-396, which
enables the skilled artisan to use other insertion sites or other
Advantageously, the polynucleotide to be expressed is inserted under the
control of a specific poxvirus promoter, e.g., the vaccinia promoter 7.5
kDa (Cochran et al., J. Virology, 1985, 54, 30-35), the vaccinia promoter
I3L (Riviere et al., J. Virology, 1992, 66, 3424-3434), the vaccinia
promoter HA (Shida, Virology, 1986, 150, 451-457), the cowpox promoter ATI
(Funahashi et al., J. Gen. Virol., 1988, 69, 35-47), the vaccinia promoter
H6 (Taylor J. et al., Vaccine, 1988, 6, 504-508; Guo P. et al. J. Virol.,
1989, 63, 4189-4198; Perkus M. et al., J. Virol., 1989, 63, 3829-3836),
In a particular embodiment the viral vector is an adenovirus, such as a
human adenovirus (HAV) or a canine adenovirus (CAV).
In one embodiment the viral vector is a human adenovirus, in particular a
serotype 5 adenovirus, rendered incompetent for replication by a deletion
in the E1 region of the viral genome, in particular from about nucleotide
459 to about nucleotide 3510 by reference to the sequence of the hAd5
disclosed in Genbank under the accession number M73260 and in the
referenced publication J. Chroboczek et al Virol. 1992, 186, 280-285. The
deleted adenovirus is propagated in E1-expressing 293 (F. Graham et al J.
Gen. Virol. 1977, 36, 59-72) or PER cells, in particular PER.C6 (F.
Falloux et al Human Gene Therapy 1998, 9, 1909-1917). The human adenovirus
can be deleted in the E3 region, in particular from about nucleotide 28592
to about nucleotide 30470. The deletion in the E1 region can be done in
combination with a deletion in the E3 region (see, e.g. J. Shriver et al.
Nature, 2002, 415, 331-335, F. Graham et al Methods in Molecular Biology
Vol .7: Gene Transfer and Expression Protocols Edited by E. Murray, The
Human Press Inc, 1991, p 109-128; Y. Ilan et al Proc. Natl. Acad. Sci.
1997, 94, 2587-2592; U.S. Pat. No. 6,133,028; U.S. Pat. No. 6,692,956; S.
Tripathy et al Proc. Natl. Acad. Sci. 1994, 91, 11557-11561; B. Tapnell
Adv. Drug Deliv. Rev. 1993, 12, 185-199; X. Danthinne et al Gene Thrapy
2000, 7, 1707-1714; K. Berkner Bio Techniques 1988, 6, 616-629; K. Berkner
et al Nucl. Acid Res. 1983, 11, 6003-6020; C. Chavier et al J. Virol.
1996, 70, 4805-4810). The insertion sites can be the E1 and/or E3 loci
(region) eventually after a partial or complete deletion of the E1 and/or
E3 regions. Advantageously, when the expression vector is an adenovirus,
the polynucleotide to be expressed is inserted under the control of a
promoter functional in eukaryotic cells, such as a strong promoter,
preferably a cytomegalovirus immediate-early gene promoter (CMV-IE
promoter), in particular the enhancer/promoter region from about
nucleotide -734 to about nucleotide +7 in M. Boshart et al Cell 1985, 41,
521-530 or the enhancer/promoter region from the pCI vector from Promega
Corp. The CMV-IE promoter is advantageously of murine or human origin. The
promoter of the elongation factor 1.alpha. can also be used. In one
particular embodiment a promoter regulated by hypoxia, e.g. the promoter
HRE described in K. Boast et al Human Gene Therapy 1999, 13, 2197-2208),
can be used. A muscle specific promoter can also be used (X. Li et al Nat.
Biotechnol. 1999, 17, 241-245). Strong promoters are also discussed herein
in relation to plasmid vectors. In one embodiment, a splicing sequence can
be located downstream of the enhancer/promoter region. For example, the
intron 1 isolated from the CMV-IE gene (R. Stenberg et al J. Virol. 1984,
49, 190), the intron isolated from the rabbit or human .beta.-globin gene,
in particular the intron 2 from the b-globin gene, the intron isolated
from the immunoglobulin gene, a splicing sequence from the SV40 early gene
or the chimeric intron sequence isolated from the pCI vector from Promege
Corp. comprising the human .beta.-globin donor sequence fused to the mouse
immunoglobulin acceptor sequence (from about nucleotide 890 to about
nucleotide 1022 in Genbank under the accession number CVU47120). A poly(A)
sequence and terminator sequence can be inserted downstream the
polynucleotide to be expressed, e.g. a bovine growth hormone gene, in
particular from about nucleotide 2339 to about nucleotide 2550 in Genbank
under the accession number BOVGHRH, a rabbit .beta.-globin gene or a SV40
late gene polyadenylation signal.
In another embodiment the viral vector is a canine adenovirus, in
particular a CAV-2 (see, e.g. L. Fischer et al. Vaccine, 2002, 20,
3485-3497; U.S. Pat. No. 5,529,780; U.S. Pat. No. 5,688,920; PCT
Application No. WO95/14102). For CAV, the insertion sites can be in the E3
region and /or in the region located between the E4 region and the right
ITR region (see U.S. Pat. No. 6,090,393; U.S. Pat. No. 6,156,567). In one
embodiment the insert is under the control of a promoter, such as a
cytomegalovirus immediate-early gene promoter (CMV-IE promoter) or a
promoter already described for a human adenovirus vector. A poly(A)
sequence and terminator sequence can be inserted downstream the
polynucleotide to be expressed, e.g. a bovine growth hormone gene or a
rabbit .beta.-globin gene polyadenylation signal.
In another particular embodiment the viral vector is a herpesvirus such as
a canine herpesvirus (CHV) or a feline herpesvirus (FHV). For CHV, the
insertion sites may be in particular in the thymidine kinase gene, in the
ORF3, or in the UL43 ORF (see U.S. Pat. No. 6,159,477). In one embodiment
the polynucleotide to be expressed is inserted under the control of a
promoter functional in eukaryotic cells, advantageously a CMV-IE promoter
(murine or human). In one particular embodiment a promoter regulated by
hypoxia, e.g. the promoter HRE described in K. Boast et al Human Gene
Therapy 1999, 13, 2197-2208), can be used. A poly(A) sequence and
terminator sequence can be inserted downstream the polynucleotide to be
expressed, e.g. bovine growth hormone or a rabbit .beta.-globin gene
According to a yet further embodiment of the invention, the expression
vector is a plasmid vector or a DNA plasmid vector, in particular an in
vivo expression vector. In a specific, non-limiting example, the pVR1020
or 1012 plasmid (VICAL Inc.; Luke C. et al., Journal of Infectious
Diseases, 1997, 175, 91-97; Hartikka J. et al., Human Gene Therapy, 1996,
7, 1205-1217, see, e.g., U.S. Pat. Nos. 5,846,946 and 6,451,769) can be
utilized as a vector for the insertion of a polynucleotide sequence. The
pVR1020 plasmid is derived from pVR1012 and contains the human tPA signal
sequence. In one embodiment the human tPA signal comprises from amino acid
M(1) to amino acid S(23) in Genbank under the accession number HUMTPA14.
In another specific, non-limiting example, the plasmid utilized as a
vector for the insertion of a polynucleotide sequence can contain the
signal peptide sequence of equine IGF1 from amino acid M(24) to amino acid
A(48) in Genbank under the accession number U28070. Additional information
on DNA plasmids which may be consulted or employed in the practice are
found, for example, in U.S. Pat. Nos. 6,852,705; 6,818,628; 6,586,412;
6,576,243; 6,558,674; 6,464,984; 6,451,770; 6,376,473 and 6,221,362.
The term plasmid covers any DNA transcription unit comprising a
polynucleotide according to the invention and the elements necessary for
its in vivo expression in a cell or cells of the desired host or target;
and, in this regard, it is noted that a supercoiled or non-supercoiled,
circular plasmid, as well as a linear form, are intended to be within the
scope of the invention.
Each plasmid comprises or contains or consists essentially of, in addition
to the polynucleotide encoding the FeLV immunogen or a variant, analog or
fragment thereof, operably linked to a promoter or under the control of a
promoter or dependent upon a promoter. In general, it is advantageous to
employ a strong promoter functional in eukaryotic cells. The preferred
strong promoter is the immediate early cytomegalovirus promoter (CMV-IE)
of human or murine origin, or optionally having another origin such as the
rat or guinea pig. The CMV-IE promoter can comprise the actual promoter
part, which may or may not be associated with the enhancer part. Reference
can be made to EP-A-260 148, EP-A-323 597, U.S. Pat. Nos. 5,168,062,
5,385,839, and 4,968,615, as well as to PCT Application No WO87/03905. The
CMV-IE promoter is advantageously a human CMV-IE (Boshart M. et al.,
Cell., 1985, 41, 521-530) or murine CMV-IE.
In more general terms, the promoter has either a viral or a cellular
origin. A strong viral promoter other than CMV-IE that may be usefully
employed in the practice of the invention is the early/late promoter of
the SV40 virus or the LTR promoter of the Rous sarcoma virus. A strong
cellular promoter that may be usefully employed in the practice of the
invention is the promoter of a gene of the cytoskeleton, such as e.g. the
desmin promoter (Kwissa M. et al., Vaccine, 2000, 18, 2337-2344), or the
actin promoter (Miyazaki J. et al., Gene, 1989, 79, 269-277).
Functional sub fragments of these promoters, i.e., portions of these
promoters that maintain an adequate promoting activity, are included
within the present invention, e.g. truncated CMV-IE promoters according to
PCT Application No. WO98/00166 or U.S. Pat. No. 6,156,567 can be used in
the practice of the invention. A promoter in the practice of the invention
consequently includes derivatives and sub fragments of a full-length
promoter that maintain an adequate promoting activity and hence function
as a promoter, preferably promoting activity substantially similar to that
of the actual or full-length promoter from which the derivative or sub
fragment is derived, e.g., akin to the activity of the truncated CMV-IE
promoters of U.S. Pat. No. 6,156,567 to the activity of full-length CMV-IE
promoters. Thus, a CMV-IE promoter in the practice of the invention can
comprise or consist essentially of or consist of the promoter portion of
the full-length promoter and/or the enhancer portion of the full-length
promoter, as well as derivatives and sub fragments.
Preferably, the plasmids comprise or consist essentially of other
expression control elements. It is particularly advantageous to
incorporate stabilizing sequence(s), e.g., intron sequence(s), preferably
the first intron of the hCMV-IE (PCT Application No. WO89/01036), the
intron II of the rabbit b-globin gene (van Ooyen et al., Science, 1979,
As to the polyadenylation signal (polyA) for the plasmids and viral
vectors other than poxviruses, use can more be made of the poly(A) signal
of the bovine growth hormone (bGH) gene (see U.S. Pat. No. 5,122,458), or
the poly(A) signal of the rabbit b-globin gene or the poly(A) signal of
the SV40 virus.
According to another embodiment of the invention, the expression vectors
are expression vectors used for the in vitro expression of proteins in an
appropriate cell system. The expressed proteins can be harvested in or
from the culture supernatant after, or not after secretion (if there is no
secretion a cell lysis typically occurs or is performed), optionally
concentrated by concentration methods such as ultrafiltration and/or
purified by purification means, such as affinity, ion exchange or gel
filtration-type chromatography methods.
Host cells that can be used in the present invention include, but are not
limited to, muscle cells, keratinocytes, myoblasts, Chinese Hamster ovary
cells (CHO), vero cells, BHK21, sf9 cells, and the like. It is understood
to one of skill in the art that conditions for culturing a host cell
varies according to the particular gene and that routine experimentation
is necessary at times to determine the optimal conditions for culturing an
FeLV depending on the host cell. For example, the vector encoding an FeLV
immunogen can be transformed into myoblasts (which can be obtained from
muscle tissue from the animal in need of treatment), and the transformed
myoblasts can be transplanted to the animal. In another example,
keratinocytes can also be transformed with a vector encoding a FeLV
immunogen and transplanted into the animal, resulting in secretion of a
FeLV immunogen into circulation.
A "host cell" denotes a prokaryotic or eukaryotic cell that has been
genetically altered, or is capable of being genetically altered by
administration of an exogenous polynucleotide, such as a recombinant
plasmid or vector. When referring to genetically altered cells, the term
refers both to the originally altered cell and to the progeny thereof.
Polynucleotides comprising a desired sequence can be inserted into a
suitable cloning or expression vector, and the vector in turn can be
introduced into a suitable host cell for replication and amplification.
Polynucleotides can be introduced into host cells by any means known in
the art. The vectors containing the polynucleotides of interest can be
introduced into the host cell by any of a number of appropriate means,
including direct uptake, endocytosis, transfection, f-mating,
electroporation, transfection employing calcium chloride, rubidium
chloride, calcium phosphate, DEAE-dextran, or other substances;
microprojectile bombardment; lipofection; and infection (where the vector
is infectious, for instance, a retroviral vector). The choice of
introducing vectors or polynucleotides will often depend on features of
the host cell.
In an advantageous embodiment, the invention provides for the
administration of a therapeutically effective amount of a formulation for
the delivery and expression of a FeLV immunogen in a target cell.
Determination of the therapeutically effective amount is routine
experimentation for one of ordinary skill in the art. In one embodiment,
the formulation comprises an expression vector comprising a polynucleotide
that expresses a FeLV immunogen and a pharmaceutically or veterinarily
acceptable carrier, vehicle or excipient. In an advantageous embodiment,
the pharmaceutically or veterinarily acceptable carrier, vehicle or
excipient facilitates transfection and/or improves preservation of the
vector or protein.
The pharmaceutically or veterinarily acceptable carriers or vehicles or
excipients are well known to the one skilled in the art. For example, a
pharmaceutically or veterinarily acceptable carrier or vehicle or
excipient can be a 0.9% NaCl (e.g., saline) solution or a phosphate
buffer. Other pharmaceutically or veterinarily acceptable carrier or
vehicle or excipients that can be used for methods of this invention
include, but are not limited to, poly-(L-glutamate) or
polyvinylpyrrolidone. The pharmaceutically or veterinarily acceptable
carrier or vehicle or excipients may be any compound or combination of
compounds facilitating the administration of the vector (or protein
expressed from an inventive vector in vitro); advantageously, the carrier,
vehicle or excipient may facilitate transfection and/or improve
preservation of the vector (or protein). Doses and dose volumes are herein
discussed in the general description and can also be determined by the
skilled artisan from this disclosure read in conjunction with the
knowledge in the art, without any undue experimentation.
The cationic lipids containing a quaternary ammonium salt which are
advantageously but not exclusively suitable for plasmids, are
advantageously those having the following formula
-- see Original Patent.
Among these cationic lipids, preference
is given to DMRIE
ammonium; WO96/34109), advantageously associated with a neutral lipid,
advantageously DOPE (dioleoyl-phosphatidyl-ethanol amine; Behr J. P.,
1994, Bioconjugate Chemistry, 5, 382-389), to form DMRIE-DOPE.
Advantageously, the plasmid mixture with the adjuvant is formed
extemporaneously and advantageously contemporaneously with administration
of the preparation or shortly before administration of the preparation;
for instance, shortly before or prior to administration, the
plasmid-adjuvant mixture is formed, advantageously so as to give enough
time prior to administration for the mixture to form a complex, e.g.
between about 10 and about 60 minutes prior to administration, such as
approximately 30 minutes prior to administration.
When DOPE is present, the DMRIE:DOPE molar ratio is advantageously about
95: about 5 to about 5:about 95, more advantageously about 1: about 1,
The DMRIE or DMRIE-DOPE adjuvant:plasmid weight ratio can be between about
50: about 1 and about 1: about 10, such as about 10: about 1 and about
1:about 5, and advantageously about 1: about 1 and about 1: about 2, e.g.,
1:1 and 1:2.
The polymers of acrylic or methacrylic acid are preferably crosslinked, in
particular with polyalkenyl ethers of sugars or polyalcohols. These
compounds are known under the term carbomer (Pharmeuropa vol. 8, No. 2,
June 1996). Persons skilled in the art can also refer to U.S. Pat. No.
2,909,462 describing such acrylic polymers crosslinked with a
polyhydroxylated compound having at least 3 hydroxyl groups, preferably
not more than 8, the hydrogen atoms of at least three hydroxyls being
replaced with unsaturated aliphatic radicals having at least 2 carbon
atoms. The preferred radicals are those containing 2 to 4 carbon atoms,
e.g. vinyls, allyls and other ethylenically unsaturated groups. The
unsaturated radicals may themselves contain other substituents, such as
methyl. The products sold under the name Carbopol.RTM. (BF Goodrich, Ohio,
USA) are particularly appropriate. They are crosslinked with an allyl
sucrose or with allylpentaerythritol. Among them, there may be mentioned
Carbopol.RTM. 974P, 934P and 971 P.
Among the copolymers of maleic anhydride and of alkenyl derivative, the
EMA.RTM. copolymers (Monsanto) which are copolymers of maleic anhydride
and of ethylene, which are linear or crosslinked, for example crosslinked
with divinyl ether, are preferred. Reference may be made to J. Fields et
al., Nature, 186: 778-780, Jun. 4, 1960.
The proportions of adjuvant which are useful are well known and readily
available to the one skilled in the art. By way of example, the
concentration of polymers of acrylic or methacrylic acid or of anhydride
maleic and alkenyl copolymers in the final vaccine composition will be
from 0.01% to 1.5% W/V, more particularly from 0.05 to 1% W/V, preferably
from 0.1 to 0.4% W/V.
Optionally the vaccine used according to the method of the invention may
contain a cytokine. The cytokine may be present as a protein or as a gene
encoding this cytokine inserted into a recombinant viral vector. The
cytokines may be selected among the feline cytokines, e.g. feline
interleukine 18 (flL-18) (Taylor S. et al., DNA Seq., 2000, 10(6),
387-394), flL-16 (Leutenegger C. M. et al., DNA Seq., 1998, 9(1), 59-63),
flL-12 (Fehr D. et al., DNA Seq., 1997, 8(1-2), 77-82; Imamura T. et al.,
J. Vet. Med. Sci., 2000, 62(10), 1079-1087) and feline GM-CSF
(Granulocyte-Macrophage Colony-Stimulating Factor) (GenBank AF053007).
In a specific embodiment, the pharmaceutical composition is directly
administered in vivo, and the encoded product is expressed by the vector
in the host. The methods of in vivo delivery a vector encoding a FeLV
immunogen (see, e.g., U.S. Pat. No. 6,423,693; patent publications EP
1052286, EP 1205551, U.S. patent publication 20040057941, WO 9905300 and
Draghia-Akli et al., Mol Ther. December 2002;6(6):830-6; the disclosures
of which are incorporated by reference in their entireties) can be
modified to deliver the FeLV immunogen of the present invention to a cat.
The in vivo delivery of a vector encoding the FeLV immunogen described
herein can be accomplished by one of ordinary skill in the art given the
teachings of the above-mentioned references.
Advantageously, the pharmaceutical and/or therapeutic compositions and/or
formulations according to the invention comprise or consist essentially of
or consist of an effective quantity to elicit a therapeutic response of
one or more expression vectors and/or polypeptides as discussed herein;
and, an effective quantity can be determined from this disclosure,
including the documents incorporated herein, and the knowledge in the art,
without undue experimentation.
In the case of therapeutic and/or pharmaceutical compositions based on a
plasmid vector, a dose can comprise, consist essentially of or consist of,
in general terms, about in 1 mg to about 2000 mg, advantageously about 50
mg to about 1000 mg and more advantageously from about 100 .mu.g to about
800 .mu.g of plasmid expressing a FeLV immunogen. When the therapeutic
and/or pharmaceutical compositions based on a plasmid vector is
administered with electroporation the dose of plasmid is generally between
about 0.1 .mu.g and 1mg, advantageously between about 1 .mu.g and 100 .mu.g,
advantageously between about 2 .mu.g and 50 .mu.g. The dose volumes can be
between about 0.1 and about 2 ml, advantageously between about 0.2 and
about 1 ml. These doses and dose volumes are suitable for the treatment of
felines and other mammalian target species such as equines and canines.
The therapeutic and/or pharmaceutical composition contains per dose from
about 10.sup.4 to about 10.sup.11, advantageously from about 10.sup.5 to
about 10.sup.10 and more advantageously from about 10.sup.6 to about
10.sup.9 viral particles of recombinant adenovirus expressing a FeLV
immunogen. In the case of therapeutic and/or pharmaceutical compositions
based on a poxvirus, a dose can be between about 10.sup.2 pfu and about
10.sup.9 pfu. The pharmaceutical composition contains per dose from about
10.sup.5 to 10.sup.9, advantageously from about 10.sup.6 to 10.sup.8 pfu
of poxvirus or herpesvirus recombinant expressing a FeLV immunogen.
The dose volume of compositions for target species that are mammals, e.g.,
the dose volume of feline compositions, based on viral vectors, e.g.,
non-poxvirus-viral-vector-based compositions, is generally between about
0.1 to about 2.0 ml, preferably between about 0.1 to about 1.0 ml, and
more preferably between about 0.5 ml to about 1.0 ml.
t should be understood by one of skill in the art that the disclosure
herein is provided by way of example and the present invention is not
limited thereto. From the disclosure herein and the knowledge in the art,
the skilled artisan can determine the number of administrations, the
administration route, and the doses to be used for each injection
protocol, without any undue experimentation.
The present invention contemplates at least one administration to an
animal of an efficient amount of the therapeutic composition made
according to the invention. The animal may be male, female, pregnant
female and newborn. This administration may be via various routes
including, but not limited to, intramuscular (IM), intradermal (ID) or
subcutaneous (SC) injection or via intranasal or oral administration. The
therapeutic composition according to the invention can also be
administered by a needleless apparatus (as, for example with a Pigjet,
Biojector or Vitajet apparatus (Bioject, Oreg., USA)). Another approach to
administer plasmid compositions is to use electroporation (see, e.g. S.
Tollefsen et al. Vaccine, 2002, 20, 3370-3378; S. Tollefsen et al. Scand.
J. Immunol., 2003, 57, 229-238; S. Babiuk et al., Vaccine, 2002, 20,
3399-3408; PCT Application No. WO99/01158). In another embodiment, the
plasmid is delivered to the animal by gene gun or gold particle
bombardment. In an advantageous embodiment, the animal is a vertebrate. In
a more advantageous embodiment, the vertebrate is a cat.
Liquid jet needle-free injectors are devices performing injections of a
certain amount of liquid under high pressure through a minute orifice.
Mechanical specifications of the injector may be adjusted or selected in
order to control the depth of penetration into tissues. Administrations of
a liquid using a syringe or a needle-free injector end up in a different
distribution of the liquid in the tissues. Using a syringe end up in a
localized bolus or pool. Using an injector end up in a diffused
distribution in the layers of the targeted tissues, as illustrated in
The depth of penetration is mainly controlled by the liquid pressure. This
liquid pressure is depending upon the mechanical specifications of the
injector, such as the strength of spring or any other propulsion means and
the diameter of the piston and the nozzle orifice. This is readily
available to the one skilled in the art.
The depth of injection may be easily determined by the dissection of the
tissue at the injection site (corresponding preferably to the location
where the vaccine is going to be administered, and the test is
advantageously performed on an animal of the same species and age than the
population to be vaccinated) after the administration of a colored liquid
having preferably the same viscosity than the intended vaccine. This test
may be performed directly with the intended vaccine containing further a
dye. This test allows the one skilled in the art to adjust the mechanical
specifications of an injector.
The needle-free injector may be equipped with a head comprising one or
several nozzles. The use of several nozzles allows to increase the
dispersion pattern of the vaccine over a larger area. There can be from 1
to 10 nozzles, preferably from 1 to 6.
Several injectors are available in the commerce. The Vitajet.TM.3 (Bioject
Inc.) is particularly adapted to the method according to the invention.
It is advantageous to use an injector equipped with means allowing to fit
to the injector directly a standard vial or ampoule. In addition, the
vaccine vial may comprise several vaccine doses allowing several shots of
vaccine and/or vaccination of several animals using the injector and the
same vial. Thus, the injector is preferably able to perform successive
injections from a same vial.
The invention also relates to a method to stimulate the immune response of
a vertebrate. In one embodiment, the vertebrate is a bird, cat, cow, dog,
fish, goat, horse, human, mouse, monkey, pig, rat or sheep. In a more
advantageous embodiment, the vertebrate is a cat.
In one aspect of the invention, vaccination against FeLV can be associated
with a vaccination against another feline disease. The vaccine comprises
the viral vector according to the invention and a vaccine component able
to protect against these other diseases, notably feline herpesvirus
disease, feline calicivirus disease, feline panleukopenia, parainfluenza
virus type 2 disease, feline immunodeficiency disease, and chlamydiosis.
The volume of dose injected may be from about 0.1 ml to about 1.0 ml,
preferably about 0.1 ml to about 0.8 ml, more preferably from about 0.2 ml
to about 0.5 ml, and in a preferred use the volume of dose injected may be
0.25 ml. By definition, the volume of one dose means the total volume of
vaccine administered at once to one animal.
The vaccine may contain from about 10.sup.4.5to about 10.sup.8.0
TCID.sub.50/dose (50% tissue culture infective dose per dose of vaccine)
and preferably from about 10.sup.5.5 to about 10.sup.6.5 TCID.sub.50/dose.
Optionally, the administration can be repeated, as booster administration,
at suitable intervals if necessary or desirable, e.g. about from 2 to
about 8 weeks after the first administration, and preferably about from 3
to about 5 weeks after the first administration. A booster administration
can also be repeated every year.
Another object of the invention is the use of an efficient amount of a
recombinant viral vector encoding and expressing at least one FeLV
immunogen as described above and of an acceptable vehicle or diluent, for
the preparation of a liquid recombinant viral vaccine designed to be
administered essentially in the dermis and the hypodermis of an animal of
the felidae family using a liquid jet needle-free injector as described
above, and resulting in eliciting a safe and protective immune response
Another object is a vaccination kit or set, comprising such a liquid jet
needle-free injector and at least one vaccine vial containing a FeLV
recombinant vaccine based on a viral vector as described above,
operatively assembled to perform the administration of the vaccine to an
animal of the felidae family. The distribution of the vaccine is
essentially done in the dermis and the hypodermis.
Such vaccination kit or set is able to elicit a safe and protective immune
response against FeLV.
Claim 1 of 18 Claims
1. A method of eliciting a safe and
protective immune response against FeLV comprising administering a vaccine
comprising an effective amount of a recombinant avipox vector containing
and expressing an exogeneous nucleotide sequence encoding at least one
FeLV immunogen and an acceptable vehicle or diluent, to a felidae with a
liquid jet needle-free injector, wherein the vaccine comprises from about
10.sup.4.5 to about 10.sup.7.0 TCID.sub.50/dose.
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