NZ620566B2 - Recombinant feline leukemia virus vaccine containing optimized feline leukemia virus envelope gene - Google Patents
Recombinant feline leukemia virus vaccine containing optimized feline leukemia virus envelope gene Download PDFInfo
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- NZ620566B2 NZ620566B2 NZ620566A NZ62056612A NZ620566B2 NZ 620566 B2 NZ620566 B2 NZ 620566B2 NZ 620566 A NZ620566 A NZ 620566A NZ 62056612 A NZ62056612 A NZ 62056612A NZ 620566 B2 NZ620566 B2 NZ 620566B2
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- felv
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Abstract
Discloses a composition comprising an expression vector comprising a first polynucleotides encoding an optimised Feline Leukemia Virus (FeLV) envelope (ENV) polypeptide and a second polynucleotide encoding an FeLV GAG/PRO polypeptide, wherein the optimised FeLV ENV polypeptide comprises a mutation at amino acid position 527 or equivalent corresponding amino acid position of an FeLV ENV protein, and wherein the mutation comprises a substitution of arginine (R), aspartic acid (D), or methionine (M) for glutamic acid (E), and wherein the polynucleotide encodes an optimised FeLV ENV polypeptide having the sequence as set forth in SEQ ID NO: 2, 4, 27, 28, 29, 30, 31, 32, 33 or 34, wherein the sequences are as defined in the complete specification. t amino acid position 527 or equivalent corresponding amino acid position of an FeLV ENV protein, and wherein the mutation comprises a substitution of arginine (R), aspartic acid (D), or methionine (M) for glutamic acid (E), and wherein the polynucleotide encodes an optimised FeLV ENV polypeptide having the sequence as set forth in SEQ ID NO: 2, 4, 27, 28, 29, 30, 31, 32, 33 or 34, wherein the sequences are as defined in the complete specification.
Description
RECOMBINANT FELINE LEUKEMIA VIRUS VACCINE CONTAINING OPTIMIZED
FELINE LEUKEMIA VIRUS ENVELOPE GENE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]
This application claims priority to U.S. provisional application 61/509,912 filed July
, 2011.
FIELD OF THE INVENTION
100021 The present invention relates to compositions or vaccines for combating feline
leukemia virus infections in animals. Specifically, the present invention provides vectors that
contain and express
in vivo or in vitro optimized feline leukemia virus envelope antigens that
elicit an immune response in animals against feline leukemia virus, including compositions
comprising said vectors, methods of vaccination against feline leukemia virus, and kits for use
with such methods and compositions.
BACKGROUND OF THE INVENTION
Feline Leukemia Virus (FeLV) is a common cause of infection of domestic cats
throughout the world and a cause of significant morbidity and mortality. The prevalence of
antigenaemia may vary from
I to 5 percent in healthy cats to 15 to 30 percent in sick cats (Hosie
M.J. et al.,
Veterinary Records, 1989, 128, 293-297; Braley J., Feline Practice, 1994, 22, 25-29;
Malik R. et aL, Australian Veterinary Journal, 1997, 75, 323-327; Arjona A. Journal
et al., of
Clinical Microbiology, 2000, 38, 3448-3449). The virus may establish a life-long infection
characterized by a persistent viraemia and a fatal outcome. Most FeLV-related diseases occur
persistently in infected animals, and they arc always serious and most likely fatal. Among the
most frequently diagnosed conditions are lymphomas, myeloid leukaemias, immunodeficiency
and non-regenerative anaemia. The infection can be controlled by the identification and isolation
of persistently viraemic cats, which are the source of the infection. Vaccines have also helped to
prevent the virus spreading. Several FeLV vaccines are available. Most of them contain either
inactivated virus or recombinant subunits. Their efficacy is controversial (Sparkes A.H., Journal
of Small Animal
Practice, 1997, 38, 187-194). Vaccine breakdowns have been observed.
An alternative way
would be to use recombinant viral vector, The canarypox virus
al.,
commercial recombinant FeLV vaccine is also available (EURIFEL® FeLV, Merial).
The FeLV genome codes for three genes: a GAG gene coding for the major structural
components of the virus, an ENV gene which codes for the envelope glycoprotein, and a POL gene
cndoing the polymerase protein (Thomsen D.R., et al., Journal of General Virology, 73, 1819-1824,
transmembrane protein p15E (DeNoronha, F., et al., 1978, Virology 85:617-621; Nunberg, J.H., et al.,
gammaretroviruses with immunosuppressive properties (Mathes, L.E. et al., 1978, Nature). FeLV
envelope glycoprotein is one of the major immunogens and is the target of FeLV-specific cytotoxic T cell
responses as well as neutralizing antibodies (Flynn, IN., et al., 2002, J. Virol.). US patent application US
2008/0008683 discussed a polypeptide that is capable of modulating the immunosuppressive properties
of a viral protein against the host in which it is expressed. The FeLV GAG gene encodes a precursor
polyprotein which is cleaved by the protease (FeLV PRO gene) to generate the capsid proteins. The
capsid proteins are also a major immunogen inducing FeLV-specific cytotoxic T cell responses as well as
neutralizing antibodies (Flynn, J.N., et al., 2002, J. Virol.). The POL gene encodes three proteins:
gives rise to all three proteins of the POL region (Thomsen D.R., et al., 1992).
There is a general need for an improvement in efficacy and safety of the FeLV vaccines and
SUMMARY OF THE INVENTION
vaccines as well as methods for treatment and prophylaxis of infection by FeLV.
vector and especially the ALVAC vector have been tested for the expression of FeLV genes (Tartaglia J.
Journal of Virology, 1993, 67, 2370-2375; Poulet H. et al., Veterinary Record, 2003, 153, 141-145). A
1992). The FeLV envelope (ENV) gene encodes a gp85 precursor protein which is proteolytically
processed by cellular enzymes(s) to yield the major envelope glycoprotein gp70 and the associated
1983, PNAS 81:3675-3679). The transmembrane protein pl5E contains a sequence conserved among
protease (PRO), reverse transcriptase and integrase. Autoprocessing by the protease portion of the gene
for more effective protection in field conditions.
A preferred embodiment of this invention can be any one or all of providing recombinant
vectors or viruses as well as methods for making such viruses, and providing compositions and/or
The invention provides a recombinant vector, such as a recombinant virus, e.g., a
recombinant poxvirus, that contains and expresses at least one exogenous nucleic acid molecule
and, the at least one exogenous nucleic acid molecule may comprise a nucleic acid molecule
encoding an immunogen or epitope of interest from FeLV proteins, such as FeLV ENV
and/or FeLV GAG/PRO.
In particular, the present invention provides a recombinant vector, such as a
recombinant virus, e.g., a recombinant poxvirus, that contains and expresses at least one
exogenous nucleic acid molecule and, the at least one exogenous nucleic acid molecule may
comprise FeLV polypeptides and/or variants or fragments thereof.
The invention further provides compositions or vaccine comprising such an
expression vector or the expression product(s) of such an expression vector.
[0010A] The invention provides a composition comprising an expression vector
comprising a first polynucleotides encoding an optimized Feline Leukemia Virus (FeLV)
envelope (ENV) polypeptide and a second polynucleotide encoding an FeLV GAG/PRO
polypeptide, wherein the optimized FeLV ENV polypeptide comprises a mutation at amino
acid position 527 or equivalent corresponding amino acid position of an FeLV ENV protein,
and wherein the mutation comprises a substitution of arginine (R), aspartic acid (D), or
methionine (M) for glutamic acid (E), and wherein the polynucleotide encodes an optimized
FeLV ENV polypeptide having the sequence as set forth in SEQ ID NO: 2, 4, 27, 28, 29, 30,
31, 32, 33 or 34.
The invention further provides methods for inducing an immunological (or
immunogenic) or protective response against FeLV, as well as methods for preventing FeLV
or disease state(s) caused by FeLV, comprising administering the expression vector or an
expression product of the expression vector, or a composition comprising the expression
vector, or a composition comprising an expression product of the expression vector.
The invention also relates to expression products from the virus as well as
antibodies generated from the expression products or the expression thereof in vivo and uses
for such products and antibodies, e.g., in diagnostic applications.
These and other embodiments are disclosed or are obvious from and
encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF DRAWINGS
[0014] The following detailed description, given by way of example, and which is not
intended to limit the invention to specific embodiments described, may be understood in
conjunction with the accompanying figures, incorporated herein by reference, in which:
Figure 1 provides a table identifying the SEQ ID NO assigned to the
polynucleotide and protein sequence.
Figure 2 depicts a plasmid map of pH6C5env (208.2).
Figure 3 provides the sequences for plasmid pCXL208.2 (pH6C5env) fragment
containing FeLV ENV DNA and left and right arms (SEQ ID NO:36) and FeLV ENV
protein (SEQ ID NO:7) from plasmid pHCMV-ENV FeLV.
[0018] Figure 4 provides the restriction map for plasmid pPB713.
Figure 5 provides the sequence alignments of the FeLV ENV DNA and proteins.
Figure 6 provides the plasmid pPB712 restriction map.
(SEQ ID NO:11) and codon-optimized GAG/PRO DNA (SEQ ID NO:10).
Figure 8 provides the cloning scheme.
[0023]
Figure 11 shows the nucleotide sequence of the pJY1874.1 DNA fragment containing
the arms and insert (SEQ ID NO:38).
[0027] Figure 13 shows the vCP2294 plasmid C3 region map with primer locations.
Figure 16 shows the vCP2296 plasmid C5 region map with primer locations.
Figure 19 is a graph showing the evolution of the mean proviremia per group after
status after challenge.
[0035]
Figure 23 shows the FeLV specific (ENV peptide pool No. 1)
Figure 25 shows the FeLV specific (GAG/PRO peptide pools) — IL-20 response on
D126.
(ENV stimulation) — IL-10 response on D126.
IL-10 response on
D126.
Figure 7 shows the DNA sequence alignment between wild-type GAG/PRO DNA
Figure 9 provides the restriction map of plasmid pJY1874.1.
Figure 10 provides the FeLV GAG-PRO protein sequence.
Figure 12 provides the cloning scheme for making vCP2294 plasmid.
Figure 14 depicts the vCP2294 plasmid sequence (annotated).
Figure 15 provides the cloning scheme for making vCP2296 plasmid.
Figure 17 provides the cloning scheme for making vCP2295 plasmid.
Figure 18 depicts the vCP2295 plasmid sequence.
challenge.
Figure 20 is a graph showing the evolution of the mean proviremia per group and p27
Figure 21 is a graph showing the proviremia in marrow correlating to p27 status.
Figure 22 shows the FeLV specific- IFNy response on D35.
IFNy response on D35.
Figure 24 shows the FeLV specific (ENV peptide pools) IL-10 response on D35.
D35.
Figures 26a-b show the FeLV specific (ENV stimulation) — IFNy/IL-10 ratio on D35.
Figure 27 shows the FeLV specific (GAG/PRO stimulation) - IFNy response on
Figure 28a shows the FeLV
specific
Figure 28b shows the FeLV specific (GAG/PRO stimulation)
Figure 29 shows the FeLV specific IFNI/AL-10 ratio FeLV ENV and GAG/PRO
DETAILED DESCRIPTION
[0044]
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.
Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V.
02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1569-8).
indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context
The term "FeLV ENV polypeptide or DNA" refers to any native or
FeLV ENV DNA may be optimized to produce a single amino acid mutation in the FeLV
as used herein may be selected from the group consisting of equine (e.g., horse), canine (e.g.,
big cats, and other felines including cheetahs and lynx), bovine (e.g., cattle), porcine (e.g., pig),
peptide pools on D35.
It is noted that in this disclosure and particularly in the claims, 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
Unless otherwise noted, technical terms are used according to conventional usage.
published by Oxford University Press, 1994 (ISBN 0854287-9); Kendrew et al. (eds.), The
Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0
The singular terms "a," "an," and "the" include plural referents unless context clearly
clearly indicate otherwise. The word "or" means any one member of a particular list and also
includes any combination of members of that list.
optimized/mutated FeLV ENV polypeptide or DNA, and their derivatives and variants. For
example, the optimized/mutated FeLV ENV DNA may be codon-optimized FeLV DNA, the
polypeptide. The optimized/mutated FeLV ENV polypeptide may comprise a single amino acid
mutation, or a double amino acid mutation, or a multiple amino acid mutation.
The term "animal" is used herein to include all mammals, birds and fish. The animal
dogs, wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wild cats, other
pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g., prosimian,
tarsier, monkey, gibbon, ape), humans, and fish. The term "animal" also includes an individual
The terms "polypeptide" and "protein" are used interchangeably herein to refer to a
polymer of consecutive amino acid residues.
100501 The term "nucleic acid", "nucleotide", and "polynucleotide" refers to RNA or DNA
(e.g. by chemical synthesis, by gene cloning etc.) and can take various forms (e.g. linear or
genomic sequence, or just the coding sequences as in cDNAs , such as an open reading frame
such as transcription initiation, translation and transcription termination. Thus, also included are
between 60 and 250 nucleotides upstream of the start codon of the coding sequence or gene;
Doree S M et al.; Pandher K Chung J Y
terminator is located within approximately 50 nucleotides downstream of the stop codon of the
Gene or polynucleotide also refers to a nucleic acid
fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which
The term "immunogenic polypeptide" or "immunogenic fragment" as used herein
molecule and induce a cytotoxic T lymphocyte ("CTL") response, and/or a B (for
ovine (e.g., sheep, goats, lamas, bisons), avian (e.g., chicken, duck, goose, turkey, quail,
animal in all stages of development, including embryonic and fetal stages.
[0049]
and derivatives thereof, such as those containing modified backbones. It should be appreciated
that the invention provides polynucleotides comprising sequences complementary to those
described herein. Polynucleotides according to the invention can be prepared in different ways
branched, single or double stranded, or a hybrid thereof, primers, probes etc.).
100511
The term "gene" is used broadly to refer to any segment of polynucleotide associated
with a biological function. Thus, genes or polynucleotides include introns and exons as in
(ORF), starting from the start codon (methionine codon) and ending with a termination signal
(stop codon). Genes and polynucleotides can also include regions that regulate their expression,
promoters and ribosome binding regions (in general these regulatory elements lie approximately
et al.; et al.), transcription terminators (in general the
coding sequence or gene; Ward C K et al.).
includes regulatory sequences.
[00521
refers to a polypeptide or a fragment of a polypeptide which comprises an allele-specific motif,
an epitope or other sequence such that the polypeptide or the fragment will bind an MHC
cell response
example, antibody production), and/or T-helper lymphocyte response, and/or a delayed type
hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide
or the immunogenic fragment is derived. A DTH response is an immune reaction in which T
cell-dependent macrophage activation and inflammation cause tissue injury. A DTH reaction to
the subcutaneous injection of antigen is often used as an assay for cell-mediated immunity.
the sense that once administered to the host, it is able to evoke an immune response of the
humoral (B cells) and/or cellular type (T cells). These are particular chemical groups or peptide
peptide sequence in a polypeptide, a tri- to penta-glycoside sequence in a polysaccharide. In the
animal most antigens will present several or even many antigenic determinants simultaneously.
Such a polypeptide may also be qualified as an immunogenic polypeptide and the epitope may
be identified as described further.
An "isolated" biological component (such as a nucleic acid or protein or organelle)
instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.
Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified
[0055]
embodiments at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%,
or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for
By definition, an epitope is an antigenic determinant that is immunologically active in
sequences on a molecule that are antigenic. An antibody specifically binds a particular antigenic
epitope on a polypeptide. Specific, non-limiting examples of an epitope include a tetra- to penta-
refers to a component that has been substantially separated or purified away from other
biological components in the cell of the organism in which the component naturally occurs, for
by standard purification methods. The term also embraces nucleic acids and proteins prepared
by recombinant technology as well as chemical synthesis.
The term "purified" as used herein does not require absolute purity; rather, it is
intended as a relative term. Thus, for example, a purified polypeptide preparation is one in
which the polypeptide is more enriched than the polypeptide is in its natural environment. A
polypeptide preparation is substantially purified such that the polypeptide represents several
of the total polypeptide content of the preparation. The same applies to polynucleotides. The
polypeptides disclosed herein can be purified by any of the means known in the art.
A recombinant polynucleotide is one that has a sequence that is not naturally
occurring or has a sequence that is made by an artificial combination of two otherwise separated
segments of sequence. This artificial combination is often accomplished by chemical synthesis
example, by genetic engineering techniques. In one embodiment, a recombinant poly -nucleotide
In one aspect, the present invention provides optimized or mutated polypeptides from
FeLV. In another aspect, the present invention provides optimized or mutated FeLV ENV
NOs: 2, 4, 6, 27, 28, 29, 30, 31, 32, 33, 34, or 43 or amino acid position 533 of SEQ ID NO:7. In
32, 33, 34, or 43, or amino acid position 533 of SEQ ID NO:7, It is appreciated by a person
skilled in the art that based on sequence alignment, the described mutation encompasses the
amino acid position 533 of SEQ ID NO:7. The protein sequence alignment of some of the FeLV
ENV polypeptides is exemplified in Figure id. In one embodiment, the optimized or mutated
embodiment, the optimized or mutated FeLV ENV polypeptide comprises the amino acid
substitution of R, D or M for E at amino acid position 527 of SEQ ID NO:6 or at the
corresponding amino acid position of FeLV ENV polypeptide. In yet another embodiment, the
optimized or mutated FeLV ENV polypeptide comprises the amino acid substitution of R for E
at amino acid position 527 of SEQ ID NO:6 or at the corresponding amino acid position of FeLV
ENV polypeptide. In yet another embodiment, the mutated FELV ENV polypeptide has the
sequence as set forth in SEQ ID NO:2, 4, 7, or 43 .
Moreover, homologs of polypeptides from FeLV are intended to be within the scope
paralogs. The tern "anologs" refers to two polynucleotides or polypeptides that have the same or
encodes a fusion protein.
polypeptides. In yet another aspect, the present invention provides an optimized FeLV ENV
protein wherein a mutation occurs at, but not limited to, the amino acid position 527 of SEQ ID
yet another aspect, the mutation is a substitution of arginine (R), aspartic acid (D), or methionine
(M) for glutamic acid (E) at amino acid position 527 of SEQ ID NOs: 2, 4, 6, 27, 28, 29, 30, 31,
mutation at the corresponding amino acid position in other FeLV ENV polypeptides which are
not listed in the present application, wherein the corresponding amino acid position is equivalent
to the amino acid position 527 of SEQ ID NOs: 2, 4, 6, 27, 28, 29, 30, 31, 32, 33, 34, or 43, or
FeLV ENV polypeptide comprises an amino acid mutation at amino acid position 527 of SEQ ID
NO:6 or at the corresponding amino acid position of FeLV ENV proteins. In yet another
of the present invention. As used herein, the term "homologs" includes orthologs, analogs and
similar function, but that have evolved separately in unrelated organisms. The term "orthologs"
refers to two polynucleotides or polypeptides from different species, but that have evolved from
a common ancestral gene by speciation. Normally, orthologs encode polypeptides having the
same or similar functions. The term "paralogs" refers to two polynucleotides or polypeptides that
are related by duplication within a genome. Paralogs usually have different functions, but these
functions may be related. Analogs, orthologs, and paralogs of a wild-type FeLV polypeptide can
differ from the wild-type FeLV polypeptide by post-translational modifications, by amino acid
sequence differences, or by both. In particular, homologs of the invention will generally exhibit
at least 80-85%, 85-90%, 90-95%, or 95%, 96%, 97%, 98% , 99% sequence identity, with all or
part of the wild-type FeLV polypeptide or polynucleotide sequences, and will exhibit a similar
function.
In another aspect, the present invention provides an optimized or mutated FeLV ENV
polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide having a sequence as set forth
in SEQ
ID NO: 2, 4, 6, 27, 28, 29, 30, 31, 32, 33, or 34.
In yet another aspect, the present invention provides fragments and variants of the
optimized or mutated FeLV ENV polypeptides identified above, which may readily be prepared
by one of skill in the art using well-known molecular biology techniques.
Variants are homologous polypeptides having an amino acid sequence at least 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence as set forth
in SEQ ID
NO: 2, 4, 6, 27, 28, 29, 30, 31, 32, 33, or 34.
Variants include allelic variants. The term "allelic variant" refers to a polynucleotide
or a polypeptide containing polymorphisms that lead to changes in the amino acid sequences of a
protein and that exist within a natural population (e.g., a virus species or variety). Such natural
allelic variations can typically result in 1- 5% variance in a polynucleotide or a polypeptide.
Allelic variants can be identified by sequencing the nucleic acid sequence of interest in a number
of different species, which can be readily carried out by using hybridization probes to identify
the same gene genetic locus in those species. Any and all such nucleic acid variations and
resulting amino acid polymorphisms or variations that are the result of natural allelic variation
and that do not alter the functional activity of gene if interest, are intended to be within the scope
of the invention.
0063]
As used herein, the term "derivative" or "variant" refers to a polypeptide, or a nucleic
acid encoding a polypeptide, that has one or more conservative amino acid variations or other
minor modifications such that (I) the corresponding polypeptide has substantially equivalent
polypeptide is immunoreactive with the wild-type polypeptide. These variants or derivatives
polypeptide primary amino acid sequences that may result in peptides which have substantially
further contemplates deletions, additions and substitutions to the sequence, so long as the
polypeptide functions to produce an immunological response as defined herein. The
modifications may be any amino acid change at amino acid positions other than position 527 of
10064]
basic--lysine, arginine, histidine; (3) non-polar--alanine, valine, leucine, isoleucine, proline,
cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes
classified as aromatic amino acids. Examples of conservative variations include the substitution
hydrophobic residue, or the substitution of one polar residue for another polar residue, such as
asparagine, and the like; or a similar conservative replacement of an amino acid with a
structurally related amino acid that will not have a major effect on the biological activity.
possessing minor amino acid substitutions that do not substantially affect the immunogenicity of
variation" also includes the use of a substituted amino acid in place of an unsubstituted parent
function when compared to the wild type polypeptide or (2) an antibody raised against the
include polypeptides having minor modifications of the optimized or mutated FeLV ENV
equivalent activity as compared to the unmodified counterpart polypeptide. Such modifications
may be deliberate, as by site-directed mutagenesis, or may be spontaneous. The term "variant"
SEQ ID NOs: 2, 4, 6, 27, 28, 29, 30, 31, 32, 33, 34, or 43, or amino acid position 533 of SEQ ID
NO:7.
The term "conservative variation" denotes the replacement of an amino acid residue
by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid
sequence such that the encoded amino acid residue does not change or is another biologically
similar residue. In this regard, particularly preferred substitutions will generally be conservative
in nature, i.e., those substitutions that take place within a family of amino acids. For example,
amino acids are generally divided into four families: (1) acidic--aspartate and glutamate; (2)
phenylalanine, methionine, tryptophan; and (4) uncharged polar--glycine, asparagine, glutamine,
of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another
the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for
Proteins having substantially the same amino acid sequence as the reference molecule but
the protein are, therefore, within the definition of the reference polypeptide. All of the
polypeptides produced by these modifications are included herein. The term "conservative
acids, or at least 30 amino acids of an FeLV ENV polypeptide having a sequence as set forth in
SEQ ID NO: 2, 4, 6, 7, 27, 28, 29, 30, 31, 32, 33, 34, or 43, or variants thereof. In another
embodiment, a fragment of an FeLV ENV polypeptide includes a specific antigenic epitope
1985; Van der Zee R. et al.; et al.;
T. Parker K. et al.),
non-limiting examples of epitopes include a tetra- to penta- peptide sequence in a polypeptide, a
protein fragment of a larger molecule it will have substantially the same immunological activity
100671
ENV polypeptide. In another aspect, the present invention provides an FeLV ENV
43, or amino acid position 533 of SEQ ID NO:7. In yet another aspect, the FeLV ENV
amino acid position 527 of SEQ ID NOs: 2, 4, 6, 7, 28, 29, 30, 31, 32, 33, 34, or 43, or amino
amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with
the unsubstituted polypeptide.
An immunogenic fragment of an FeLV ENV polypeptide includes at least 8, 10, 15,
or 20 consecutive amino acids, at least 21 amino acids, at least 23 amino acids, at least 25 amino
found on a full-length FeLV ENV polypeptide.
Procedures to determine fragments of polypeptide and epitope such as, generating
to overlapping peptide libraries (Hemmer a et al.), Pepscan (Geysen H. M. et 41984; Geysen H.
M. et al., Geysen H. M.) and algorithms (De Groot A.
Hoop
et aL; can be used in the practice of the invention, without undue
experimentation. Generally, antibodies specifically bind a particular antigenic epitope. Specific,
tri- to penta glycoside sequence in a polysaccharide. In animals most antigens will present
several or even many antigenic determinants simultaneously. Preferably wherein the epitope is a
as the total protein.
In one aspect, the present invention provides a polynucleotide encoding an FeLV
polynucleotide encoding an optimized or mutated FeLV ENV polypeptide, wherein the mutation
occurs at the amino acid position 527 of SEQ ID NOs: 2, 4, 6, 27, 28, 29, 30, 31, 32, 33, 34, or
polynucleotide encodes an optimized or mutated FeLV ENV polypeptide wherein the mutation is
a substitution of arginine (R), aspartic acid (D), or methionine (M) for glutamic acid (E) at the
acid position 533 of SEQ ID NO:7. In yet another aspect, the FeLV ENV polynucleotide encodes
an optimized or mutated FeLV ENV polypeptide having an amino acid mutation at amino acid
position 527 of SEQ ID NO:6 or at the corresponding amino acid position of FeLV ENV
proteins. In another aspect, the FeLV ENV polynucleotide encodes an optimized or mutated
FeLV ENV polypeptide having the amino acid change of E to R, D or M at amino acid position
527 of SEQ ID NO:6 or at the corresponding amino acid position of FeLV ENV polypeptide, In
yet another aspect, the FeLV ENV polynucleotide encodes an optimized or mutated FeLV ENV
polypeptide having the amino acid change of E to R at amino acid position 527 of SEQ ID NO:6
or at the corresponding amino acid position of FeLV ENV polypeptide. In yet another
embodiment, the FeLV ENV polynucleotide encodes an FeLV ENV polypeptide having the
sequence as set forth in SEQ ID NO:2, 4, 7, or 43. In yet another embodiment, the FeLV ENV
polynucleotide encodes an FeLV ENV polypeptide having at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a
polypeptide having a sequence as set forth in SEQ ID NO: 2, 4, 6, 7, 27, 28, 29, 30, 31, 32, 33,
34, or 43, or a conservative variant, an allelic variant, a homolog or an immunogenic fragment
comprising at least eight or at east ten consecutive amino acids of one of these polypeptides, or a
combination of these polypeptides.
[00681
In another aspect, the present invention provides an FeLV GAG-PRO polypeptide
having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%,
97%, 98% or 99% sequence identity to a polypeptide having a sequence as set forth in SEQ ID
NO: 12.
[00691
In another aspect, the present invention provides an FeLV ENV polynucleotide
having a nucleotide sequence as set forth in SEQ ID NO: 1, 3, or 5, or a variant thereof. In yet
another aspect, the present invention provides an FeLV ENV polynucleotide having at least 70%,
at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, 96%, 97%, 98%
or 99% sequence identity to a polynucleotide having a sequence as set forth in SEQ ID NO: 1, 3,
or 5, or a variant thereof
[00701 In yet another aspect, the present invention provides an FeLV GAG-PRO
polynucleotide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least
95%, at least 95%, 96%, 97%, 98% or 99% sequence identity to a polynucleotide having a
sequence as set forth in SEQ ID NO: 10, or 11, or a variant thereof.
These polynucleotides may include DNA, eDNA, and RNA sequences that encode
FeLV ENV or GAG-PRO polypeptides. It is understood that all polynucleotides encoding FeLV
ENV or GAG-PRO polypeptides are also included herein, as long as they encode a polypeptide
with the recognized activity, such as the binding to an antibody that recognizes the polypeptide,
the induction of an immune response to
the polypeptide, or an effect on survival of Leukemia
sign or a symptom of FeLV infection.
result of the genetic code, e.g., optimized codon usage for a specific host. As used herein,
"optimized" refers to a polynucleotide that is genetically engineered to increase its expression in
comprise codons preferred by highly expressed genes in a particular species; 2) comprise an A+T
or G+C content in nucleotide base composition to that substantially found in said species; 3)
form an initiation sequence of said species; or 4) eliminate sequences that cause destabilization,
structure hairpins or RNA splice sites. Increased expression of FeLV protein in said species can
be achieved by utilizing the distribution frequency of codon usage in eukaryotes and prokaryotes,
There are 20 natural amino acids, most of which are specified by more than one codon.
as the
functionally unchanged.
[0073] The sequence identity between two amino acid sequences may be established by the
NCBI (National Center for Biotechnology Information) pairwise blast and the blosum62 matrix,
in Altschul and thus, this document speaks of using the algorithm or the BLAST or
available via the
Internet at sites thereon such as the NCBI site.
disease when administered to a subject exposed to the parasite or who undergoes a decrease in a
The polynucleotides of the disclosure include sequences that are degenerate as a
a given species. To provide optimized polynucleotides coding for an FeLV ENV or GAG-PRO
polypeptide, the DNA sequence of the FeLV ENV or GAG-PRO gene can be modified to 1)
inappropriate polyadenylation, degradation and termination of RNA, or that form secondary
or in a particular species. The term "frequency of preferred codon usage" refers to the preference
exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid.
Therefore, all degenerate nucleotide sequences are included in the disclosure as long
amino acid sequence of the FeLV polypeptide encoded by the nucleotide sequence is
using the standard parameters (see, e.g., the BLAST or BLASTX algorithm available on the
"National Center for Biotechnology Information" (NCBI, Bethesda, Md., USA) server, as well as
et al.;
BLASTX and BLOSUM62 matrix by the term "blasts").
Sequence identity between two nucleotide sequences also may be determined using
the "Align" program of Myers and Miller, ("Optimal Alignments in Linear Space", CABIOS 4,
11-17, 1988) and available at NCBI, as well as the same or other programs
Alternatively or additionally, the term "identity", for instance, with respect to a
nucleotide or amino acid sequence, may indicate a quantitative measure of homology between
two sequences. The percent sequence homology may be calculated as:
(Nref Ndif)*100/N
100761 ref , wherein Nof is the total number of non-identical residues in
the two sequences when aligned and wherein
Nref is the number of residues in one of the
sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with
the sequence AATCAATC (Nr f = 8; Ndr2).
Alternatively or additionally, "identity" with respect to sequences can refer to the
number of positions with identical nucleotides or amino acids divided by the number of
nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two
sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur and
Lipman), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and
a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data
including alignment can be conveniently performed using commercially available programs
(e.g., Intel ligeneticsTm Suite, Intelligenetics Inc. CA). When RNA sequences are said to be
similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T)
in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA
sequences are within the scope of the invention and can be derived from DNA sequences, by
thymidine (T)
in the DNA sequence being considered equal to uracil (U) in RNA sequences.
[0078] The sequence identity or sequence similarity of two amino acid sequences, or the
sequence identity between two nucleotide sequences can be determined using Vector NTI
software package (Invitrogen, 1600 Faraday Ave., Carlsbad, CA).
The FeLV ENV or GAG-PRO polynucleotides may include a recombinant DNA
which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the
genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (for example,
a eDNA) independent of other sequences.
100801
Recombinant vectors disclosed herein may include a polynucleotide encoding a
polypeptide, a variant thereof or a fragment thereof. Recombinant vectors may include plasmids
and viral vectors and may be used for in vitro or
in vivo expression. Recombinant vectors may
include further a signal peptide. Signal peptides are short peptide chain (3-60 amino acids long)
that direct the post -translational transport
of a protein (which are synthesized in the cytosol) to
certain organelles such as the nucleus, mitochondrial matrix, endoplasmic reticulurn, ehloroplast,
apoplast and peroxisome. Typically, the naturally occurring FeLV ENV proteins may be
translated as precursors, having an N-terminal signal peptide sequence and a "mature" protein
domain. The signal peptide may be cleaved off rapidly upon translation. The signal sequence
may be the natural sequence from the FeLV ENV protein or a peptide signal from a secreted
protein e.g. the signal peptide from the tissue plasminogen activator protein (tPA), in particular
the human tPA (S. Friezner Degen et al.; R. Rickles et al.; D. Berg. et al.), or the signal peptide
from the Insulin-like growth factor I (IGF1), in particular the equine IGF1 (K. Otte et al.), the
canine IGF I (P. Delafontaine et at), the feline IGF1 (W003/022886), the bovine IGF1 (S. Lien
et al.), the porcine IGF1 (M. Muller
et al.), the chicken IGF1 (Y. Kajimoto et al.), the turkey
IGF I (GenBank accession number AF074980). The signal peptide from IGF1 may be natural or
optimized which may be achieved by removing cryptic splice sites and/or by adapting the codon
usage. Upon translation, the unprocessed polypeptide may be cleaved at a cleavage site to lead to
the mature polypeptide. The cleavage site may be predicted using the method of Von Heijne
(1986).
A plasmid may include a DNA transcription unit, for instance a nucleic acid sequence
that permits it to replicate in a host cell, such as an origin of replication (prokaryotic or
eukaryotic). A plasmid may also include one or more selectable marker genes and other genetic
elements known in the art. Circular and linear forms of plasmids are encompassed in the present
disclosure.
In a further aspect, the present invention relates to an in vivo expression vector
comprising a polynucleotide sequence, which contains and expresses
in viva in a host the
optimized or mutated FeLV ENV polypeptides and/or variants or fragments thereof. The
expression vector may further comprise a polynucleotide encoding an FeLV GAG-PRO
polypeptide and/or variants or fragments thereof.
in vivo expression vector may include any transcription unit containing a
polynucleotide or a gene of interest and those essential elements for its in vivo expression. These
expression vectors may be plasmids or recombinant viral vectors. For in vivo expression, the
promoter may be of viral or cellular origin. In one embodiment, the promoter may be the
cytomegalovirus (CMV) early promoter (CMV-IE promoter), the SV40 virus early or late
promoter or the Rous Sarcoma virus LTR promoter, a promoter of a cytoskeleton gene, such as
the desmin promoter (Kwissa M, et al.), or the actin promoter (Miyazaki J. et al.). When several
genes are present in the same plasmid, they may be provided in the same transcription unit or in
different units.
As used herein, the term "plasmid" may include 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. The plasmids may also comprise other transcription-regulating
elements such as, for example, stabilizing sequences of the intron type. In several embodiments,
the plasmids may include the first intron of CMV-IE (WO 89/01036), the intron II of the rabbit
beta-globin gene (van Ooyen et al.), the signal sequence of the protein encoded by the tissue
plasminogen activator (tPA; Montgomery et al.), and/or a polyadenylation signal (polyA), in
particular the polyA of the bovine growth hormone (bGH) gene (US 5,122,458) or the polyA of
the rabbit beta-globin gene or of SV40 virus.
100851 In a further aspect, the present invention relates to a composition comprising: a) an in
vivo
expression vector, wherein the vector comprises a polynucleotide encoding one or more
polypeptide selected from the group consisting of an FeLV ENV polypeptide, a variant or
fragment of the FeLV ENV polypeptide, and a mixture thereof; and b) a pharmaceutically or
veterinary acceptable vehicle, diluent or excipient.
[00861 In another aspect, the present invention relates to a composition comprising: a) an in
vivo
expression vector, wherein the vector comprises a polynucleotide encoding one or more
polypeptide selected from the group consisting of an FeLV ENV polypeptide, an FeLV
GAG/PRO polypeptide, a variant or fragment of the FeLV ENV polypeptide, and a mixture
thereof; and b) a pharmaceutically or veterinary acceptable vehicle, diluent or excipient.
100871 In yet another aspect, the present invention relates to a composition comprising: a) an
in vivo expression vector, wherein the vector comprises a polynucleotide encoding an FeLV
ENV polypeptide, an FeLV GAG/PRO polypeptide; and b) a pharmaceutically or veterinary
acceptable vehicle, diluent or excipient.
The FeLV ENV and FeLV GAG/PRO polypeptides are described above.
In one embodiment, the present invention relates to a composition comprising: a) an
in vivo
expression vector, wherein the vector comprises a polynucleotide encoding an optimized
or mutated FeLV ENV having the amino acid substitution of R, D or M for E at amino acid
position 527 of SEQ ID NO:6 or at the corresponding amino acid position of FeLV polypeptide
and a polynucleotide encoding an FeLV GAG/PRO polypeptide having at least 90% sequence
identity to a polypeptide having the sequence as set forth in SEQ ID NO:12; and b) a
pharmaceutically or veterinary acceptable vehicle, diluent or excipient. In yet another
embodiment, the composition of the present invention comprises: a) an expression vector
comprising a first polynucleotide encoding an FeLV ENV polypeptide having an amino acid
sequence as set forth in SEQ ID NO:2 or 4 and a second polynucleotide encoding an FeLV
GAG/PRO polypeptide having an amino acid sequence as set forth in SEQ ID NO:12; and b) a
pharmaceutically or veterinary acceptable vehicle, diluent or excipient.
100901 The term "composition" comprises any vaccine or immunological composition, once
it has been injected to a host, including canines, felines and humans, that induces an immune
response in the host, and/or protects the host from leukemia, and/or which may prevent
implantation of the parasite, and/or which may prevent disease progression in infected subjects,
and/or which may limit the diffusion of runaway parasites to internal organs. This may be
accomplished upon vaccination according to the present invention through the induction of
cytokine secretion, notably IFN-gamma secretion (as example of a method of measurement of
IFN-gamma secretion, the Quantikine® immunoassay from R&D Systems Inc. (catalog number#
CAIF00) could be used (Djoba Siawaya JF et al.)).
[0091] The pharmaceutically acceptable vehicles or excipients of use are conventional.
Remington's Pharmaceutical Sciences,
by E. W. Martin, Mack Publishing Co., Easton, PA, 15th
Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of
the polypeptides, plasmids, viral vectors herein disclosed. In general, the nature of the vehicle or
excipient will depend on the particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that include pharmaceutically and
physiologically acceptable fluids such as water, physiological saline, balanced salt solutions,
aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, freeze-
dried pastille, powder, pill, tablet, or capsule forms), conventional non-toxic solid vehicles or
excipients can include, for example, pharmaceutical grades of mannitol, lactose, starch, or
magnesium stearate. In addition to biologically neutral vehicles or excipients, immunogenic
compositions to be administered can contain minor amounts of non-toxic auxiliary substances,
such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for
100921
[00931 Multiple insertions may be done in the same vector using different insertion sites or
head-to- head, tail-to-head, or head-to-tail. IRES elements (Internal Ribosome Entry Site, see EP
0803573) can also be used to separate and to express multiple inserts operably linked to the same
in vivo expression vector, or an
polynucleotide aforementioned. The expression vector may be an
in vivo
10095]
P.) in vivo in a
in vivo expression.
100961
et al.; pVR2001-TOPA (or pVR2001-TOPO) (Oliveira F.
pAB110 (US 6,852,705) can be utilized as a vector for the insertion of a polynucleotide
increases the likelihood of producing a secreted protein, (see Figure 1 in Oliveira F.
100971 Each plasmid may comprise or contain or consist essentially of, the polynucleotide
example sodium acetate or sorbitan monolaurate.
The compositions or vaccines according to the instant invention may include vectors
encoding any polynucleotide according to the present invention as described above.
using the same insertion site. When the same insertion site is used, each polynucleotide insert,
which may be any polynucleotide of the present invention aforementioned, may be inserted
under the control of the same and/or different promoters. The insertion can be done tail-to-tail,
and/or different promoters.
100941 In one embodiment, the present invention relates to an expression vector comprising a
in vitro expression vector.
More generally, the present invention encompasses expression vectors
including any plasmid (EP-A2-1001025; Chaudhuri containing and expressing
host the polynucleotide or gene of FeLV ENV polypeptide, variant thereof or fragment thereof
and elements necessary for its
In a specific, non-limiting example, the pVR1020 or pVR1012 plasmid (VICAL Inc.;
Hartikka J.
Luke C. et al.), et al.) or
sequence. The pVR1020 plasmid is derived from pVR1012 and contains the human tPA signal
sequence. The pVR1020 is a plasmid backbone available from Vical, Inc., (San Diego, CA)
which has been previously used, see, e.g., US Patent Nos. 6,451,769 and 7,078,507. As described
in Oliveira et al., plasmid pVR2001-TOPO (or pVR2001-TOPA) is pVR1020 modified by the
addition of topoisomerases flanking the cloning site and containing coding for and expressing a
signal secretory peptide, for example, tissue plasminogen activator signal peptide (tPA), that
et al.).
according to the present invention, operably linked to a promoter or under the control of a
promoter or dependent upon a promoter, wherein the promoter may be advantageously adjacent
to the polynucleotide for which expression is desired. In general, it is advantageous to employ a
strong promoter that is functional in eukaryotic cells. One example of a useful promoter may be
the immediate early cytomegalovirus promoter (CMV-IE) of human or murine origin, or it may
optionally have another origin such as from rat or guinea pig, The CMV-IE promoter may
viral or a cellular origin. A strong viral promoter other than CMV-IE that may be usefully
promoter of the Rous sarcoma virus. A strong cellular promoter that may be usefully employed
promoter (Kwissa M. et al.), or the actin promoter (Miyazaki J. et al.). Functional sub fragments
of these promoters, i.e., portions of these promoters that maintain adequate promoter activity, are
and/or the enhancer portion of the full-Length promoter, as well as derivatives and/or sub
Advantageously, the plasmids comprise or consist essentially of other expression
sequence(s), for example, the first intron of the hCMV-1E (WO 89/01036), the intron 11 of the
rabbit 0-globin gene (van Ooyen
plasmids and viral vectors other than poxviruses, use can be made of the poly(A) signal of the
comprise the actual promoter part, which may or may not be associated with the enhancer part.
Reference can be made to EP 260 148, EP 323 597, US 5,168,062, 5,385,839, and 4,968,615, as
well as to WO 87/03905. The CMV-IE promoter may advantageously be a human CMV-IE
(Boshart M. et al.) or murine CMV-IE. In more general terms, the promoter may have either a
ta employed in the practice of the invention is the early/late promoter of the SV40 virus or the LTR
in the practice of the invention is the promoter of a gene of the cytoskeleton, such as the desmin
included within the present invention, e.g. truncated CMV-IE promoters according to WO
98/00166 or US 6,156,567 and may be used in the practice of the invention. A promoter useful in
the practice of the invention consequently may include derivatives and/or sub fragments of a full-
length promoter that maintain adequate promoter activity and hence function as a promoter, and
which may advantageously have promoter activity that is 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 US 6,156,567 in comparison to the activity of
full-length CMV-IE promoters. Thus, a CMV-IE promoter in the practice of the invention may
comprise or consist essentially of or consist of the promoter portion of the full-length promoter
fragments thereof.
100981
control elements. It is especially advantageous to incorporate stabilizing sequence(s), e.g., intron
et al.). As to the polyadenylation signal (polyA) for the
bovine growth hormone (bGH) gene (see US 5,122,458), or the poly(A) signal of the rabbit 13-
globin gene or the poly(A) signal of the SV40 virus.
100991 More generally, the present invention encompasses
in vivo expression vectors
including any recombinant viral vector containing a polynucleotide or gene encoding one or
more FeLV ENV and/or variants or fragments thereof, including any elements necessary for its
in vivo expression.
[01001 Said recombinant viral vectors could
be selected from, for example, the poxviruses,
especially avipox viruses, such as fowlpox viruses or canarypox viruses. In one embodiment, the
fowlpox virus is a TROVAC (see
WO 96/40241). In another embodiment, the canarypox vector
is
an ALVAC. The use of these recombinant viral vectors and the insertion of polynucleotides or
genes of interest are fully described in US 5,174,993; US 5,505,941 and US 5,766,599 for
fowlpox, and in US 5,756,103 for canarypox. More than one insertion site inside the viral
genome could be used for the insertion of multiple genes of interest.
[01011 In one embodiment the viral vector is an adenovirus, such as a human adenovirus
(HAV) or a canine adenovirus (CAV).
In another embodiment the viral vector is a human adenovirus, specifically a scrotype
adenovirus, rendered incompetent for replication by a deletion in the El region of the viral
genome, especially 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 Chroboczek et al, 1992. The deleted adenovirus is propagated in El-
expressing 293 (Graham et al., 1977) or PER cells, especially PER.C6 (Falloux et al,
1998). The
human adenovirus can additionally or
alternatively be deleted in the E3 region, especially from
about nucleotide 28592 to about nucleotide 30470. The deletion in the E 1 region can be done in
combination with a deletion in
the E3 region (see, e.g. Shriver et al.; Graham et al.; Ilan et al.;
U.S. Patent Nos. 6,133,028 and 6,692,956; Tripathy
et al.; Tapnell; Danthinne et al.; Berkner;
Berkner et al.; Chavier et al.).
The insertion sites can be the El and/or E3 loci (region)
eventually after a partial or complete deletion of the El 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,
advantageously a cytonnegalovirus immediate-early gene promoter (CMV-IE promoter),
especially the enhancer / promoter region from about nucleotide —734 to about nucleotide +7 in
et al.,
Boshart
sequence can be located downstream of the enhancer / promoter region. For example, the intron
1 isolated from the CMV-IE gene (Stenberg
P-globin gene, especially the intron 2 from the P-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. A poly(A) sequence and terminator
sequence can be inserted downstream the polynucleotide to be expressed, e.g. a bovine growth
with GenBank accession No. BOVGHRH, a rabbit P-globin gene or a SV40 late gene
polyadenylation signal.
U.S. Patent Nos. 5,529,780 and 5,688,920; WO 95/14102). For CAV, the
insertion sites can be in the E3 region and /or in the region located between the E4 region and the
is under the control of a promoter, such as a cytornegalovirus immediate-early gene promoter
sequence and terminator sequence can be inserted downstream the polynucleotide to be
In another embodiment, the viral vector is a herpesvirus such as a feline herpesvirus
For recombinant vectors based on a poxvirus vector, a vaccinia virus or an attenuated
passages of the Ankara vaccine strain on chicken embryo fibroblasts; see Stick' & Hochstein-
Mintzel; Sutter
see US 5,494,807, and U.S.
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 la can also be used. A muscle specific promoter can also be used (Li et al.). Strong
promoters are also discussed herein in relation to plasmid vectors. In one embodiment, a splicing
et al.), the intron isolated from the rabbit or human
hormone gene, especially from about nucleotide 2339 to about nucleotide 2550 of the sequence
In another embodiment the viral vector is a canine adenovirus, especially a CAV-2
(see, e.g. Fischer et al,;
right ITR region (see U.S. Patent Nos. 6,090,393 and 6,156,567). In one embodiment the insert
(CMV-IE promoter) or a promoter already described for a human adenovirus vector. A poly(A)
expressed, e.g. a bovine growth hormone gene or a rabbit P-globin gene polyadenylation signal.
10104]
(FHV). 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).
A poly(A) sequence and terminator sequence can be inserted downstream the polynucleotide to
be expressed, e.g. bovine growth hormone or a rabbit p-globin gene polyadenylation signal.
vaccinia virus, (for instance, MVA, a modified Ankara strain obtained after more than 570
et al.;
available as ATCC VR-1508; or NYVAC,
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 WO 96/40241), an avipox virus or an attenuated
avipox virus (e.g., canarypox, fowlpox, dovepox, pigeonpox, quailpox, ALVAC or TROVAC;
see, e.g., U.S. Patent Nos. 5,505,941, 5,494,807) can be used. Attenuated canarypox viruses are
described in US 5,756,103 (ALVAC) and WO 01/05934. Reference is also made to US5,766,599
which pertains to the attenuated fowlpox strain TROVAC. Reference is made to the canarypox
available from the ATCC under access number VR-111. Numerous fowlpox virus vaccination
strains are also available, e.g. the DIFTOSEC CT strain marketed by MERIAL and the NOBILIS
VARIOLE vaccine marketed by INTERVET. For information on the method used to generate
recombinants thereof and how to administer recombinants thereof, the skilled artisan can refer
documents cited herein and to WO 90/12882, e.g., as to vaccinia virus, mention is made of U.S.
Patents Nos. 4,769,330, 4,722,848, 4,603,112, 5,110,587, 5,494,807, and 5,762,938 inter alia; as
to fowlpox, mention is made of U.S. Patents Nos. 5,174,993, 5,505,941 and 5,766,599
inter alia;
as to canarypox, mention is made of U.S. Patent No. 5,756,103 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 fowlpox,
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
are advantageously as in various publications, including Carroll M. W. et al.; Stittelaar K. J.
al.; Sutter G. et aL;
and, in this regard it is also noted that the complete MVA genome is
described in Antoine G., Virology, which enables the skilled artisan to use other insertion sites or
other promoters. 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.), the
vaccinia promoter 13L (Riviere
et al.), the vaccinia promoter HA (Shida), the cowpox promoter
ATI (Funah .ashi et al.),
the vaccinia promoter H6 (Taylor J. et al.; Guo P. et al. J.; Perkus M. et
al.), inter alia.
101061
Any of the polynucleotides disclosed here may be expressed in vitro by DNA transfer
or expression vectors into a suitable host cell. The host cell may be prokaryotic or eukaryotic.
The term "host cell" also includes any progeny of the subject host cell. Methods of stable
transfer, meaning that the foreign polynucleotide is continuously maintained in the host cell, are
known in the art. Host cells may include bacteria (for example,
Escherichia coil), yeast, insect
cells, and vertebrate cells. Methods of expressing DNA sequences in eukaryotic cells are well
known in the art. As a method for in vitro expression, recombinant Baculovirus vectors (for
example, Autographa California Nuclear Polyhedrosis Virus (AcNPV)) may be used with the
nucleic acids disclosed herein. For example, polyhedrin promoters may be utilized with insect
cells (for example, Spodoptera,frugiperda
cells, like Sf9 cells available at the ATCC under the
Accession number CRL 1711, or Sf21 cells) (see for example, Smith et al.;
Pennock et al.;
Vialard et al.; Verne A.; O'Reilly
et al.; Kidd I. M. & Emery V.C.; EP 0370573; EP 0265785;
US 4,745,051). For expression, the BaculoGold Starter Package (Cat # 21001K) from
Pharmingen (Becton Dickinson) may be used. As a method for in vitro expression, recombinant
E. coil
may be used with a vector. For example, when cloning in bacterial systems, inducible
promoters such as arabinose promoter, pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac
hybrid promoter), and the like may be used. Transformation of a host cell with recombinant
DNA may be carried out by conventional techniques are well known to those skilled in the art.
Where the host is prokaryotic, such as
E. coli, competent cells which are capable of DNA uptake
can be prepared from cells harvested after exponential growth phase and subsequently treated by
the CaCl2 method using procedures well known in the art. Alternatively, MgCl2 or RbC1 can be
used. Transformation can also be performed by electroporation. When the host is a eukaryote,
such methods of transduction of DNA as calcium phosphate coprecipitates, conventional
mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in
liposomes, or virus vectors may be used. Eukaryotic cells may also be cotransformed with L.
longipalpis
polynucleotide sequences, and a second foreign DNA molecule encoding a selectable
phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector (see above), such as a herpes virus or adenovirus (for example, canine
adenovirus 2), to transiently transduce eukaryotic cells and express the protein (Gluzman EA). In
addition, a transfection agent can be utilized, such as dioleoyl-phosphatidyl-ethanolamme
(Don).
Isolation and purification of recombinantly expressed polypeptide may be carried out
by conventional means including preparative chromatography (for example, size exclusion, ion
exchange, affinity), selective precipitation and ultra-filtration. Examples of state of the art
techniques that can be used, but not limited to, may be found in "Protein Purification
Applications", Second Edition, Edited by Simon Roe and available at Oxford University Press.
Such a recombinantly expressed polypeptide is part of the present disclosure. The methods for
production of any polypeptide according to the present invention as described above are also
encompassed, in particular the use of a recombinant expression vector comprising a
polynucleotide according to the disclosure and of a host cell.
The vaccines containing recombinant viral vectors according to the invention may be
freeze-dried, advantageously with a stabilizer. Freeze-drying can be done according to well-
known standard freeze-drying procedures. The pharmaceutically or veterinary acceptable
stabilizers may be carbohydrates (e.g. sorbitol, mannitol, lactose, sucrose, glucose, dextran,
trehalose), sodium glutamate (Tsvetkov T et al.), proteins such as peptone,
albumin, lactalbumin or casein, protein containing agents such as skimmed milk (Mills C K
al.; et al.), and buffers (e.g. phosphate buffer, alkaline metal phosphate buffer). An
adjuvant may be used to make soluble the freeze-dried preparations.
Any vaccine composition according to the invention can also advantageously contain
one or more adjuvant.
The plasmid-based vaccines may be formulated with cationic lipids, advantageously
with DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propananunonium ;
W096/34109), and advantageously in association with a neutral lipid, for example DOPE
(dioleoyl-phosphatidyl-ethanolamine ; Behr J. P.), in order to form DMRIE-DOPE. In one
embodiment, the mixture is made extemporaneously, and before its administration it is
advantageous to wait about 10 min to about 60 min, for example, about 30 min, for the
appropriate mixture. When DOPE is used, the molar ratio of DMRIE/DOPE can be from 95/5 to
/95 and is advantageously 1/1. The weight ratio plasmid/DMRIE or DMRIE-DOPE adjuvant is,
for example, from 50/1 to 1/10, from 10/1 to 1/5 or from 1/1 to 1/2.
Optionally a cytokine may be added to the composition, especially GM-CSF or
cytokines inducing Thl (e.g. IL12). These cytokines can be added to the composition as a
plasmid encoding the cytokine protein. In one embodiment, the cytokines are from canine origin,
et al.; Israeli E
Wolff E
[0HO]
similar to what was made in WO 00/77210.
The recombinant viral vector-based vaccine may be combined with fMLP (N-formyl-
groups, advantageously not more than 8, the hydrogen atoms of at least three hydroxyls being
replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. For example, the
- -C
(CH2) x --
(CH2) y
COON
methyl. The products sold under the name CARBOPOL® (BF Goodrich, Ohio, USA) are
Among them, there may be advantageously mentioned CARBOPOL® 974P, 934P and 971P.
Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers
EMA® (Monsanto) which are copolymers of maleic anhydride and ethylene, linear or cross-
linked, for example cross-linked with divinyl ether, are advantageous. Reference may be made to
et al.
for example, of basic units of the following formula in which:
- R1 and
- x = 0 or 1, preferably x = 1
- y = 1 or 2, with x + y = 2
e.g. canine GM-CSF which gene sequence has been deposited at the GenBank database
(accession number S49738). This sequence can be used to create said plasmid in a manner
methionyl-leucyl-phenylalanine; US 6,017,537) and/or Carbomer adjuvant (Phameuropa Vol. 8,
No. 2, June 1996). Persons skilled in the art can also refer to US 2,909,462, which describes such
acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl
radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically
unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as
CCOH
appropriate. The products are cross-linked with an ally1 sucrose or with ally! pentaerythritol.
J. Fields
The polymers of acrylic or rnethacrylic acid and the copolymers EMA® are formed,
R2, which are identical or different, represent H or CH3
For the copolymers EMA®, x = 0 and y = 2. For the carbomers, x = y =1.
The dissolution of these polymers in water leads to an acid solution, which is
neutralized, advantageously to physiological pH, in order to provide the adjuvant solution into
which the vaccine itself is incorporated. The carboxyl groups of the polymer are then partly in
C00 - form.
In one embodiment, a solution of adjuvant, especially of carbomer (Phanneuropa,
vol. 8, No.2, June 1996), is prepared in distilled water, advantageously in the presence of sodium
chloride, the solution obtained being at an acidic pH. This stock solution is diluted by adding it
to the desired quantity (for obtaining the desired final concentration), or a substantial part
thereof, of water charged with NaCl, advantageously physiological saline (NaCI 9 g/1) all
at once
in several portions with concomitant or subsequent neutralization (pH 7.3 to 7.4),
advantageously with NaOH. This solution at physiological pH is used for mixing with the
vaccine, which may be especially stored in freeze-dried, liquid or frozen form.
The polymer concentration in the final vaccine composition can be from 0.01% to 2%
w/v, from 0.06 to 1% w/v, or from 0.1 to 0.6% w/v.
[0118]
The sub-unit vaccine may be combined with adjuvants, like oil-in-water, water-in-oil-
in-water emulsions based on mineral oil and/or vegetable oil and non ionic surfactants such as
block copolymers, TWEEN®, SPAN®. Such emulsions are notably those described in page 147
of "Vaccine Design — The Subunit and Adjuvant Approach", Pharmaceutical Biotechnology,
1995, or TS emulsions, notably the TS6 emulsion, and LF emulsions, notably LF2 emulsion (for
both TS and LF emulsions, see WO 04/024027). Other suitable adjuvants are for example
vitamin E, saponins, and CARBOPOL® (Noveon; see WO 99/51269; WO 99/44633),
aluminium hydroxide or aluminium phosphate ("Vaccine Design, The subunit and adjuvant
approach", Pharmaceutical Biotechnology, vol. 6, 1995), biological adjuvants (i.e. C4b, notably
murine C4b (Ogata R T et al.)
or equine C4b, GM-CSF, notably equine GM-CSF (US
6,645,740)), toxins (i.e. cholera toxins CTA or CTB,
Escherichia colt heat-labile toxins LTA or
et al.;
LTB (Olsen C W Fingerut E et al.; Zurbriggen R et al. Peppoloni S et al.),
and CpG (i.e.
CpG #2395 (see Jurk M
et al.), CpG #2142 (see SEQ. ID. NO; 890 in EP 1,221,955).
101191
The composition or vaccine may also contain or comprise one or more FeLV
antigens, for example, ENV, or ENV and GAG, or ENV and GAG and PRO gene.
[0120]
The composition or vaccine may also be associated with at least one FeLV antigen,
for example inactivated FeLV. In a particular embodiment, the FeLV strain may be an FeLV
type A strain, or a combination of FeLV type A and type B, or a combination of FeLV type A
and type C, or a combination of type A, type B and type C strains. These strains of FeLV may
be inactivated by chemical or physical methods. The chemical methods are notably BPL,
formaldehyde. The physical methods may notably be sonication. One method for inactivating
FeLV for use in a vaccine is described in R. Cordeiro Giunchetti
et al., Vaccine, 2007. The
inactivated FeLV vaccine may be combined with adjuvants, like those described previously for
sub-unit vaccines.
Another aspect of the present invention relates to methods of vaccinating a host
against FeLV using the vaccine compositions disclosed herein.
[0122] The host may be any one or all of felines (for example, domesticated cats, kittens, big
cats and wild cats). In one embodiment, the host is a feline.
The routes of administration may be, for example, intramuscular (IM) or intradermal
(ID) or transdermal (TD) or subcutaneous (SC). The means of administration may be, for
example, a syringe with a needle, or needle free apparatus, or a syringe with a needle coupled to
eleetrotransfer (ET) treatment, or needle free apparatus coupled to ET treatment.
101241
Another aspect of the invention relates to the use of a plasmid-based vaccine
according to the present invention for administration to a host, wherein this administration is
coupled to ET treatment. The administration of a plasmid-based vaccine is advantageously
intramuscular. The means of administration is, for example, a syringe and a needle. One or
several injections may be administered successively. In the case of several injections, they may
be carried out 2 to 6 weeks apart, for example, about 3 weeks apart. In one embodiment, a semi-
annual booster or an annual booster is further administered.
For plasmid-based vaccines, advantageous routes of administration may be ID or IM.
This administration may be through use of a syringe with a needle or with a needle free
apparatus like Dermojet or Biojector (Bioject, Oregon, USA) or VetjetTM (Merial) or ViajetTM
(Bioject Inc.), see US 2006/0034867. The dosage may be from 50 pig to 500 )1g per plasmid.
When
DMRIE-DOPE is added, 100 p.g per plasmid may be utilized. When GM-CSF or other
cytokines are used, the plasmid encoding this protein may be present at a dosage of from about
200 pg to about 500 lag and may be 200 jug.
The volume of doses can be between 0.01 ml and
0.5 ml, for example, 0.25 ml. Administration may be provided with multiple points of injection.
Alternatively, plasmid-based vaccines may be administered via the IM route coupled
to electrotransfer (ET) treatment. The ET treatment may be performed using an apparatus for
electrotransfer and the specifications of the manufacturer (i.e. Sphergen G250 generator
(Sphergen SARL, Evry Genopole, France); MedPulser® DNA electroporation system (Innovio
Biomedical Corporation, San Diego, California, USA)). In one embodiment, the apparatus for
electrotransfer has a unipolar field. The field intensity may be from about 50 to about 250 V/cm,
from about 50 to about 200 V/em, or from about 50 to about 175 V/cm. The pulse duration may
be from about 1 to about 50 msec, or from about 15 to about 25 msec. The frequency may be
from about 1 to about 50 Hz, or from about 5 to about 15 Hz. The interpulse interval may be
from about 1 to 1000 msec, or from about 1 to about 200 msec. The number of pulses may be
from 1 to 20, or from 5 to 10. The Ultra tissular intensity may advantageously be up to about 2 A.
The distance between electrodes may be from about 0.2 to about 1 cm, or from about 0.2 to
about 0.5 cm.
For recombinant viral vector-based vaccines, the routes of administration may
advantageously be SC or IM or TD or ID. This administration may be made by a syringe with a
needle or with a needle free apparatus like Dermojet or Biojector (Bioject, Oregon, USA) or
VetjetTM (Merial) or VitajetTM (Bioject Inc.). The dosage may be from about 10
3 pfu to about 10 9
pfu per recombinant poxvirus vector. When the vector is a canarypox virus, the dosage may be,
for example, from about 10 5
pfu to about 10 9 pfu, from about 10 6 pfu to about 10 8 pfu, or from
about 10 6 pfu to about 10 7
pfu. The volume of doses may be from about 0.01 ml to 0.2 ml, and is
advantageously 0.1 ml. Administration may comprise multiple points of injection.
101281
For the IM route the volume of the vaccine provided may be from 0.2 to 2 ml, in
particular from about 0.5 to 1 ml. The same dosages are utilized for any of the vectors of the
present invention.
[0129]
For sub-unit vaccines, the route of administration may advantageously be via SC or
IM or TD or ID. This administration may be made by a syringe with a needle or with a needle
free apparatus like Dermojet or Biojector (Bioject, Oregon, USA) or VetjetTM (Merial) or
Vitajetim (Bioject Inc.). The dosage may be from about 50 to about 500 p.g, in particular from
about 50 to about 150 iug, and more particularly from about 50 to about 100 lig. The volume of
the sub-unit vaccine provided is from 0.2 to 2 ml, in particular from about 0.5 to 1 ml.
on a prime-boost administration regimen, where the primo-administration and the boost
administration regimen, comprising a primo-administration of a vaccine comprising a
in vivo
pharmaceutically or veterinary acceptable vehicle, diluent or excipient, an expression
vector containing a polynucleotide sequence for expressing,
variants or fragments thereof, followed by a boost administration of a vaccine comprising a
pharmaceutically or veterinary acceptable vehicle or excipient, an
in vivo,
booster vaccine. The primo-administration may comprise one or more administrations. Similarly,
the boost administration may comprise one or more administrations.
The routes of administration, doses and volumes are as previously disclosed herein.
The prime-boost administrations may be advantageously carried out 2 to 6 weeks
prime-administration of a plasmid-based vaccine according to the present invention and at least
In another aspect, the present invention relates to a vaccine strategy, which is based
administration(s) utilize a composition comprising a pharmaceutically or veterinary acceptable
excipient, diluent or vehicle and an in vivo expression vector comprising a polynucleotide
sequence, that contains and expresses the FeLV polypeptide and/or variants or fragments thereof.
The present invention relates to the use of in vivo expression vectors in a prime-boost
in vivo, FeLV polypeptides and/or
in vivo expression vector
containing a polynucleotide sequence for expressing, FeLV polypeptides and/or variants
or fragments thereof as described above, to protect a host from FeLV and/or to prevent disease
progression in infected hosts.
A prime-boost regimen comprises at least one primo-administration and at least one
boost administration using at Least one common polypeptide and/or variants or fragments thereof.
The vaccine used in primo-administration may be different in nature from those used as a later
[0133]
apart, for example, about 3 weeks apart. According to one embodiment, a semi-annual booster or
an annual booster, advantageously using the viral vector-based vaccine, is also envisaged. The
animals may be at least 6 to 8 weeks old at the time of the first administration.
In one embodiment, the prime-boost administration regimen comprises at least one
one boost-administration of a recombinant viral vector-based vaccine according to the present
invention.
In another embodiment, the prime-boost administration regimen comprises at least
one prime-administration of a recombinant viral vector-based vaccine according to the present
invention and at least one boost-administration of a sub-unit vaccine according to the present
In another embodiment, the prime boost administration regimen comprises at least
one prime-administration of a recombinant viral vector-based vaccine according to the present
invention and at least one boost-administration of a plasmid-based vaccine according to the
present invention.
to or veterinary acceptable vehicle, diluent or excipient, a plasmid containing a polynucleotide for
expressing, in vivo, an FeLV polypeptide, a variant or fragment of the FeLV polypeptide,
pharmaceutically or veterinary acceptable vehicle or excipient, a recombinant viral vector
comprising a polynucleotide for expressing, in vivo, the same FeLV polypeptide(s), variant
in infected subject.
In another embodiment, the present invention relates to a method vaccinating a
pharmaceutically or veterinary acceptable vehicle, diluent or excipient, a recombinant viral
vector comprising a polynucleotide for expressing,
plasmid containing a polynucleotide for expressing, in vivo, the FeLV polypeptide(s), variant
thereof, fragment thereof, to protect the subject from FeLV and/or to prevent disease progression
in infected subject.
regiment comprises a prime-administration of a vaccine or composition comprising, in a
invention.
In one embodiment, the present invention relates to a method of vaccinating a subject
susceptible to FeLV comprising a prime-boost administration regimen wherein said regiment
comprises a prime-administration of a vaccine or composition comprising, in a pharmaceutically
or a
mixture thereof, followed by a boost administration of a vaccine comprising, in a
thereof, fragment thereof, to protect the subject from FeLV and/or to prevent disease progression
subject susceptible to FeLV comprising a prime-boost administration regimen wherein said
regiment comprises a prime-administration of a vaccine or composition comprising, in a
in vivo, an FeLV polypeptide, a variant or
fragment of the FeLV polypeptide, or a mixture thereof, followed by a boost administration of a
vaccine comprising, in a pharmaceutically or veterinary acceptable vehicle or excipient, a
In yet another embodiment, the present invention related to a method of vaccinating a
subject susceptible to FeLV comprising a prime-boost administration regimen wherein said
pharmaceutically or veterinary acceptable vehicle, diluent or excipient, a recombinant viral
vector comprising a polynucleotide for expressing, in vivo, a an FeLV polypeptide, a variant or
FeLV polypeptide(s), variant thereof, fragment thereof, to protect the subject from FeLV and/or
Another aspect of the present invention relates to a kit for prime-boost vaccination
invention.
In another embodiment, the kit may comprise two vials, one containing a recombinant
viral vector-based vaccine for the prime-vaccination according to the present invention, the other
In another embodiment, the kit may comprise two vials, one containing a recombinant
viral vector-based vaccine for the prime-vaccination according to the present invention, the other
10146]
examples are to be construed as merely illustrative, and not limitations of the preceding
fragment of the FeLV polypeptide, or a mixture thereof, followed by a boost administration of a
vaccine comprising, in a pharmaceutically or veterinary acceptable vehicle or excipient, the same
to prevent disease progression in infected subject.
according to the present invention. The kit may comprise at least two vials: a first vial containing
a vaccine for the prime-vaccination according to the present invention, and a second vial
containing a vaccine for the boost-vaccination according to the present invention. The kit may
advantageously contain additional first or second vials for additional prime-vaccinations or
additional boost-vaccinations.
In one embodiment, the kit may comprise two vials, one containing a plasmid-based
vaccine for the prime-vaccination according to the present invention, the other vial containing a
recombinant viral vector-based vaccine for the boost-vaccination according to the present
vial containing a sub-unit vaccine for the boost-vaccination according to the present invention.
vial containing a plasmid-based vaccine for the boost-vaccination according to the present
invention.
The invention will now be further described by way of the following non-limiting
examples.
EXAMPLES
Without further elaboration, it is believed that one skilled in the art can, using the
preceding descriptions, practice the present invention to its fullest extent. The following detailed
disclosure in any way whatsoever. Those skilled in the art will promptly recognize appropriate
variations from the procedures both as to reactants and as to reaction conditions and techniques.
Construction of DNA inserts, plasmids and recombinant viral vectors was carried out
using the standard molecular biology techniques described by J. (Molecular
isolated using the "Geneclean" kit
Construction of pH6C5env plasmid pPB713
ENV/ALVAC(2) recombinants
An ALVAC(1) recombinant virus which contains FeLV ENV inserted at C5 locus
and GAG/POL (-FT5NT) inserted at C3 locus (Merial proprietary material) was used to amplify
the FeLV ENV gene. Primers 7862CXL and 7847CXL were used for the PCR amplification.
7862CXL: ACG CCG CTC GAG CGG GGA TCT CTT TAT TCT ATA CTT A
(SEQ ID NO:25)
Xho I H6 promoter
Barn HI T5NT stop
101491 The amplified PCR fragment (2.1Kb) contains the FeLV ENV gene, H6 promoter
The PCR fragment was then digested with Xho1/BamHI and ligated to XhoI/BamHI digested
pH6C5ALVAC donor plasmid (Merial proprietary material) to generate pCXL208.2, which was
[01501
Construction of pH6C5env plasmid pPB713
mutation is the substitution of Arg for Glu at position 527 of the FeLV ENV gene.
[01521
provided contains 5 mutations (in nucleotides) by comparison with the reference sequence
Sambrook et al.
Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York, 1989). All the restriction fragments used for the present invention were
(B10 101 Inc., La Jolla, Calif.).
Example 1
Construction of pH6C5env - pCXL208.2, a C5 insertion plasmid for the generation of FeLV-
7847CXL: CTC GGA TCC AGAAAAA TCA TGG TCG GTC CGG ATC (SEQ ID NO:26)
immediately upstream of the ENV and a T5NT sequence followed by stop codon of the ENV.
sequence confirmed.
The plasmid map of pCXL208.2 and its sequence are shown in Figures 2 and 3.
FeLV ENV is glycosylated and cleaved to produce glycoprotein gp70 ENV and pl5E
ENV. The protein sequence of mutated FeLV ENV gene of strain 82K is shown in Figure 5. The
Plasmid pHCMV- ENV FeLV was received from Institut Gustave-Roussy
(Villejuif, France). The sequence of the mutated FeLV ENV fragment (SEQ ID NO:3)
(Glasgow, GenBank accession No. M12500, SEQ ID NO:35). Among the five nucleotide
mutations, two mutations are silent mutations (no amino-acid change), but introduced a new
restriction site (= FspI); three mutations introduced a mutation in the amino-acid sequence of
FeLV ENV (Arg in place of Glu; as shown in Figure 5, SEQ ID NO:4).
Plasmid phCMV-ENV FeLV was digested with Rsril/Sacli to generate an RsrII-
SacIl fragment (fragment B: 520 bp). Plasmid pCXL208.2 was digested with RsrII/SacII to
generate a Rsr11-Saell fragment (fragment A: 6231 bp). Fragments A and B were ligated to
generate plasmid pPB713 (6756 bp). The identity of pPB713 was confirmed by an FspI
digestion. The restriction map of pPB713 and the pPB713 sequences are shown in Figure 4.
Construction of pH6C5env plasmid pPB7I2
Plasmid PhCMV-ENV FeLV was digested with
RsrIESaclI to generate an RsrII-
SacII fragment (fragment A: 520 bp). Plasmid pPB575 (Merial proprietary material) was
digested with
Rsril/Sacil to generate an RsrII-SacII fragment (fragment B: 5971 bp).
Fragments A and B were ligated to generate plasmid pPB712 (6496 bp). The identity of
pPB7I2 was confirmed by an EcoRI digestion. The sequence of the mutated region of FeLV
present in pPB712 clone was controlled by DNA sequencing (Cogenics, France) with
universal M13 primer and reverse M13 primer. Two candidates were selected (n°1 and n°2).
The sequences of the 2 clones were identical but were different from SEQ ID NO:4 (single
amino acid mutation Glu to Arg). There are eight nucleotide mutations, leading to only one
amino acid change. The DNA and protein sequence comparisons between the mutated FeLV
(SEQ ID NO:1) in pPB712 and the mutated FeLV (SEQ ID NO:3) in pHCMV-ENV FeLV are
shown in Figure 5. The sequence comparison of FeLV ENV proteins of different strains is
shown in Figure 5.
Example 2
Construction of C3 ALVAC donor plasmid for generation of an ALVAC
recombinant expressing FeLV codon optimized GAG-PRO
FeLV (Feline leukemia virus) codon optimized
GAG-PRO gene was used in making
the vCP2294. FeLV GAG-PRO
gene was optimized for gene expression in mammalian cells.
The sequence comparison at the DNA level between the codon-optimized
GAG-PRO gene (SEQ
ID NO:10) and the wild-type gap-pro gene (Genbank accession No. M18247, SEQ 113 NO:11) is
show in Figure 7.
The construction scheme is outlined in Figure 8. The plasmid pJY1320.1 (Merial
proprietary material) containing H6p-FeLV codon optimized
GAG-PRO cassette was used as a
template for PCR amplification. H6p is Vaccinia virus H6 promoter. Primers 13301JY and
13302JY were used for the PCR amplification. The PCR fragment was cloned to a pCR2,
TOPO vector. The resulting plasmid pJY1857.5 was sequenced and confirmed to have
correct sequences of H6p-FeLV
GAG-PRO. In order to construct pC3 FeLV 146p-GAG-PRO,
an NruI/SpeI DNA fragment, which contains 3'-partial H6 promoter and full-length GAG-PRO,
was isolated from pJY1857.5 and ligated to Nru I/Spe I digested pJY1738.2 (Merial proprietary
material) to create p1Y1874.1 (as shown in Figures 9, 10 and 11), which was confirmed to have
the correct sequences.
Primer forward 13301JY (SEQ
ID NO:13)
1 H6p (SEQ ID NO :15)
' ATTA TCGCGA! TATCCGTTAAGTTTGTATCGTA ATG GGA CAG ACC ATC ACC ACC
CCC CTG T
Primer reverse 13302JY (SEQ ID NO :14)
' ATTA ACTAGT CAAGAAAAA TCA TTA CAG CAC CTG CAG GGG CAG TCC TCT
In FeLV infected cells,
GAG-PRO is produced by readthrough. GAG is further
cleaved to MA (p15), CA (p30) and NC proteins during the later stage of virus assembly.
Example 3. Generation and characterization of ALVAC recombinant containing H6p FeLV
codon optimized GAG-PRO inserted in C3 locus of ALVAC (vFP2294)
The IVR (in vitro recombinant) was performed by transfection of Primary chicken
embryo fibroblast cells (1°CEF) with 10 tg of Not I-linearized donor plasmid p.TY1874.1 using
FuGENE-6® reagent (Roche). The primary chicken embryo fibroblast cells (1°CEF) used for in
vitro
recombination were grown in 10% FBS (JRH: y-irradiated # 12107-500M), DMEM
(BRL/Gibco#11960-051 or 11960-044) supplemented with 4 mM Glutamine
(BRL/Gibco#25030-081) and 1 inM Sodium Pyruvate (BRL/Gibco#11360-070) in the presence
of lx antibiotics/antimycotics (P/S/A/A, BRL/Gibco#15240-062).The transfected cells were
subsequently infected with ALVAC as rescue virus at MOI (multiplicity of infection) of 10
(ALVAC #HM1372 07 Apr 04). After 24 hours, the transfected-infected cells were harvested,
Recombinant plaques were screened based on the plaque lift hybridization method
using a 1.4 kb FeLV GAG specific probe labeled with horse radish peroxidase (HRP) according
to the manufacturer's protocol (Amersham Cat# RPN3001). After five sequential rounds of
plaque purification, the recombinant designated as vCP2294.1.1.I.1.1 was generated and
confirmed by hybridization as 100% positive for the FeLV GAG insert and 100% negative for
round of plaque purification, and expanded to
obtain P1 (lx T25 flask), P2 (1xT75 flask) and P3 (6 x roller bottles). The infected cell culture
fluid from the roller bottles was harvested and concentrated to produce a virus stock
vCP2294,1.1.1.1.1.
The scheme to generate recombinant vCP2294 is depicted in Figure 12.
Analysis of recombinant: the following analyses were performed on the P3 stocks.
Confirmation of genetic purity
The P3 stocks were re-confirmed by hybridization, as 100% positive for the FeLV
GAG and 100% negative for the C3 ORF.
Genomic analysis
Genomic DNA from vCP2294.1.1.1.1.1 was extracted, digested with BamHI, HindIII
and run on 0.8% agarose gel. The gel with BamHI, or Pstl
DNA was transferred to a nylon membrane and Southern blot analysis was performed by probing
indicating the correct insertion of FeLV GAG-PRO gene into the C3 locus.
4152 4885
Pst I
681 2444
sonicated and used for recombinant virus screening.
the C3 ORF.
Single plaque was selected from the 5 th
or Pst I
Hind111 digested genomic
with the 1.4 kb FeLV GAG probe. Multiple bands were observed at the expected sizes,
Restriction enzyme Fragment (bp)
Barn HI
13961
Hind III 17783
12041
Expression analysis
1) Western blot
Primary CEF cells were infected with the P3 stock of vCP2294.1.1.1.1.1at MO1 of 10
and incubated at 37°C for 24 hrs. The culture supernatant and cells were then harvested. Cell
pellet was lysed with Reporter Gene Assay Lysis Buffer manufactured by Roche (Cat. 1 897
675). Both Supernatant and lysate were prepared with the NuPage® System with antioxidant
added. Proteins were separated on a NuPage® 10% Bis-Tris Pre-cast gel, and then transferred to
a PVDF membrane. Anti FeLV GAG antibodies revealed a —70kDa protein detected in both
supernatant and cell pellet, and a —57 kDa protein, which was detected only in the cell pellet.
2) Immunoplaque assay
The homogeneity of the population was 100% positive to the FeLV GAG protein for
recombinant vCP2294.1.1.1.1.1 as evidenced by an immunoplaque assay, using anti-FeLV GAG
antibodies.
Sequence analysis
A more detailed analysis of the P3 stock genomic DNA was performed by PCR
amplification and sequence analysis of the flanking arms of the C3 locus and the FeLV insert.
Primers 8103JY and 8104JY, located beyond the arms of the C3 locus in the ALVAC genome
were used to amplify the entire C3L-FeLV-C3R fragment. The results showed that the sequences
of the FeLV insert and C3L and C3R of ALVAC are correct.
Primers for amplifying the FeLV GAG probe:
11369JY: 5' ATGATGAACGTGGGCTGGCCT 3'
(SEQ ID NO:17)
11377.1Y: 5' TCTCCTAAGTTGAGCAGGGTG 3'
(SEQ ID NO:18)
[01681 Primers for PCR amplification of C3L-FeLV GAG-PRO
cassette-C3R:
8103JY: 5' GAGGCATCCAACATATAAAGAAGACTAAAG 3' (SEQ ID NO:19)
8104JY: 5' TAGTTAAATACTCATAACTCATATCTG 3' (SEQ ID NO:20)
101691 Figure 13 shows the vCP2294 C3 region map showing primer locations. The
vCP2294 sequence is depicted in Figure 14.
Example 4 Generation and characterization of ALVAC recombinant containing FeLV modified
ENV gene inserted at C5 locus of vCP2294, ALVAC C3 H6p FeLV codon optimized GAG-PRO
— vCP2296
[0170]
The IVR was performed by transfection of 1°CEF cells with 10 [ig of Not I-linearized
donor plasmid pPB713 using FuGENE-6® reagent (Roche). The transfeeted cells were
subsequently infected with vCP2294 (ALVAC C3 H6p FeLV codon optimized GAG-PRO,
Recombinant plaques were screened based on the plaque lift hybridization method
using a 503 by FeLV ENV specific probe labeled with horse radish peroxidase (HRP) according
confirmed by hybridization as
culture fluid from the roller bottles was harvested and concentrated to produce a virus stock
The construction of vCP2296 is depicted in Figure 15.
100%
Expression analysis
Primary CEF cells were infected with the P3 stock of vCP2296.6.1.1.2 at MOI of 10
Example 2) as rescue virus at MOI of 10. After 24 hours, the transfected infected cells were
harvested, sonicated and used for recombinant virus screening.
to the manufacturer's protocol (Amersham Cat# RPN3001). After four sequential rounds of
plaque purification, the recombinant designated as vCP2296.6.1.1.2 was generated and
100% positive for the FeLV ENV insert and 100% negative for
the empty C5 sites.
[0172] Single plaque was selected from the 4 round of plaque purification, and expanded to
obtain P1 (lx T25 flask), P2 (1xT75 flask) and P3 (6 x roller bottles) stocks. The infected cell
vCP2296.6.1.1.2.
P3 stocks.
Analysis of recombinant: the following analyses were performed on the
Confirmation of genetic purity
The P3 stocks were re-confirmed by hybridization, as 100% positive for both FeLV
GAG and FeLV ENV and
negative for both C3 and C5 ORF.
1) Western blot:
and incubated at 37 C for 24 hrs. The culture supernatant and cells were then harvested. Cell
pellet was lysed with Reporter Gene Assay Lysis Buffer manufactured by Roche (Cat. 1 897
675). Both supernatant and lysate were prepared with the NuPage® System with antioxidant
added. Proteins were separated on a NuPage® 10% Bis-Tris Pre-cast gel, and then transferred to
a PVDF membrane. Anti FeLV GAG antibodies revealed a —70kDa protein detected in both
supernatant and cell pellet, and a —80 kDa protein was also expressed in both the supernatant and
cell pellet by incubating with anti FeLV ENV antibody.
2) Immunoplaque assay:
The homogeneity of the population was 100% positive to the FeLV ENV protein for
recombinant vCP2296.1.1.2 as evidenced by an immunoplaque assay, using anti-FeLV ENV
antibody (see IP confirmation scan picture in attachment vCP2296 Immunoplaque,doc).
Insertion of the FeLV ENV gene at the C5 sites of vCP2296.6.1.1.2 was amplified by
PCR. Primers 7931DC and 7932DC, located beyond the arms of the C5 locus in the ALVAC
genome (see Figure 16), were used to amplify the entire C5L-FeLV-05R fragment,
Primers for amplifying the FeLV ENV probe:
Primers for PCR amplification of C5L-FeLV ENV cassette-05R:
7931DC
'TGATTATAGCTATTATCACAGACTC 3'
Example 5 Generation and characterization of ALVAC recombinant containing FeLV native
ENV gene inserted at C5 locus of vCP2294, ALVAC C3 H6p FeLV
codon optimized GAG-PRO — vCP2295
The donor plasmid pCXL208.2 contains the native ENV gene (SEQ ID NO:5).
donor plasmid pCXL208.2 using FuGENE-6® reagent (Roche). The transfected cells were
subsequently infected with vCP2294 (Example 2) as rescue virus at MOI of 10. After 24 hours,
the transfected-infected cells were harvested, sonicated and used for recombinant virus
Recombinant plaques were screened based on the plaque lift hybridization method
using a 503bp FeLV ENV specific probe labeled with horse radish peroxidase (HRP) according
to the manufacturer's protocol (Arnersham Cat# RPN3001). After four sequential rounds of
plaque purification, the recombinant designated as vCP2295.2.2.2.1 was generated and
confirmed by hybridization as 100% positive for the FeLV ENV insert and 100% negative for
Single plaque was selected from the 4 round of plaque purification, and expanded to
fluid from the roller bottles was harvested and concentrated to produce a virus stock
scheme to generate recombinant vCP2295 is shown in Figure 17.
Sequence analysis
7900CXL 5 'AGGAGGGCTTTAGTCCCTGTTCCGA 3' (SEQ ID NO:21)
7934CXL 5 'ACTAAAGACTGTTGGCTCTGCCTG 3' (SEQ ID NO:22)
'GAATCTGTTAGTTAGTTACTTGGAT 3' (SEQ ID NO:23)
(SEQ ID NO:24)
7932DC
The IVR was performed by transfection of 1°CEF cells with 10 j.tg of Not I-linearized
screening.
the empty C5 sites.
obtain PI (lx T25 flask), P2 (1xT75 flask) and P3 (6 x roller bottles). The infected cell culture
vCP2295.2.2.2.1.
Analysis of recombinant: the following analyses were performed on the P3 stocks.
Confirmation of genetic purity
The P3 stocks were re-confirmed by hybridization, as 100% positive for both FeLV
GAG and FeLV ENV and 100% negative for both C3 and C5 ORF.
Expression analysis
1) Western blot
Primary CEF cells were infected with the P3 stock of vCP229.5.2.2.2.1at MOI of 10
and incubated at 37°C for 24 hrs. The culture supernatant and cells were then harvested. Cell
pellet was lysed with Reporter Gene Assay Lysis Buffer manufactured by Roche (Cat. 1 897
675). Both Supernatant and lysate were prepared with the NuPage® System with antioxidant
added. Proteins were separated on a NuPage® 10% Bis-Tris Pre-cast gel, and then transferred to
a PVDF membrane. Anti FeLV gag antibodies revealed a —70kDa protein detected in both
supernatant and cell pellet, and a —80 kDa protein was also expressed in both the supernatant
and cell pellet by incubating with anti FeLV ENV antibody.
2) Immunoplaque assay:
The homogeneity of the population was 100% positive to the FeLV ENV protein for
recombinant vCP2295.2.2.2.1as evidenced by an immunoplaque assay, using anti-FeLV ENV
antibody.
Sequence analysis
[0185] A detailed analysis of the P3 stock genomic DNA was performed by PCR
amplification and sequence analysis of the flanking arms of the C5 locus and the FeLV insert.
Primers 7931DC and 7932DC, located beyond the arms of the C5 locus in the ALVAC genome,
were used to amplify the entire C5L-FeLV-05R fragment. The results showed that the sequences
of the FeLV insert and C5L and C5R of ALVAC are correct.
[0186] Recombinant vCP2295 sequence is depicted in Figures 18.
Example 6 Efficacy Evaluation of Canarypox Vectored Vaccine (vCP2296, FeLV ENV)
Administered Subcutaneously Via a Vaccination/Challenge Model
Materials/Methods
[0187]
Forty-four cats, male and female, between 57 and 63 days of age at first vaccination
(average 58 days; standard deviation 1.3 days) were randomly allocated into two groups of
twenty-two animals. Cats in Group 1 were vaccinated subcutaneously (SQ) on Days 0 and 21
6'2 Tissue Culture Dose50
(TCID50)/ml. Cats in Group 2 received two doses of lml of the Placebo Vaccine containing
42 and 43 (3 weeks following the 2nd vaccination), all cats were challenged with 1 ml of a
virulent strain of FeLV (61-E) suspension containing 10 5 and 104 ' 50/m1; (Days 42 and 43
(prior to challenge), and at approximately 3 weeks post-challenge and at weekly intervals for up
to 12 consecutive weeks (Days 62-Day 146) and the sera tested for FeLV antigenemia (FeLV
p27 protein).
Clinical evaluation was conducted starting 2 days prior to the 1st vaccination up to
Day 42. Rectal temperature was recorded daily on Days 0 (prior to vaccination), 1-2, 19-21
Results: Persistence of FeLV p27 antigenemia after challenge
[01891
(86.4%) from the placebo group became persistently FeLV antigenemic in comparison to 5/21
(23.R%) of the vaccinated group. The incidence of cats with persistent FeLV antigenemia
following results:
1. Upon challenge, the test vaccine was shown to be effective in preventing persistent FeLV
with 1ml of the FeLV-canarypox vector vaccine (vCP2296) at 10
Sterile Physiological Saline Solution on Days 0 and 21 and served as negative controls, On Days
4' 7 TC1D
respectively) administered by the oro-nasal route. Blood samples were collected on Days -6, 42
(prior to vaccination) and 22-23. In addition, injection sites were assessed the first 2 days
following each vaccination and at weekly intervals post-vaccination until the day of challenge
and included the evaluation for swelling, redness and pain upon palpation.
A cat was considered as having persistent FeLV p27 antigenemia when it was tested
FeLV p27 positive for 3 consecutive weeks or 5 non-consecutive weeks. Nineteen out of 22 cats
attributable to the FeLV challenge was significantly lower (p=0.00005) in the vaccinated group
than in the placebo group. The estimated prevented fraction was 72.43% with a 95% confidence
interval of 43.04% to 89.78%. Thus, there was a 72% reduction in the chance of an animal
becoming persistent FeLV antigenemic in a vaccinated Placebo
animal compared to that of a
animal.
Conclusion
Two doses of Medal's FeLV-Canarypox Vectored Vaccine (vCP2296) administered
by the SQ route were found to be efficacious against an FeLV challenge as evidenced by the
antigenemia in 16 out of the 21 (76.2%) vaccinated-challenged cats with a significantly lower
number of vaccinated cats developing a persistent antigenemia as compared to controls
2. An effective challenge was validated, as evidenced by the development of persistent FeLV
3. None of the vaccinated cats showed local or systemic reactions following vaccination.
Example 7 Comparison of the efficacy of the recombinant canarypox-FeLV with native ENV
Total of thirty SPF (specific pathogen free) kittens, 15 male and 15 female, aged
kittens according to their sex, litter and age.
Table I. Experimental design of the study
Group Vaccination DO — D28 Challenge
vaccine Target Route D44
volume
SC**
B 1 0 tmL
vCP2296 Glasgow-1
Not vaccinated
* group C: # of cats = 9 from DI to the end due to the death of one cat on DI
** in log 1 OCCID50/mL
BS: blood sampling
Cats from groups A and B were then vaccinated under general anesthesia by subcutaneous
injection in inter-scapular area. On D44, the challenge strain was thawed at 37 °
were mixed with 8mL of F15 medium with 10% foetal calf serum and kept on crushed ice before
inoculation. All cats underwent general anesthesia. Then each cat was inoculated via the oro-
(p=0.00005; prevented fraction 72%; primary efficacy variable).
antigenemia in 86% (19/22) of the control cats.
gene (vCP2295) and the recombinant canarypox-FeLV with optimized ENV gene (vCP2296) by
challenge in cats
Materials/Methods
between 8 and 12 weeks (9 weeks on average on DO), were randomly assigned to 3 groups of 10
# of cats
titre**
vCP2295 6.0 FeLV-A-
Oro-Nasal
route
SC: subcutaneous
DO and D28, prior to vaccination, all kittens were monitored for body condition.
C, 32mL of strain
nasal route with ImL of inoculum (0.25mL in each nasal cavity) and 0.5mL orally (tongue,
pharynx and tonsil).
Results
Blood samplings were performed on vigil cats on DO, D5, D7, D15, D26, D35, D49,
D70, D77, DB4, D91, D96, D105, D112, D133 and under general anesthesia (0.1 to 0.2naLlkg of
Zoletll" 50, Intramuscular route) on D44, D56, D63, D119, D126, DI40 and D147.
1...4 tztigetionia test
Blood samples were collected in dry tubes on DO, before the vaccination, on D44
before the challenge and every week from the third week post challenge, i.e., on D63, D70, D77,
D84, D91, D98, D105, D112, D119, D126, D133, D140 and D147 for FeLV p27 antigen
titration with Witness FeLV kit (Synbiotics Corporation, MO, USA). The response was a binary
one (presence/absence). Three categories of response were defined: a) 0: no antigenemia (all the
titrations were negative), b) 1: transient antigenemia (less than three positive consecutive
ti.trations and less than five positive titrations), c) 2: persistent antigenemia (positive on at least
five occasions or at least three positive consecutive titrations).
In the vCP2295-vaccinated group (group A), 40% of cats were protected against
persistent antigenemia: 4/10 cats were never found positive and 6/10 cats presented a persistent
antigenemia. In the vCP2296-vaccinated group (group 8), 60% of cats were protected against
p27 persistent antigenemia. 5/10 were never found positive and 1/10 cat presented a transient
antigenemia: p27 could be detected in the serum of this cat on D63 and D84, 4/10 cats presented
a persistent antigenemia. In the control group (group C), 100% of cats had persistent
antlgencmia. The results are shown in Table 2.
Table 2. p27 antigenemia results (rates)
Group
Persistent Transient No positive Protection*
antigenemia antigenemia antigenemia
rate
A 6/10** 0/10 4/10 4/10
vCP2295 vaccinated 60%
0% 40% 40%
4/10 1/10 5/10
6/10
vCP2296 vaccinated 40%
% 50% 60%
C 9/9 0/9
0/9 NA
control 100%
** One cat which died during the study was found positive 4 consecutive times
0), transient (antigenemia = 1) or persistent (antigenemia = 2) antigenemy gave a significant p-
value ("Fisher's exact test": p = 0.028). A trend to the significance was evidenced between group
and group C (adjusted p-value with Bonferroni's method: A vs C: p = 0.260, B vs C: p = 0.056,
Proviremia
10197] Leukocyte counts were used to express proviremia in provirus copy number / 50,000
cells) using a quantitative PC II_ Due to the repeated measurement nature of the criterion and the
Figure 19 displays the evolution of the mean proviremia per group after challenge.
after challenge. In both vaccinated groups, p27 antigenemia was well correlated to proviremia
Immune response was monitored by EllSpot after stimulation of PBMC by dendritic cells (DC)
* Number of non persistently infected cats / Number of cats
NA: not applicable: control group
The comparison of the 3 groups on the frequency of cats presenting no (antigenemia
A vs B: p 1).
2. test
WBC (white blood cell). Blood samples were collected in EDTA tubes on D44 before the
challenge and every 3 weeks after the challenge, i.e., on D63, D84, D105, D126 and D147 for
leukocyte count and FeLV proviremla monitoring on PBMC (peripheral blood mononucleated
individual random effect, the proviremia data was analyzed using a mixed model with repeated
measurements,
a) Proviremia in blood
Figure 17 displays the evolution of the mean proviremia per group and p27 antigenemia status
(Figure 20).
b) proviremia in marrow
The level of proviremia in marrow of p27 negative cats was between 3 and 5 log10
whereas reached 8 to 9 log10 in p27 positive cats. The level of proviremia was well correlated
with the p27 antigenemia individual status and with individual blood proviremia (as shown in
Figure 21).
3. Cellular immune response
Blood samples were collected on heparin treated tubes on. D5, D7, Dl5, D28, D35,
D49, D56, D63, D119, and.
D126 for FeLV immunological monitoring. IFN-y -Cell Mediated
loaded with FeLV pools of peptides on D35 and D126. IL 10 mediated Immunity was monitored
Regulatory T cells were monitored on D5, D15, D35, D49, D63 and 13126.
[0201]
30minutes without brake). PBMCs were washed twice in sterile PBS (Phosphate-buffered saline)
(centrifugation 400g for 10minutes) and subsequently counted with a robotized ABX Pentra 120
cell counter. The cells were washed one last time in PBS and resuspended at concentration of
/m1 in sterile complete RPMI (=RPMI + Penicillin-Streptomycine (PS) +13Mercaptoethanol
b). Dendritic cells generation
adherent cells were removed and fresh completed medium supplemented with feline IL-4 and
feline GM-CSF was added to wells. The differentiation of monocytes into DC lasted 7 days.
c). IFNy ELISpot assay:
[02031
animals was quantified by utilizing IFNy ELISPOT assays. HA ELISPOT plates were coated
overnight at +4°C with 100pliwell of purified Anti-canine IFNy mAb diluted (1/25) in
sterile PBS and unoccupied sites were blocked with sterile complete RPMI 10% FCS for 2h at
Room Temperature (RT).
proteins at D+15, D+35 and D+126. Briefly, 100.10
peptide pools n 1 and 2 for FeLV ENV or peptide pools No. 2, 3, 6 and 8 FeLV GAG-PRO at
transferred into ELISpot plates and 500.10
3 PBMCs were added into each well. Dendritic cells
were loaded with an irrelevant peptide as a negative control. Cells were stimulated during 20-24h
at 37°C + 5% CO2. Cells were then eliminated and to allow cellular lysis. Cold distilled water
by ELISpot after stimulation of PBMC by FeLV pools of peptides on D35,1363 and D126.
A) Methods
Feline PBMCs isolation
PBMCs were isolated by PANCOLL® density-gradient centrifugation (600g for
.10 6
(OM)) + 10% of fetal calf serum (FCS).
102021 Ficoll-Isolated PBMCs were cultivated during 20 hours in flat 6-wells plates. Non
The intensity of FeLV-specific cellular immune responses in the different groups of
carbonate/bicarbonate buffer (0. 2M, pH9.6). The coated plates were washed three times in
Dendritic cells were loaded with peptide pools encoding for FeLV ENV and GAG
3 DC were re-stimulated individually by
1p.g/m1 in a final volume of 100p1 completed RPMi 10% FCS. Loaded den dritic cells were
was added to each well (2000) for 5min at RT. The plates were then washed three times in PBS-
0.05% Tween and incubated at +4°C with 100 pi of biotinylated Anti-feline yIFN MAb (diluted
at 1/100 in PBS-0.05% Tween). The plates were then washed three times in PBS-0.05% Tween
and 1001.1.1 of diluted HRP-Streptavidine solution were added to each well for 1 h at 37°C. Plates
were then washed three times in P13S-0.05%Tween and incubated for 15minutes at RT in dark
with the AEC substrate solution. The plates were extensively washed with tap water and dried.
The spots were counted with a CCD camera system (Microvision, Redmond, WA, USA) . The
frequency of peptide-specific IFNy-spot forming cells (SFC) was calculated as follow: number of
peptide-specific 1FNy SFC = number of IFNy SFC upon individual FeLV peptide pool re-
stimulation - number of IFNy SFC upon irrelevant peptide pool re-stimulation. Results were
expressed as the log10.
d). 1L-10 ELI Spot assay
102051
The ELISpot IL-10 was performed according to the manufacturer Instructions (R&D
systems, Minneapolis, MN, USA). 500.103 purified PBMCs were directly re-stimulated using
overlapping peptide pools encoding for FeLV ENV and GAG-PRO sequences, at li.tg/m1 in a
final volume of 2000 completed RPMI 10% FCS, and set down in ELIspot IFNy coated plates.
500.103 PBMCs were re-stimulated with an irrelevant peptide as a negative control. The
frequency of peptide-specific IL-10 spot forming cells (SFC) was calculated as follow:
number of peptide pool-specific 1L-10 SFC = number of IL-10 SFC upon individual FeLV
peptide pool re-stimulation - number of IL-10 SFC upon irrelevant peptide re-stimulation.
Results were expressed as the log10.
B) Results
a) Cellular immune response after vaccination
i) Monitoring of FeLV-specific IFNy secreting cell responses after vaccination
The ability of PBMCs to produce IFNy In response to re-stimulation with FeLV ENV
and GAG-PRO peptide pools-loaded DC was analyzed using an IFNy-ELISpot assay. Analysis of
the sum of IFNy +
SFC (spots forming cells) induced upon in vitro activation with dendritic cells
loaded with peptide pools encoding for FeLV ENV and GAG-PRO sequences showed that
vCP2296 vaccination induced a higher frequency of FeLV-specific IFNy secreting cells at day35
compared to vCP2295 vaccination. The non-vaccinated groups did riot induce any IFNy
secreting cells (Figure 22).
The differences between vCP2295 and vCP2296 in their ability to induce IFNy-
producing cells were clearer when focusing on Fel..V ENV pools No.1 and No.2 specific
response. Analysis of the frequency of IFN y SFC within PBMCs upon in vitro activation with
dendritie cells loaded with peptide pool No. 1 of FeLV ENV (encoding for the beginning of the
vaccination (group A) at day 35, in blood. The non-vaccinated groups did not induce any IFNy
secreting cells (Figure 23).
ii) Monitoring of FeLV-specific IL-10 secreting cells after vaccination
FeLV-specific IL-10 secreting cells monitoring: analysis of FeLV EN V-specific responses
102081 At day 35 post-vaccination, the ability of PBMCs to produce IL-TO in response to
vaccination induced a higher frequency of FeLV ENV-specific IL-10 secreting cells in
in blood
At day35 post-vaccination, the ability of PBMCs to produce IL-10 in response to
vCP2295 vaccination tended to induce more FeLV GAG-PRO specific IL .l0 secreting cells than,
vCP2296 vaccination (Figure 25).
102101 In conclusion, vCP2295 vaccination (group A) induced a higher frequency of FeLV
B) and control group (group C).
In order to further evaluate the two recombinant vaccines and the balance between
'T'h 1 response and regulatory response, the ratio between the number of FeLV-specific IFNy SFC
each vaccinated group was calculated. Comparison of the FeLV-specific TT:Ny IL-10 SR.' ratio
for each group demonstrated that vCP2296 vaccination induced a more balanced response as
compared to the immune response induced by vCP2295 vaccination which was biased toward
1L-10 response. 'This difference was more apparent in response to FeLV ENV re-stimulation than
to GAG-PRO re-stimulation (Figures 26a and 26b).
FeLV ENV sequence) showed a difference between vCP2296 (group B) and vCP2295
blood
FeLV ENV peptide pools re-stimulation was analyzed using an IL-10 Filspot assay. vCP2295
comparison to vCP2296 vaccination and control group (Figure 24).
FeLV-specific IL-10 secreting cells monitoring: analysis of FeLV GAG-PRO specific responses
FeLV GAG-PRO peptides pools re-stimulation was analyzed using an IL-10 ELlspot assay.
specific IL-10 secreting cells in peripheral blood, in comparison to vCP2296 vaccination (group
iii) FeLV-specific IFNy and IL-10 producing cells ratio after vaccination.
and the number of FeLV specific IL-10 SFC after ENV or GAG-PRO in vitro re-stimulation for
b) Cellular immune response monitoring after experimental challenge
i) Monitoring of FeLV-specific IFNy secreting cell responses after challenge
After the challenge (D126) the ability of PBMCs to produce IFNy in response to re-
stimulation with FeV ENV and GAG-PRO peptide pools-loaded OC was analyzed using an
specific IFNy secreting cells in PBMCs lately after the challenge (D126) as compared to
vCP2295-vaccinated cats. No FeLV GAG-PRO-specific IFNy secreting cells could be observed
at this time point, for any group (Figure 27).
ii) Monitoring of FeLV-specific IL-10 secreting cell responses after challenge
After the challenge (D126), the ability of PBMCs to produce IL-10 in response to
FeLV ENV or GAG-PRO peptides pools re-stimulation was analyzed using an IL-10 ELlspot
assay. FeLV challenge specifically boosted the FeLV ENV-specific IL-10 cell response in all
groups, as compared to the response at day 35, with no difference between the 3 groups (Figure
28a). The challenge did not affect the antigen-specific response directed against FeLV GAG-
PRO region, and vCP2295-vaccinated cats maintained their FeLV GAG-PRO -specific IL-10
response (Figure 28b). After the challenge, vCP2295 vaccinated cats (group A) exhibited only a
FeLV-specific IL-10 immune response whereas vCP2296-vaccinated cats (group B) developed a
FeLV-specific IL-10 immune response but also maintained their FeLV-specific IFNy response.
c) Frequency of FeLV-specific IFNy and IL-10 producing cells in protected and infected animals
Protected and Infected animals were identified according to p27 antigenemia results.
Protected and infected animals were separated within each group (Figure 29) and the IFNy/IL-I0
ratio for each sub-group was calculated to evaluate if the IFNy/IL-I0 SFC ratio after the
vaccination could be indicative of protection.
of 10 did not present any IFNy or IL10 response and were protected. Four cats out of 10
presented a low IFNy/11,10 ratio related to a high IL-10 response. Three of these cats presented a
high IFNy response and one of them did not present any IFNy response. These cats were not
protected.
102161 In the vCP2295 vaccinated group: eight cats out of 10 presented a lowIFNy/IL-1.0
ratio related to a high IL-10 response and a low IFNy response. Six of them were infected and
IFNy-El.,Ispot assay. vCP2296-vaccinated cats maintained a higher frequency of FeLV ENV-
In the vCP2296 vaccinated group: four cats out of 10 presented a high IFNy/IL-10
ratio related to a high IFNy response and a low IL- I 0 response and were protected. Two cats out
two of them were protected. Two cats out of 10 presented both IFNy and 11.-10 responses and a
high IFNy/1L-10 ratio. These cats were protected.
102171 Protected cats either from vCP2295- or vCP2296-vaccinated group displayed a higher
IFNy/IL-10 ratio in blood (Figures 26) as compared to infected cats, Moreover, protected cats
from vCP2296-vaccinated group have a higher :IFNy/IL-10 SFC ratio as compared to protected
eats from vCP2295-vaccinated group.
10218]
Protection was correlated with an increased IFN7/1L-10 ratio and protected cats from
vCP2296 vaccination developed a FeLV-specific cell mediated immunity biased toward 1FNy
production as compared to vCP2295-vaccinated cats.
Conclusion
102191 Sixty percent of cats vaccinated with vCP2296 (optimized ENV gene) were protected
against persistent antigenemia and 40% of cats vaccinated with vCP2295 (native ENV gene)
were protected against persistent antigenemia. The comparison of the three groups displayed a
significant difference of protection between vaccinated and non-vaccinated groups and a trend to
a significant difference between group B vaccinated with the optimized ENV gene (vCP2296)
and group A vaccinated with the native ENV gene (vCP2295).
102201
Proviremia and antigenemia results were well correlated: cats with persistent
antigenemia had a strong and sustained proviremia until the end of the study. Non-antigenemia
cats had lower and regressing proviremia. P27 negative cats were able to control the proviremia.
Differences between vCP2295 and vCP2296 vaccination, according to the induction of FeLV
specific IFNy and IL-10 producing cells during the vaccination and challenge phases were
evidenced. The induction of FeLV-specific IFNy producing cells by FeLV canarypox vaccines
especially when the ENV gene was mutated in its immunosuppressive sequence (vCP2296) was
demonstrated. Interestingly, these IFNy producing FeLV-specific cells induced by vCP2296
vaccination were still detected more than 100 days after challenge demonstrating that the
vCP2296 vaccination induced the generation of FeLV-specific memory T cells. Conversely,
vCP2295 was more potent to induce the differentiation of FeLV-specific IL-JO-producing cells.
The frequency of FeLV-specific IL-10 producing cells was higher in vCP2295 vaccinated cats as
compared to vCP2296 and non-vaccinated control cats after the vaccination. IL-1.0 is known for
its regulatoty properties, participating either in the inhibition of the immune response or in its
termination. The higher FeLV-specific IFNy/IL-10 SFC ratio after the vaccination was correlated
to protection (evaluated by antigenemia). All cats presenting a high IFNy/II.,-10 ratio and a low
IL-10 response were protected. This observation was in line with the potentially immune-
cells. Modification of the ENV gene in the vCP2296 vaccine decreased the imrnunosuppressive
properties of the construct and provided an immunological advantage to this construct as
compared to the native ENV gene in vCP2295.
10221] This study showed that the modification of the ENV gene of FeLV resulted in a
different quality of the immune response associated with a better protection against persistent
antigenemia. The modification of the ENV gene of FeLV allows the canarypox-FeLV to work at
lower dose than the same construct with native ENV FeLV gene.
102221 It will be apparent that the precise details of the methods described may be varied or
modified without departing from the spirit of the described disclosure. We claim all such
modifications and variations that fall within the scope and spirit of the claims below.
I0223j
All documents cited or referenced herein ("herein cited documents"), and all
documents cited or referenced in herein cited documents, together with any manufacturer's
instructions, descriptions, product specifications, and product sheets for any products mentioned
herein or in any document incorporated by reference herein, are hereby incorporated herein by
reference, and may be employed in the practice of the invention.
suppressive role of the ILproducing cells and with an anti-viral function of IFNy-producing
Claims (23)
1. A composition comprising an expression vector comprising a first polynucleotides encoding an optimized Feline Leukemia Virus (FeLV) envelope (ENV) polypeptide and a second polynucleotide encoding an FeLV GAG/PRO polypeptide, wherein the 5 optimized FeLV ENV polypeptide comprises a mutation at amino acid position 527 or equivalent corresponding amino acid position of an FeLV ENV protein, and wherein the mutation comprises a substitution of arginine (R), aspartic acid (D), or methionine (M) for glutamic acid (E), and wherein the polynucleotide encodes an optimized FeLV ENV polypeptide having the sequence as set forth in SEQ ID NO: 2, 4, 27, 28, 29, 30, 31, 32, 10 33 or 34.
2. The composition of claim 1, wherein the mutation comprises a substitution of arginine (R) for glutamic acid (E).
3. The composition of claim 1 or claim 2, wherein the polynucleotide has at least 70% sequence identity to the sequence as set forth in SEQ ID NO:1, 3, or 5. 15
4. The composition of any one of claims 1 to 3, wherein the second polynucleotide encodes an FeLV GAG/PRO polypeptide having at least 80% sequence identity to the sequence as set forth in SEQ ID NO:12.
5. The composition of claim 1, wherein the first polynucleotide encodes an optimized FeLV ENV polypeptide having an amino acid sequence as set forth in SEQ ID NO:2 or 4, and 20 wherein the second polynucleotide encodes an FeLV GAG/PRO polypeptide having an amino acid sequence as set forth in SEQ ID NO:12.
6. The composition of claim 1, wherein the polynucleotide encoding the optimized FeLV ENV polypeptide has the sequence as set forth in SEQ ID NO:1, 3, or 5, and the polynucleotide encoding FeLV GAG/PRO polypeptide has the sequence as set forth in 25 SEQ ID NO: 10 or 11.
7. The composition of any one of claims 1-6, wherein the expression vector is an avipox vector.
8. The composition of any one of claims 1-7, wherein the composition is administered to an animal at a dosage range from about 10 pfu to about 10 pfu. 30
9. An expression vector comprising a first polynucleotide encoding an optimized FeLV ENV polypeptide and a second polynucleotide encoding an FeLV GAG/PRO polypeptide, wherein the optimized FeLV ENV polypeptide comprises a mutation at amino acid position 527, and wherein the mutation comprises a substitution of arginine (R), aspartic acid (D), or methionine (M) for glutamic acid (E), and wherein the polynucleotide encodes an optimized FeLV ENV polypeptide having the sequence as set forth in SEQ ID NO: 2, 4, 27, 28, 29, 30, 31, 32, 33 or 34. 5
10. The expression vector of claim 9, wherein the second polynucleotide encodes an FeLV GAG/PRO polypeptide having at least 80% sequence identity to the sequence as set forth in SEQ ID NO:12.
11. The expression vector of claim 9 or 10, wherein the first polynucleotide encodes an FeLV ENV polypeptide having an amino acid sequence as set forth in SEQ ID NO:2 or 10 4, and wherein the second polynucleotide encodes an FeLV GAG/PRO polypeptide having an amino acid sequence as set forth in SEQ ID NO:12.
12. The expression vector of claim 9 or 10, wherein the polynucleotide encoding FeLV ENV polypeptide has the sequence as set forth in SEQ ID NO:1, 3, or 5, and the polynucleotide encoding FeLV GAG/PRO polypeptide has the sequence as set forth in 15 SEQ ID NO: 10 or 11.
13. The expression vector of any one of claims 9-12, wherein the expression vector is an avipox vector.
14. A method of vaccinating a non-human animal comprising at least one administration of the composition or expression vector of any one of claims 1-13. 20
15. The method of claim 14, wherein the method comprises a prime-boost administration regime.
16. The method of claim 14 or 15, wherein the composition is administered at a dosage range from about 10 pfu to about 10 pfu.
17. Use of the composition or expression vector of any one of claims 1 to 13 in the 25 preparation of a medicament for vaccinating an animal.
18. A kit for prime-boost vaccination comprising at least two vials: a first vial containing a composition or expression vector of any one of claims 1-13 for the prime-vaccination, and a second vial containing a composition or expression vector of any one of claims 1- 13, or a subunit FeLV vaccine, or a plasmid FeLV vaccine for the boost-vaccination. 30
19. The kit of claim 18, wherein the first vial contains the composition of claim 5 and the second vial contains the composition of claim 5, and wherein the composition is administered at a dosage range from about 10 pfu to about 10 pfu.
20. A composition according to any one of claims 1 to 8, substantially as described herein with reference to the examples and/or figures.
21. A method or use according to any one of claims 14 to 17, substantially as described herein with reference to the examples and/or figures. 5
22. An expression vector of any one of claims 9 to 13, substantially as described herein with reference to the examples and/or figures.
23. A kit for prime-boost vaccination of claim 18 or 19 substantially described herein with reference to examples and/or figures.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161509912P | 2011-07-20 | 2011-07-20 | |
US61/509,912 | 2011-07-20 | ||
PCT/US2012/023658 WO2013012446A1 (en) | 2011-07-20 | 2012-02-02 | Recombinant feline leukemia virus vaccine containing optimized feline leukemia virus envelope gene |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ620566A NZ620566A (en) | 2016-02-26 |
NZ620566B2 true NZ620566B2 (en) | 2016-05-27 |
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