U.S. patent application number 13/046038 was filed with the patent office on 2011-09-01 for method for enhancing an immune response.
This patent application is currently assigned to TAPIMMUNE, INC.. Invention is credited to Judi Barbara Alimonti, Genc Basha, Susan Shu-Ping Chen, Kyung Bok Choi, Wilfred Arthur Jefferies, Timothy Z. Vitalis, Quian-Jin Zhang.
Application Number | 20110212131 13/046038 |
Document ID | / |
Family ID | 39467373 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110212131 |
Kind Code |
A1 |
Jefferies; Wilfred Arthur ;
et al. |
September 1, 2011 |
METHOD FOR ENHANCING AN IMMUNE RESPONSE
Abstract
A vaccine composition for combating Pox viridae viral infections
in living organisms such as mammals (including humans) comprises
TAP-1 and/or TAP-2 to augment the antigen processing capability of
infected cells and hence their immunogenicity. The composition may
be used alone or, preferably, as an immunogenicity-enhancing
adjuvant with a pox antigen-based vaccine, especially in the
treatment or prophylaxis of viral infections such as smallpox.
Inventors: |
Jefferies; Wilfred Arthur;
(Surrey, CA) ; Vitalis; Timothy Z.; (Vancouver,
CA) ; Zhang; Quian-Jin; (Richmond, CA) ;
Alimonti; Judi Barbara; (Winnipeg, CA) ; Chen; Susan
Shu-Ping; (Vancouver, CA) ; Basha; Genc;
(Vancouver, CA) ; Choi; Kyung Bok; (Vancouver,
CA) |
Assignee: |
TAPIMMUNE, INC.
Seattle
WA
|
Family ID: |
39467373 |
Appl. No.: |
13/046038 |
Filed: |
March 11, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12474331 |
May 29, 2009 |
7976850 |
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13046038 |
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Current U.S.
Class: |
424/232.1 |
Current CPC
Class: |
A61K 2039/605 20130101;
A61K 2039/55516 20130101; A61P 31/20 20180101; A61K 39/12 20130101;
A61K 38/1709 20130101; A61K 2300/00 20130101; A61K 39/285 20130101;
A61K 38/1709 20130101; C12N 2710/24134 20130101; A61K 2039/55555
20130101; A61K 39/275 20130101; A61K 2039/5256 20130101; A61P 31/12
20180101; C12N 2710/24143 20130101; C12N 2710/10343 20130101; A61K
2039/53 20130101 |
Class at
Publication: |
424/232.1 |
International
Class: |
A61K 39/275 20060101
A61K039/275; A61P 31/20 20060101 A61P031/20 |
Claims
1. A pharmaceutical composition for administration to a living
organism for the treatment or prophylaxis of Pox viridae
infections, the composition consisting essentially of a Pox viridae
viral antigen, and a naked DNA plasmid encoding TAP-1 and/or
TAP-2.
2-5. (canceled)
6. The composition of claim 1 wherein the Pox viridae antigen is a
smallpox antigen.
7. The composition of claim 6 wherein the smallpox antigen is a
Modified Vaccinia Ankara (MVA) antigen.
8. (canceled)
9. A method of treatment or prophylaxis of a patient to combat Pox
viridae infections in the patient, which comprises administering to
the patient an effective amount of a pharmaceutically acceptable
composition comprising the composition of claim 1.
10-14. (canceled)
15. The method of claim 9 wherein the Pox viridae antigen is a
smallpox antigen.
16. The method of claim 15 wherein the smallpox antigen is a
Modified Vaccinia Ankara (MVA) antigen.
17-18. (canceled)
Description
[0001] This application is a National Stage Application of
PCT/CA2006/001945 filed Nov. 30, 2006, the disclosure of all of
which are incorporated herein by reference in their entirety as if
fully set forth herein.
BACKGROUND OF THE INVENTION
[0002] This invention relates to vaccines, vaccine compositions,
methods of preparation of vaccines, and uses thereof in the
prophylaxis and treatment of viral infections in mammalian patients
(including humans). More specifically it relates to vaccines and
vaccine compositions useful in combating a virus of the family Pox
viridae, such as vaccinia virus ("VV") and smallpox virus.
[0003] Vaccines against Pox viridae viruses, especially VV and
small pox, are known, and have been used for many years with a
large degree of success. However, adverse responses to standard
doses of inocula is a problem quite frequently encountered in
vaccination against VV and small pox. As a result, conventional
vaccines cannot be administered to a significant fraction of the
population who are either immune suppressed or who would otherwise
react adversely to the establish vaccine protocols. This can amount
to as many as 20% of the individuals targeted to be inoculated.
More efficient inocula compositions, allowing smaller effective
does, would be a significant advantage in this respect.
Ineffectively vaccinated patients pose a severe threat of spread of
the infectious disease
[0004] As vaccination against a variety of pathogens becomes more
widespread, needs develop to increase the efficiency of the
inocula, while reducing the sizes of the batches of vaccine
required for vaccination of an entire population. This is
particularly important during times of acute need (bio-terrorist
attacks, emergent epidemic, for example) when rapid responses are
required.
[0005] To increase vaccine potency and efficiency, a variety of
adjuvants have been developed. These include Freund's complete
adjuvant (FCA) which is an emulsion containing heat killed
mycobacterium tuberculosis, which has proved to be too toxic for
use in humans; cytokines such as IL-2 and IL-12, which elicit a
Th-I response conducive to cytotoxic mechanisms in the immune
system; and oil emulsion/aluminum salts containing immune
stimulators (proinflammatory bacterial products). Many of these are
too toxic for use in humans. Others cannot be implemented because
their mode of action is obscure.
[0006] Preferred, effective vaccines and vaccine-adjuvant
combinations should elicit both a humoral response and a cell
mediated response in the mammalian subject. The humoral response
causes the raising of antibodies specific to the antigens of the
invading pathogen and results in lasting defense against future
invasions of the same pathogen. The cell mediated response involves
destruction of infected cells by killer T-cells, i.e. cytotoxic
T-lymphocytes ("CTLs").
[0007] It is known that the cytotoxic T-lymphocyte cell (CTL)
response is a major component of the immune system, active in
immune surveillance and destruction of infected cells and invading
organisms expressing foreign antigen on their surface. A peptide
fragment of the antigen binds to major histocompatibility complex
molecules (MHC) to form the ligand of the antigen specific T-cell
receptor. Cytotoxic T-lymphocytes recognize peptide bound to MHC
Class 1 molecules on the surface of the infected cells, and destroy
them. For this to occur, a ternary complex must be formed in the
cell and transported to the cell surface. The formation of the
ternary complex is thought to involve the transport of peptides,
generated by protein degradation in the cytoplasm of the cell, into
the lumen of the endoplasmic reticulum (ER). This transport
involves two genes located in the MHC region which encode proteins
of the ATP binding cassette (ABC) family, call TAP-I and TAP-2
(Deverson, E. V. et al, "Nature" 348:738, 1990).
SUMMARY OF THE INVENTION
[0008] The present invention is based upon the discovery that
augmenting the presence of TAP-I or TAP-2 in a Pox viridae virally
infected cell, e.g. by enhancing TAP expression in the cell,
substantially increases the MHC Class 1-antigen presentation of the
cells so as to effect a substantial increase in immogenicity of the
infected cells. This manifests itself in a substantially increased
cytotoxic T-lymphocyte response and consequent increased
destruction of the infected cell by the CTL.
[0009] Thus according to one aspect of the present invention, there
is provided a vaccine composition for the treatment or prophylaxis
of Pox viridae infections in living organisms, comprising TAP-I
and/or TAP-2, or precursors thereof. As used herein, "TAP" is an
abbreviation for "transporters associated with antigen
processing".
[0010] According to another aspect, the invention provides an
adjuvant for administration to a living organism for the treatment
or prophylaxis of Pox viridae infections in conjunction with a Pox
viridae viral vaccine containing live or attenuated Pox viridae
virus, the adjuvant comprising a pharmaceutically acceptable
composition comprising TAP-I and/or TAP-2, or precursors
thereof.
[0011] A further aspect of the present invention provides a method
of treatment or prophylaxis of a living organism to combat Pox
viridae infections, which comprises administering to the patient an
effective amount of a pharmaceutically acceptable composition
comprising TAP-I and/or TAP-2, or precursors thereof optionally in
conjunction with a Pox viridae viral vaccine containing live or
attenuated Pox viridae virus.
[0012] The invention also provides, from another aspect, the
preparation or manufacture of a pharmaceutical composition for the
treatment or prophylaxis of Pox viridae infections in living
organisms, the composition comprising TAP-I and/or TAP-2, or
precursors thereof.
BRIEF REFERENCE TO THE DRAWINGS
[0013] FIG. 1 and FIG. 2 are graphical representations of the
results of experiments as shown in Example 1 below;
[0014] FIG. 3 is a graphical representation of the results of
experiments shown in Example 2 below;
[0015] FIG. 4 and FIG. 5 are graphical representations of the
results of experiments shown in Example 3 below;
[0016] FIG. 6 is a graphical representation of the results of
experiments shown in Example 4 below;
[0017] FIG. 7 is a graphical representation of the results of
experiments shown in Example 5 below;
[0018] FIGS. 8a and 8b are graphical representations of the results
of experiments shown in Example 7 below.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0019] The over-expression of TAP, or the presence of excess TAP,
in living organisms, in proper functional relationship with an
antigen of a Pox viridae virus in a cell capable of presenting the
antigen, increases antigen presentation, and therefore enhances
antigen specific cytotoxicity in response to a Pox viridae viral
infection. The TAP-I and/or TAP-2 protein may be generated in situ
in the living mammalian cells, or may be introduced into the cells
as a preformed protein, in any manner which ensures that at least
some of the TAP protein(s) enter into and participate in the
antigen presenting pathway of the cell. A preferred way of causing
the participation of TAP in the antigen presenting pathway of the
cells is to arrange TAP over-expression therein, e.g. by
administration to the living organism an expression system carrying
TAP-I and/or TAP-2 genes, which expresses TAP in the cells.
[0020] Accordingly, preferred vaccine compositions of the present
invention comprise one or both of the TAP-I or TAP-2 genes, in
expressible form, for injection into a Pox viridae infected living
organism or a living organism at risk of similar infection. The
TAP-I and TAP-2 genes are suitably provided as components of a
recombinant viral vector such as a vaccinia viral vector, or
adenohuman viral vector along with an appropriate promoter, signal
and expression sequences, so that after injection as a vaccine
itself, or as a vaccine adjuvant along with a live or attenuated
pox antigen-containing vaccine, TAP-I and/or TAP-2 is thus
over-expressed in the antigen presenting cells. This significantly
increases the output of the antigen presentation pathway, by
increasing the activity of these transporters. TAP over-expression
can act as an adjuvant for increasing responses against Pox viridae
virus, especially smallpox virus, in immuno-competent and
immuno-compromised hosts.
[0021] Another way of practicing the present invention is the
preparation and administration to living organisms of a naked DNA
plasmid coding for the TAP-I and/or the TAP-2 gene, along with the
antigen against which immunity is to be developed, e.g. smallpox
antigen. The antigen may be encoded in the DNA plasmid for
expression in cells of the organism after administration, or added
to the cells as a separate entity from the TAP-encoding
plasmid.
[0022] In the alternative, the TAP-I and/or TAP-2 proteins
themselves, expression products of the TAP-I and TAP-2 genes, and
obtainable by cell cultivation techniques and in other ways can be
used directly as a vaccine or vaccine adjuvant. When the TAP
protein(s) themselves are used, alone or as an adjuvant with a pox
virus-based vaccine, they should be administered in a form
facilitating their entry into the cells. One suitable such way is
administration of the proteins encapsulated in liposomes.
Techniques for accomplishing this are known in the art.
[0023] The viral antigen to which the immune response is to be
generated in accordance with the invention is preferably smallpox
virus, since this is the commonest, most serious viral infection of
members of the Pox viridae family. However, it is applicable to
other members of the Pox viridae family which includes the
subfamilies Chordopoxyirinae and Entomopoxvirinae. The subfamily
Chordopoxyirinae includes the genuses: variola virus (smallpox
virus); avipoxvirus (which includes species canary poxvirus; fowl
pox virus; Hawaiian goose poxvirus; pigeon pox virus; and vulture
gryphus poxvirus); capripoxvirus (which includes species
capripoxvirus strain Rapine; goat pox virus; lumpy skin disease
virus; and sheep pox virus); leporipoxvirus (which includes species
malignant rabbit fibroma virus; myxoma virus; rabbit fibroma virus
and Shope fibroma virus); molluscipoxvirus (which includes species
molluscum contagiosum virus); orthopoxvirus (which includes species
aracatuba virus; BeAn 58058 virus; Buffalo pox virus; camel pox
virus; cantagalo orthopoxvirus; cowpox virus; ectromelia virus;
elephant pox virus; monkey pox virus; rabbit pox virus; raccoon pox
virus; skunk pox virus; tarapox virus; vaccinia virus; and volepox
virus); parapoxvirus (which includes species bovine popular
stomatitis virus; orf virus; pseudocowpox virus; red deer
parapoxvirus; and seal pox virus); suipoxvirus (which includes
species swinepox virus) and yatapoxvirus (which includes species
tanapox virus; yaba monkey tumor virus; and yaba-like disease
virus).
[0024] Thus, the present invention relates to the treatment of
viral infections in living organisms infected with any of the above
viruses, the living organism being a mammal (including humans), a
bird or any other organism in which the virus is a natural
infectant or to which it has transfected. It also relates to
prophylaxis against the spread of such infections in any of these
living organisms.
[0025] When the method of the invention is used in a prophylactic
therapy or a vaccine, the target cell is essentially a normal cell
(expressing normal TAP levels) that may not have been otherwise
exposed to the pox antigen. In such a case, the agent that augments
TAP is coadministered with the pox antigen to which one wishes to
generate an immune response. Substantially all of the Pox viridae
viral species listed above are sufficiently similar to smallpox
virus that vaccination with any of them, accompanied by TAP
over-expression in the antigen-presenting cells, will confer an
effective degree of immunity against smallpox on the treated
organism. When the method of the invention is used as a
therapeutic, the target cell may be previously infected with pox
virus.
[0026] A particularly preferred use of the present invention is in
connection with the strain of vaccinia known as Modified Vaccinia
Ankara (MVA), which is an attenuated strain of vaccinia and was
used in the late stages of the smallpox eradication program
undertaken by Western government authorities in the late 1970s.
This has a superior safety profile, but a lower degree of
immunogenicity than the widely commercially used "dryvax" strain.
Increasing the immunogenicity of MVA by use in combination with TAP
and TAP expression systems in accordance with the present
invention, without detracting from its very good safety profile,
has the potential to provide a superior smallpox vaccine for human
patients, especially immunocompromised such patients.
[0027] In a preferred embodiment, the method of the invention
involves administering a recombinant viral vector, which comprises
a nucleic acid molecule, encoding a TAP molecule in order to
augment the level of TAP expression in the target cell. Thus in one
preferred embodiment, the present invention provides a method of
enhancing an immune response to a pox antigen comprising
administering an effective amount of a nucleic acid molecule
comprising a sequence encoding a TAP molecule in expressible form
to a living organism such as a mammal in need thereof.
[0028] The nucleic acid molecule encoding the TAP molecule can be
administered to the animal in vivo where the TAP molecule will be
expressed in vivo. When administered in vivo, the TAP molecule can
be administered by any route including, but not limited to,
intraperitoneally, intravenously, subcutaneously, orally,
scarfocially, intramuscularly or intradermally. As an alternative,
the TAP molecule can be administered to the target cells ex vivo
where the TAP molecule will be expressed in the cells in vitro and
then the target cells expressing TAP can be administered to the
animal.
[0029] The nucleic acid molecule comprising a sequence encoding
TAP-1 and/or TAP-2 under the control of a suitable promoter may be
readily synthesized using techniques known in the art. A sequence
encoding TAP-1 includes a sequence encoding a protein having the
amino acid sequence as set out in Trowsdale, J. et al., Nature
348:741, 1990 and international Patent Application No.
PCT/US91/06105 published on Mar. 19, 1992. A nucleic acid molecule
comprising a sequence encoding TAP-1 may be isolated and sequenced,
for example, by synthesizing cDNA's from RNA using rapid
amplification of cDNA ends (RACE, Frohman, et al., 1986) using
oligonucleotides specific for TAP-1 and analyzing the sequences of
the clones obtained following amplification. Oligonucleotides
specific for TAP-1 may be identified by comparing the nucleic acid
sequence of the nucleic acid molecules of the invention to known
sequences of TAP-1. Nucleic acid molecules used in the method of
the invention encoding TAP-1 or Tap-2 may also be constructed by
chemical synthesis and enzymatic ligation reactions using
procedures known in the art. The sequence encoding TAP-1 or TAP-2
may also be prepared using recombinant DNA methods.
[0030] The method of the invention not only contemplates the use of
the known TAP-1 and TAP-2 sequences, but also includes the use of
sequences that have substantial sequence homology to the known TAP
sequences, sequences that hybridize to the known TAP sequences, as
well as all analogs or modified forms of the known TAP sequences.
The term "sequence that has substantial sequence homology" means
those nucleic acid sequences which have slight or inconsequential
sequence variations from the known TAP sequences i.e., the
sequences function in substantially the same manner and can be used
to augment an immune response. The variations may be attributable
to local mutations or structural modifications. Nucleic acid
sequences having substantial homology include nucleic acid
sequences having at least 65%, more preferably at least 85%, and
most preferably 90-95% identity with the known nucleic acid
sequences of TAP.
[0031] The term "sequence that hybridizes" means a nucleic acid
sequence that can hybridize to a TAP sequence under stringent
hybridization conditions. Appropriate "stringent hybridization
conditions" which promote DNA hybridization are known to those
skilled in the art, or may be found in Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
For example, the following may be employed: 6.0.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by a
wash of 2.0.times.SSC at 50.degree. C.; 0.2.times.SSC at 50.degree.
C. to 65.degree. C.; or 2.0.times.SSC at 44.degree. C. to
50.degree. C. The stringency may be selected based on the
conditions used in the wash step. For example, the salt
concentration in the wash step can be selected from a high
stringency of about 0.2.times.SSC at 50.degree. C. In addition, the
temperature in the wash step can be at high stringency conditions,
at about 65.degree. C.
[0032] The term "a nucleic acid sequence which is an analog" means
a nucleic acid sequence which has been modified as compared to the
known sequence of a TAP molecule wherein the modification does not
alter the utility of the sequence as described herein. The modified
sequence or analog may have improved properties over the known
sequence. One example of a modification to prepare an analog is to
replace one of the naturally occurring bases (i.e. adenine,
quinine, cytosine or thymidine) of the known sequence with a
modified base such as xanthine, hypoxanthine, 2-aminoadenine,
6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo
cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo
uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol
adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other
8-substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiol
guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other
8-substituted guanines, other azo and deazo uracils, thymidines,
cytosines, adenines, or guanines, 5-trifluoromethyl uracil and
5-trifluoro cytosine.
[0033] Another example of a modification is to include modified
phosphorous or oxygen heteroatoms in the phosphate backbone, short
chain alkyl or cycloalkyl intersugar linkages or short chain
heteroatomic or heterocyclic intersugar linkages in the nucleic
acid molecule. For example, the nucleic acid sequences may contain
phosphorothioates, phosphotriesters, methyl phosphonates, and
phosphorodithioates.
[0034] A further example of an analog of a nucleic acid molecule of
the invention is a peptide nucleic acid (PNA) wherein the
deoxyribose or (ribose) phosphate backbone in the DNA (or RNA), is
replaced with a polyamide backbone which is similar to that found
in peptides (P. E. Nielsen, et al Science 1991, 254, 1497). PNA
analogs have been shown to be resistant to degradation by enzymes
and to have extended lives in vivo and in vitro. PNAs also bind
stronger to a complementary DNA sequence due to the lack of charge
repulsion between the PNA strand and the DNA strand. Other nucleic
acid analogs may contain nucleotides containing polymer backbones,
cyclic backbones, or acyclic backbones. For example, the
nucleotides may have morpholino backbone structures (U.S. Pat. No.
5,034,506). The analogs may also contain groups such as reporter
groups, a group for improving the pharmacokinetic or
pharmacodynamic properties of nucleic acid sequence, etc.
[0035] Some of the methods contemplated herein use nucleic acid
molecules containing sequences encoding truncated non functional
forms of TAP-1 or TAP-2, Truncated non functional forms of TAP-1
and TAP-2 may be constructed by deleting portions of the TAP-1 or
TAP-2 gene to produce fragments. Such fragments should hybridize to
the TAP-1 or TAP-2 sequences under stringent hybridization
conditions. Stringent hybridization conditions are those which are
stringent enough to provide specificity, reduce the number of
mismatches, and yet are sufficiently flexible to allow formation of
stable hybrids at an acceptable rate. Such conditions are known to
those skilled in the art and are described, for example, in
Sambrook, et al, (1989), Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor. The ability of the truncated forms of TAP-1 and
TAP-2 to transport endogenous peptides may be determined using the
methods described herein.
[0036] The invention also includes nucleic acid constructs
containing both TAP-1 and TAP-2. In such case, the nucleic acid
construct encodes a fusion protein of TAP-1 and TAP-2. The
invention also includes TAP-1 or TAP-2 which have been modified in
such a way as to decrease constitutively active phosphorylation
regulated sites or peptide bridging sites or assembly
structures.
[0037] Nucleic acid molecules having a sequence which codes for
TAP-1 and TAP-2, including the homologs and modified forms
discussed above, may be incorporated in a known manner into an
appropriate expression vector which ensures good expression of the
protein or a part thereof. Possible expression vectors include but
are not limited to cosmids, plasmids (including both naked DNA
plasmids and liposome encapsulated plasmids), or modified viruses,
so long as the vector is compatible with the target cell used.
[0038] It is contemplated that the nucleic acid molecules described
herein contain the necessary elements for the transcription and
translation of the inserted sequence. Suitable transcription and
translation elements may be derived from a variety of sources,
including bacterial, fungal, viral, mammalian, or insect genes.
Selection of appropriate transcription and translation elements is
dependent on the target cell chosen as discussed below, and may be
readily accomplished by one of ordinary skill in the art. Examples
of such elements include: a transcriptional promoter and enhancer
or RNA polymerase binding sequence, a ribosomal binding sequence,
and a translation initiation signal. Additionally, depending on the
host cell chosen and the vector employed, other genetic elements,
such as an origin of replication, additional DNA restriction sites,
enhancers, and sequences conferring inducibility of transcription
may be incorporated into the expression vector. It will also be
appreciated that the necessary transcriptional and translation
elements may be supplied by the native TAP-1 gene, TAP-2 gene
and/or their flanking regions.
[0039] The nucleic acid molecules may also contain a reporter gene
which facilitates the selection of transformed or transfected host
cells. Examples of reporter genes are genes encoding a protein such
as .beta.-galactosidase, chloramphenicol acetyltransferase, firefly
luciferase, or an immunoglobulin or portion thereof such as the Fc
portion of an immunoglobulin, preferably IgG. In a preferred
embodiment, the reporter gene is Lac Z. Transcription of the
reporter gene is monitored by changes in the concentration of the
reporter protein such as .beta.-galactosidase, chloramphenicol
acetyltransferase, or firefly luciferase. This makes it possible to
visualize and assay for expression of TAP.
[0040] Nucleic acid molecules comprising a sequence encoding TAP-1
or TAP-2 can be introduced into target cells via transformation,
transfection, infection, electroporation, etc. Methods for
transforming, transfecting, etc. host cells to express foreign DNA
are well known in the art (see, e.g., Itakura et al., U.S. Pat. No.
4,704,362; Hinnen et al., PNAS USA 75:19291933, 1978; Murray et
al., U.S. Pat. No. 4,801,542; Upshall et al., U.S. Pat. No.
4,935,349; Axel et al., U.S. Pat. No. 4,399,216; Goeddel et al.,
U.S. Pat. No. 4,784,950; Goeddel et al., U.S. Pat. No. 4,766,950;
and Sambrook et al., Molecular Cloning A Laboratory Manual,
2.sup.nd edition, Cold Spring Harbor Laboratory Press, 1989; all of
which are incorporated herein by reference in their entirety).
[0041] Suitable expression vectors for directing the expression in
mammalian cells generally include a promoter, as well as other
transcriptional and translational control sequences. Common
promoters include SV40, MMTV, metallothionein-1, adenovirus EIa,
CmV, immediate early, immunoglobulin heavy chain promoter and
enhancer, and RSV-LTR. Protocols for the transfection of mammalian
cells are well known to those of ordinary skill in the art.
[0042] The nucleic acid molecule encoding TAP is incorporated into
a suitable vehicle for delivery to a cell such as viral vectors,
plasmids, liposomes and microspheres. In a preferred embodiment,
the nucleic acid molecule is introduced into the target call in a
viral vector, preferably vaccine viral vectors, adenovirus based
vectors, lenti virus based vectors and herpes simplex virus based
vectors. The vectors may be live, attenuated, replication
conditional or replication deficient. Most preferably the viral
vectors are attenuated.
[0043] The present invention also includes pharmaceutical
compositions or vaccines for carrying out the methods of the
invention. Accordingly, the present invention provides a
pharmaceutical composition for use in enhancing an immune response
comprising an effective amount of an agent that can augment the
level of a TAP molecule in admixture with a suitable diluent or
carrier. In a preferred embodiment, the pharmaceutical composition
comprises an effective amount of a nucleic acid molecule comprising
a sequence encoding a TAP molecule in admixture with a suitable
diluent or carrier.
[0044] The above described nucleic acid molecules encoding a TAP
molecule or a vector comprising the nucleic acid molecules may be
formulated into pharmaceutical compositions for administration to
subjects in a biologically compatible form suitable for
administration in vivo. By "biologically compatible form suitable
for administration in vivo" is meant a form of the substance to be
administered in which any toxic effects are outweighed by the
therapeutic effects. The substances may be administered to living
organisms including humans and animals.
[0045] The pharmaceutical composition may be administered in a
convenient manner as by injection (subcutaneous, intravenous,
intraperitoneal, intramuscular, scarifacionally etc.), oral
administration, inhalation, transdermal application, or rectal
administration. Depending on the route of administration, the
nucleic acid molecules may be coated in a material to protect the
compound from the action of enzymes, acids and other natural
conditions which may inactivate the compound.
[0046] The compositions described herein can be prepared by per se
known methods for the preparation of pharmaceutically acceptable
compositions which can be administered to subjects, such that an
effective quantity of the active substance is combined in a mixture
with a pharmaceutically acceptable vehicle. Suitable vehicles are
described, for example, in Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., USA 1985), or Handbook of Pharmaceutical Additives
(compiled by Michael and Irene Ash, Gower Publishing Limited,
Aldershot, England (1995)). On this basis, the compositions
include, albeit not exclusively, solutions of the substances in
association with one or more pharmaceutically acceptable vehicles
or diluents, and may be contained in buffered solutions with a
suitable pH and/or be iso-osmotic with physiological fluids. In
this regard, reference can be made to U.S. Pat. No. 5,843,456, the
pertinent disclosure of which is incorporated herein by reference
in its entirety. As will also be appreciated by those skilled to
the art, the administration of substances described herein may be
by an inactive viral carrier.
[0047] The invention is further described and illustrated in the
following examples which are not intended to limit the specifically
enumerated embodiments or the scope of the appended claims. The
pertinent portions of all cited references are incorporated herein
in their entirety.
Materials and Methods
[0048] Animals, Cells and Viruses
[0049] The mouse strain C57BL/6 (H-2b) was obtained from Jackson
Laboratories and housed and bred at the Biotechnology Breeding
Facility (University of British Columbia). The mice were maintained
according to the guidelines of the Canadian Council on Animal
Care.
[0050] Mice were kept on a standard diet with water ad libitum. The
colony was routinely screened for Mycoplasma pulmonis, and
Mycoplasma arthritidis, rodent coronaviruses (including Hepatitis),
and SV using the Murine ImmunoComb Test (Charles River Labs). The
mice used in the experiments were between 6 and 12 weeks of
age.
[0051] Recombinant vaccinia virus carrying human TAP-1 and TAP-2
genes (W-IiTAP-1,2) and carrying murine TAP-1 (VV-mTAP-1) were
gifts from J. Yewdell, NIA, NIAID. Vaccinia virus encoding the
plasmid PJS-5 (W-PJS-5), which was used as a negative control, was
a gift from J. Alimonti, University of British Columbia, but is
obtainable elsewhere or constructable by methods known to those of
skill in the art. Vaccinia Virus Western Reserve strain (VV) was a
gift from S. Gillam, University of British Columbia, but is
available from scientific supply sources. VV strains were cultured
on CV-I cells (ATCC). CV-I cells were cultured in DMEM/10.degree. A
heat inactivated fetal bovine serum (FBS) (HyClone GIBCO BRL), 2 mM
L-glutamine, 100 IU/ml penicillin, 100 Bg/ml streptomycin, 20 mM
Hepes. RMA cells were cultured in RPMI/10% FBS, 2 mM L-glutamine,
100 IU/ml penicillin, 100 Bg/ml streptomycin, and 20 mM Hepes. VV
titres were determined by tissue culture infective dose (TCID)
assay or standard plaque assay (PFU) using Vero cells and CV-I
cells, respectively. T2 cells negative for both TAP-1 and TAP-2
were transfected with mouse K-2K.sup.b and stable clones were
established with standard protocols.
Example 1
[0052] Mice were co-infected with a recombinant vaccinia virus
carrying expressible TAP-I and TAP-2 genes (W-h TAP-1,2) and
vaccinia viruses. The resultant cytotoxic T-lymphocyte activity was
measured, and compared with controls. The resultant protection from
lethal virus challenge was quantified.
[0053] The recombinant vaccinia virus containing TAP-I and TAP-2
genes (W-h TAP-1,2) and a control recombinant virus vaccinia
containing no TAP genes (W-PJS-5) were prepared by standard methods
outlined herein. Groups (n=3) of mice were vaccinated with
equivalent does of VV-hTAP-1,2 or W-PJS-5, and, 12 days later, were
challenged with a lethal dose of Vaccinia virus--WR (neurotropic
strain) by the intranasal route. Mice were euthanized if weight
loss fell to 75% of pre-challenge weight.
[0054] Three different dosages, 3.times.10.sup.3 IU,
3.times.10.sup.4 IU and 3.times.10.sup.5 IU, of VV-h TAP-1,2 were
administered to three different groups of animals. A fourth group
received a sham vaccination with PBS. The results are presented
graphically on FIG. 1 of the accompanying drawings. These graphs
show that mice vaccinated with VVh TAP-1,2 were able to resist the
challenge at all doses without morbidity or mortality, compared
with the animals receiving the sham vaccination.
[0055] In a similar manner, three groups of control mice were
vaccinated with the same three different does of VV-PJS-5 and
similarly tracked. These results are presented graphically on
accompanying FIG. 2 (along with the sham vaccination result from
FIG. 1). Only at the highest vaccination doses were the mice able
to resist the challenge without morbidity or mortality. It required
100.times. fold increase in vaccination dose of W-PJS-5 (3e5 IU) to
generate the same protection as VV-h TAP-1,2 (3.times.10.sup.3
IU).
[0056] This experiment demonstrates that the recombinant W-hTAP-1,2
alone acts as an effective vaccine against Pox viridae infections
subsequently encountered. Since it is more effective than the
TAP-free VV-PJS-5 recombinant virus in this application, the extra
effectiveness can be safely concluded to be due to the presence of
TAP in the cells when the invading pox virus is subsequently
encountered, not the viral vector alone.
Example 2
[0057] To confirm that weight loss was an adequate measure of
resistance to the viral challenge, lung viremia (the presence of
virus in the blood in the animal's lungs) and weight loss was
determined 3 and 5 days after challenge with W-WR in groups of mice
vaccinated with either PBS, VV-hTAP-1,2 (3.times.10.sup.3 IU) or
VV-PJS-5 (3.times.1O.sup.3 IU). The results are presented
graphically on accompanying FIG. 3. There was a highly significant
correlation between weight loss and lung viremia.
Example 3
[0058] The infection doses required to generate minimal and maximal
(CTL) responses against VV-PJS-5 was determined.
[0059] Cytotoxic T-cell activity in mice infected with VV is
dependent on infection dose. Splenocytes from mice infected with
doses of VV were assayed for their ability to kill cellular targets
presenting MHC class 1 restricted VV antigens on their surface. A
dose response titration curve is presented as FIG. 4 herein. Low
infection doses do not stimulate W specific CTL activity. Maximal
CTL activity was generated at high doses. Having determined the
infectious doses required to generate minimal maximal CTL response
against VV-PJ5-5 (a recombinant vaccinia virus not coding for TAP),
the CTL activity was compared between mice infected with equivalent
low doses of VV-h TAP-1,2 or W-PJS-5. The results are presented
graphically on accompanying FIG. 5. The CTL activity in mice
infected with VV-hTAP-1,2 was approximately two times greater than
the activity generated by an equivalent low dose of W-PJS-5,
indicating that the increases observed were specific to the
presence of hTAP-1,2.
Example 4
[0060] A peptide translocation assay was used to measure the
accumulation of radio-labeled peptides from the cytoplasm to the
endoplasmic reticulum (ER). This is a measure of TAP activity. The
presence or absence of ATP confirmed that the accumulation of
peptide in the ER was due to active transport. The results are
presented on FIG. 6, as bar graphs. Splenocytes from TAP knockout
mice were unable to translocate peptides and confirmed that the
observed accumulation was due to TAP activity. Peptide transport
activity was higher in mice infected with VV-hTAP-1,2 than mice
infected with W-PJS-5 or normal uninfected mice indicating that the
increased activity was due to hTAP-1 expression.
Example 5
[0061] Histopathological examination of mice over expressing human
TAP were conducted. Mice were randomized into three groups as
follows: I-untreated, II-infected with VV-hTAP-1,2
(3.times.10.sup.4 IU), III-infected with W-PJS-5 (3.times.10.sup.4
IU). The mice in groups II and III were sacrificed on days 5, 15
and 30 after infection. The untreated mice (group I) were
sacrificed five days following infection of groups I and II.
Kidney, salivary gland, intestine, lung and joints were harvested
from each mouse. Tissues were fixed in 10% formalin buffer and
processed routinely for paraffin embedding. Fifteen sections from
each tissue (spaced throughout the tissue sample) were cut and
stained with hematoxylin and eosin. Histopathological alterations
were assessed by an experienced blinded observer. Each tissue was
examined for abnormal pathology as well as for inflammatory
infiltrate.
[0062] Histopathological examination for evidence of autoimmune
inflammation was performed in mice infected with VV-hTAP-1,2,
W-PJS-5, or in uninfected mice. Kidney, salivary gland, intestine,
lung and joints were graded for abnormal pathology and infiltrating
leukocytes. The results are reported in the Table below. No
differences were seen in the tissues of infected and uninfected
mice over a thirty-day period post infection. Thus no evidence of
enhanced inflammation or autoimmunity due to the over-expression of
TAP was observed, and it can be safely concluded that TAP
over-expression does not cause autoimmunity.
TABLE-US-00001 TABLE Table 1. Pathology of Tissues from VV-PJS-5
and VV-hTAP-1,2 Tissue Examined Salivary Group Lung Intestine Joint
Kidney Gland Untreated Normal Normal Normal Normal Normal Day 30
VV- Normal Normal Normal Normal Normal PJS-5 Day 15 VV- Normal
Normal Normal Normal Normal hTAP-1,2 Day 15 VV- Normal Normal
Normal Normal Normal PJS-5 Day 15 VV- Normal Normal Normal Normal
Normal hTAP-1,2 Day 30 VV- Normal Normal Normal Normal Normal PJS-5
Day 30 VV- Normal Normal Normal Normal Normal hTAP-1,2 Note: In the
above Table, "normal" is defined as no pathology or infiltrated
leukocytes in the tissue tested.
Example 6
[0063] An experiment was conducted using laboratory mice to
investigate the effect of TAP over expression in VV-infected cells,
on W specific T-cell immune response. For the generation of W
antigen specific effectors, mice were infected with infectious
doses of W-hTAP-1,2 low dose (1.times.10.sup.4 PFU), W-PJS-5 low
dose (I.times.IO.sup.4 PFU) or VV-PJS-5 high dose (5.times.10.sup.6
PFU). Six days after infection, splenocytes were removed and
separated from erythrocytes and cultured and re-stimulated in vitro
for 3 days with irradiated syngenic splenocytes infected with
VV-PJS-5 (3.4.times.10.sup.7 PFU/1.times.1O.sup.8 syngenic
splenocytes, MOI=0.335). To measure cytotoxic activity, a standard
4 hour .sup.51Cr release assay was performed by using target RMA
cells, infected overnight with W-PJS-5 (MOI=0.34). The targets were
labeled with Na 51CrO4 (70 BCi/10.sup.6 cells) for 1 hr at
37.degree. C. and then the standard 4 hour .sup.51Cr release assay
was performed. The cytotoxicity tests were done in 96 V-shaped well
plates at many effector: target ratios.
[0064] The dosages and results are presented graphically on
accompanying FIG. 7. VV antigen specific activity of T-cells from
the spleen of the mouse infected with W-hTAP1,2 (4.times.10.sup.3
PFU) is substantially higher than the activity in mice infected
with VV-PJS-5 (4.times.10.sup.3 PFU) thereby confirming that
greater T-cell responses against Poxviruses can be achieved by TAP
over-expression. This observed enhancement of W specific cytotoxic
activity is attributable to the increased activity of TAP.
Example 7
[0065] C57BL/6 naive splenocytes were stimulated in vitro with 1
Bg/ml LPS for two days and then infected overnight with either
VV-PJS-5 or W-hTAP-1,2 at two doses MOI=5.times.10.sup.4 and
MOI=0.25, respectively. The infected cells were then tested for
total surface H-2K.sup.b expression and W-antigen associated
H-2K.sup.b expression
[0066] The results of the FACS analysis are presented on FIG. 8a,
and show that the surface H-2 Kb expression does not differ between
W-hTAP-1,2 and VV-PJS-5 infected splenocytes.
[0067] The results of a VV specific CTL assay, presented
graphically as FIG. 8b, show increased killing of naive splenocyte
targets infected with VV-hTAP-1,2 compared with VV-PJS-5 infected
targets.
[0068] It is thus shown that TAP over-expression in antigen
presenting cells primes increased CTL activity by increasing VV
specific MHC class 1 but not total MHC class 1 expression in
splenocytes. This specificity towards VV indicates a particular and
especially effective potential of the TAP augmentation approach to
the development of Pox viridae vaccines and vaccine adjuvants, with
special activity and lack of side effects in this application.
* * * * *