U.S. patent application number 10/857546 was filed with the patent office on 2004-10-28 for use of parapox b2l protein to modify immune responses to administered antigens.
Invention is credited to Johnston, Stephen A., McGuire, Michael J..
Application Number | 20040213807 10/857546 |
Document ID | / |
Family ID | 28790674 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040213807 |
Kind Code |
A1 |
Johnston, Stephen A. ; et
al. |
October 28, 2004 |
Use of Parapox B2L protein to modify immune responses to
administered antigens
Abstract
The Parapox B2L virus envelope protein is used as an adjuvant to
enhance a subject's response to an administered antigen. Both
antibody and cellular immune responses can be modified. B2L protein
is particularly useful as an adjuvant for poorly immunogenic tumor
vaccines and subunit vaccines, such as those useful for preventing
and/or treating flu, tuberculosis, respiratory syncytial virus,
anthrax and HIV.
Inventors: |
Johnston, Stephen A.;
(Dallas, TX) ; McGuire, Michael J.; (Dallas,
TX) |
Correspondence
Address: |
RICHARD ARON OSMAN
SCIENCE AND TECHNOLOGY LAW GROUP
242 AVE VISTA DEL OCEANO
SAN CLEMEMTE
CA
92672
US
|
Family ID: |
28790674 |
Appl. No.: |
10/857546 |
Filed: |
May 28, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10857546 |
May 28, 2004 |
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10414609 |
Apr 15, 2003 |
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6752996 |
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10414609 |
Apr 15, 2003 |
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10123058 |
Apr 15, 2002 |
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6752995 |
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10414609 |
Apr 15, 2003 |
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PCT/US02/38971 |
Dec 6, 2002 |
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60336694 |
Dec 7, 2001 |
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Current U.S.
Class: |
424/204.1 |
Current CPC
Class: |
A61K 2039/55561
20130101; A61K 39/00 20130101; C12N 2710/24222 20130101; A61K
38/162 20130101; C07K 19/00 20130101; A61K 2039/53 20130101; C12N
2710/24234 20130101; A61K 39/275 20130101; C12N 2760/16122
20130101; C07K 14/005 20130101; A61K 39/39 20130101; C07K 2319/00
20130101; A61K 2039/55516 20130101; C12N 2710/24022 20130101; C12N
2710/24034 20130101; A61K 39/12 20130101 |
Class at
Publication: |
424/204.1 |
International
Class: |
A61K 039/12 |
Goverment Interests
[0002] This invention was funded in part by DARPA Grant No
N652369915426, Account number 36001, by which the U.S. Government
may have certain rights in this invention.
Claims
1. A method of enhancing an immune response to an antigen,
comprising the steps of: administering to a subject mammal in need
thereof (a) an effective amount of a B2L viral envelope protein of
a Parapox virus, wherein the B2L protein is unassociated with other
envelope components naturally associated with the B2L protein in
the virus, and (b) an antigen, whereby the B2L protein acts as an
adjuvant to enhance the immune response to the antigen; and
detecting a resultant enhanced, specific immune response to the
antigen.
2. The method of claim 1 wherein the B2L protein and the antigen
are administered sequentially.
3. The method of claim 1 wherein the B2L protein and the antigen
are administered simultaneously.
4. The method of claim 1 wherein the B2L protein and the antigen
are administered simultaneously as a fusion protein comprising the
B2L protein and the antigen.
5. The method of claim 1 wherein the B2L protein is administered by
means of a nucleic acid encoding the B2L protein.
6. The method of claim 1 wherein the antigen is administered by
means of a nucleic acid encoding the antigen.
7. The method of claim 1 wherein the antigen is administered as an
attenuated or killed pathogen comprising the antigen.
8. The method of claim 1 wherein the antigen is a tumor
antigen.
9. The method of claim 1 wherein the B2L protein and the antigen
are administered by injection.
10. The method of claim 1 wherein the B2L protein and the antigen
are administered intradermally.
11. The method of claim 1 wherein the Parapox virus is a Parapox
virus ovis strain selected from the group consisting of NZ2, NZ7,
NZ10, and D1701.
12. The method of claim 1 wherein the mammal is a human.
13. A pharmaceutical composition for enhancing an immune response
to an antigen, the composition comprising: a B2L viral envelope
protein of a Parapox virus, or a nucleic acid encoding said B2L
protein, wherein the B2L protein is unassociated with other
envelope components naturally associated with the B2L protein in
the virus; and an antigen or a nucleic acid encoding said antigen,
wherein administered to a subject mammal in need thereof, the B2L
protein of the pharmaceutical composition acts as an adjuvant to
enhance a specific immune response to the antigen.
14. A pharmaceutical composition for enhancing an immune response
to an antigen, the composition comprising: a PP30 protein of a
Parapox virus, or a nucleic acid encoding said PP30 protein,
wherein the PP30 protein is unassociated with other components
naturally associated with the PP30 protein in the virus; and an
antigen or a nucleic acid encoding said antigen, wherein
administered to a subject mammal in need thereof, the PP30 protein
of the pharmaceutical composition acts as an adjuvant to enhance a
specific immune response to the antigen.
15. A method of using a composition according to claim 14 to
enhance an immune response to an antigen, comprising the steps of:
administering to a subject mammal in need thereof (a) an effective
amount of a PP30 protein of a Parapox virus, wherein the PP30
protein as found in Parapox strain D1701 comprises the amino acid
sequence of SEQ ID NO:6, and wherein the PP30 protein is
unassociated with other components naturally associated with the
PP30 protein in the virus, and (b) an antigen, whereby the PP30
protein acts as an adjuvant to enhance the immune response to the
antigen; and detecting a resultant enhanced, specific immune
response to the antigen.
Description
[0001] This application is a continuation under 35USC120 of U.S.
Ser. No. 10/414,609, filed Apr. 15, 2003, which is a continuation
of U.S. Ser. No. 10/123,058, filed on Apr. 15, 2002, and is a
continuation in part of PCT Appl No. PCT/US02/38971, filed Dec. 6,
2002, which claims the benefit of U.S. provisional application Ser.
No. 60/336,694 filed Dec. 7, 2001.
FIELD OF THE INVENTION
[0003] The invention relates to the use of B2L and PP30 proteins of
a Parapox virus as a vaccine adjuvant.
BACKGROUND OF THE INVENTION
[0004] Cutaneous Parapoxvirus ovis causes recruitment of epidermal
dendritic cells to the infection site in sheep and subsequent
cell-mediated immunity (Lear et al., Eur. J. Dermatol. 6, 135-40,
1996; Haig et al., Comp Immun. Microbiol. Infect. Dis. 20 197-204,
1997). Attenuated Parapox viruses can be used to induce
Paradox-specific immunity. U.S. Pat. No. 6,162,600. In addition,
the highly attenuated strain D1701 (Baypamun HK.RTM.) is used as a
non-specific immunomodulator (Buttner et al., Immunol. Microbiol.
Infect. Dis. 16, 1-10, 1993) to promote immunity to heterologous
pathogens.
[0005] Attenuation of Parapox virus, however, is time-consuming,
taking from 100 to 200 culture passages; according to WO 95/22978,
it takes from three to five years to perform each 100 passages,
depending on the species of virus used. Attenuation can, therefore,
"encompass a period lasting from ten to twenty years." See WO
95/22978, page 9.
[0006] WO 95/22978 discloses the use of combinations of two or more
individual Parapox virus components as "multipotent paramunity
inducers" for use as adjuvant therapy for tumors and the prevention
of metastases. The components can be individual polypeptides or
detached envelopes of poxviruses. WO 95/22978, however, does not
disclose any particular viral polypeptides other than the viral
fusion protein and adsorption protein. Moreover, WO 95/22978
teaches that the disclosed paramunity inducers have virtually no
immunogenic properties.
[0007] There is a need in the art for simple, effective vaccine
adjuvants that can be used to enhance immune responses against
tumors and dysplastic lesions and against exogenous pathogens.
SUMMARY OF THE INVENTION
[0008] It is an object of the invention to provide reagents and
methods for modifying immune responses to administered antigens.
This and other objects of the invention are provided by one or more
of the embodiments described below.
[0009] One embodiment of the invention provides a method of
enhancing an immune response to a vaccine composition. The method
involves administering to a subject in need thereof (a) an
effective amount of a B2L viral envelope protein of a Parapox virus
and (b) a vaccine composition comprising an active component. The
adjuvant B2L viral envelope protein thereby enhances the immune
response to the vaccine composition.
[0010] Another embodiment of the invention provides a
pharmaceutical composition comprising a B2L viral envelope protein
of a Parapox virus and a vaccine composition comprising an active
component.
[0011] Yet another embodiment of the invention provides a
pharmaceutical composition comprising a nucleic acid molecule
encoding a B2L viral envelope protein of a Parapox virus and a
vaccine composition comprising an active component.
[0012] Thus, the invention provides pharmaceutical compositions and
methods using B2L protein to modify immune responses to
administered antigens.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The invention is based on the ability of a Parapox viral
envelope protein termed "B2L" to act as an adjuvant, i.e., to
augment or otherwise modify a subject's immune response to an
administered antigen and/or an active component of a vaccine. As
noted below, the descriptions herein are also applicable to a
second Parapox immunostimulatory polypeptide, PP30, and references
herein to B2L are understood to encompass both proteins, absent
contrary implication. Administered antigens include, but are not
limited to, cells expressing tumor antigens, attentuated or killed
pathogens and antigenic components thereof or nucleic acids
encoding the antigenic components.
[0014] Both antibody and cellular immune responses can be modified.
B2L protein is particularly useful as an adjuvant for poorly
immunogenic tumor antigens and subunit vaccines, such as those
useful for preventing and/or treating flu, tuberculosis,
respiratory syncytial virus, anthrax and HIV.
[0015] B2L is the second open reading frame in the BamHl B fragment
of the Orf virus genome (Sullivan et al., Virology 202, 968-73,
1994). Prior work teaches that, as the activity of epitopes
responsible for antigen-specific immunization decrease, adjuvant
activity of the preparations increases. See WO 95/22978, page 4.
B2L is an immunogenic protein; in fact, B2L protein is one of a few
Orf virus proteins to which a strong antibody response can be
mounted in sheep. Sullivan et al., 1994. Thus, it is surprising
that purified B2L protein itself has adjuvant activity.
[0016] B2L proteins for use in the compositions and methods
described herein are those of the Parapoxvirus genus, such as Orf
virus (OV), particularly the Parapox ovis strains NZ2, NZ7, NZ10,
and D1701. Orf viruses are reviewed in Robinson & Balassu, Vet.
Bull 51, 771, 1981; Robinson & Lyttle, in Binns & Smith,
eds., recombinant poxviruses, Chapter 9, pp. 306-17, CRC Press,
Boca Raton, 1992. An amino acid sequence for the B2L protein of OV
NZ2 is disclosed in Sullivan et al., Identification and
characterization of an orf virus homologue of the vaccinia virus
gene encoding the major envelope antigen p37K, Virology 202 (2),
968-73, 1994, and is shown in SEQ ID NO:2. A coding sequence for
SEQ ID NO:2 is shown in SEQ ID NO:1. The amino acid sequences of
the B2L proteins obtained from D1701 and NZ2 are highly conserved.
The amino acid sequence of the D1701 protein is shown in SEQ ID
NO:4. A coding sequence for SEQ ID NO:4 is shown in SEQ ID
NO:3.
[0017] Purified B2L protein is separated from other compounds that
normally associate with the B2L protein in the virus, such as other
envelope components. A preparation of purified B2L protein is at
least 80% pure; preferably, the preparations are 90%, 95%, or 99%
pure. Purity of the preparations can be assessed by any means known
in the art, such as SDS-polyacrylamide gel electrophoresis.
[0018] Purified B2L protein for use in compositions and methods of
the invention can be purified from Parapox viruses or from cells
infected by the viruses, by recombinant DNA methods, and by
chemical synthesis. Purification methods include, but are not
limited to, size exclusion chromatography, ammonium sulfate
fractionation, ion exchange chromatography, affinity
chromatography, and preparative gel electrophoresis.
[0019] B2L protein can be expressed recombinantly, after insertion
of B2L coding sequences into an expression vector that contains the
necessary elements for the transcription and translation of the
inserted coding sequence. Maintenance of orf viruses in culture is
disclosed in WO 97/37031. A preferred system for maintaining and
expressing B2L protein is HKB11 cells transfected with B2L in a
vector such as a p2ToP, pCEP4, or pcDNA3.1 vector (Invitrogen).
Recombinantly produced B2L protein can be secreted into the culture
medium and purified. Methods for producing proteins recombinantly
are well known to those skilled in the art.
[0020] A B2L protein also can be produced using chemical methods to
synthesize its amino acid sequence, such as by direct peptide
synthesis using solid-phase techniques (Merrifield, J. Am. Chem.
Soc. 85, 2149-2154, 1963; Roberge et al., Science 269, 202-204,
1995). Protein synthesis can be performed using manual techniques
or by automation. Optionally, fragments of a B2L protein can be
separately synthesized and combined using chemical methods to
produce a full-length molecule.
[0021] "B2L protein" as used herein includes both functional
portions of B2L and full-length or partial biologically active B2L
variants. Biologically active variants (i.e., variants that possess
adjuvant activity) comprise amino acid substitutions, insertions,
and/or deletions with respect to the amino acid sequences shown in
SEQ ID NOS:2 or 4. Amino acid substitutions are defined as one for
one amino acid replacements. They are conservative in nature when
the substituted amino acid has similar structural and/or chemical
properties. Examples of conservative replacements are substitution
of a leucine with an isoleucine or valine, an aspartate with a
glutamate, or a threonine with a serine. Biologically active
variants can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18,
20, 25, or 30 or more conservative amino acid substitutions as long
as adjuvant activity of the B2L variant is maintained.
[0022] Amino acid insertions or deletions are changes to or within
an amino acid sequence. They typically fall in the range of about 1
to 5 amino acids (i.e., 1, 2, 3, 4, or 5). Guidance in determining
which amino acid residues can be substituted, inserted, or deleted
without abolishing biological or immunological activity of a B2L
protein can be found using computer programs well known in the art,
such as DNASTAR software. Biological activity of a B2L protein
having an amino acid substitution, insertion, and/or deletion can
be tested, for example, as described in Example 1.
[0023] Functional portions of B2L comprising, for example, 25, 50,
75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 360,
370, 375, or 377 amino acids, also can be used in the compositions
and methods of the invention, provided that the portions of B2L
retain biological activity, e.g., the ability to enhance an immune
response and/or exert a chemotactic effect on enriched dendritic
cell populations.
[0024] Purified B2L protein can be used in pharmaceutical
compositions. Pharmaceutical compositions of the invention can be
used to boost immune responses in mammals, including laboratory
animals (e.g., mice, rats, hamsters, guinea pigs), companion
animals (e.g., dogs, cats), farm animals (e.g., horses, cows,
sheep, pigs, goats), and humans.
[0025] Pharmaceutical compositions of the invention include a
pharmaceutically acceptable carrier. Typically these will be
sterile formulations in a diluent or vehicle that is free of
pyrogenic components. Buffers, stabilizers, and the like can be
included, as is known in the art. Optionally, pharmaceutical
compositions include conventional adjuvants, such as aluminum
hydroxide and aluminum phosphate (collectively commonly referred to
as alum), saponins complexed to membrane protein antigens (immune
stimulating complexes), pluronic polymers with mineral oil, killed
mycobacteria in mineral oil, Freund's complete adjuvant, bacterial
products, such as muramyl dipeptide, and lipopolysaccharides.
[0026] If desired, B2L protein can be coupled to an antigen. Means
of making such molecules are well known in the art. For example,
coupled molecules can be synthesized chemically or produced by
covalently linking two polypeptide segments or by standard
procedures in the art of molecular biology. Recombinant DNA methods
can be used to prepare fusion proteins, for example, by making a
DNA construct which comprises B2L coding sequences in proper
reading frame with nucleotides encoding a polypeptide to be coupled
with B2L and expressing the DNA construct in a host cell, as is
known in the art. Many kits for constructing fusion proteins are
available from companies such as Promega Corporation (Madison,
Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountain View,
Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL
International Corporation (MIC; Watertown, Mass.), and Quantum
Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).
[0027] B2L-containing compositions are co-administered with a
particular antigen or vaccine composition. "Co-administration"
includes administration of B2L and the antigen or vaccine
composition separately or in the same composition.
[0028] Vaccine compositions comprise one or more active components,
e.g., a tumor antigen or an attenuated or killed pathogen or an
antigenic component thereof. Antigenic components include any
component that is recognized by cells of the immune system.
Suitable tumor antigens include, but are not limited to,
.alpha.-fetoprotein, BAGE, .beta.-HCG, CEA, ESO,, GAGE,
gangliosides, Her-2/neu, HPV E6/E7, immunoglobulins, MAGE-1,
MAGE-2, MAGE-3, MAGE-4, MAGE-12, MART-1, Melan-A, melanoma antigen
gp75, gp100, MN/G250, MUC1, MUC2, MUC3, MUC4, MUC18, PSA, PSM,
RAGE, ras, SART-1, telomerase, thyroperoxidases, tyrosinases, and
p53.
[0029] Vaccine compositions also can include a pathogen. The
pathogen can be, e.g., an attenuated or killed virus, bacterium,
mycoplasm, parasite, yeast, fungus, prion, or a protozoan. Suitable
pathogens include human immunodeficiency viruses, Herpes viruses,
hepatitis viruses, pox viruses, flu viruses, measles, mumps,
rubella, rabies, respiratory syncytial viruses, Bacillus anthracis,
Bordetella pertussis, Borrelia burgdorferi, Clostridium tetani,
Corynebacterium diphtheriae, Haemophilus influenza B, Neisseria
meningitidis, Salmonella typhi, Streptococcus pneumoniae, and
Vibrio cholerae. The active component also can be, for example, an
immunogenic fragment, extract, subunit, metabolite, or recombinant
construct of such a pathogen. Optionally, the active component can
be mixed with a pharmaceutically acceptable carrier and/or a
conventional adjuvant, as described above.
[0030] Adjuvant compositions comprising B2L protein can be
administered sequentially or simultaneously with a vaccine
composition, including a poorly immunogenic subunit vaccine. If
desired, the B2L protein can be present in the vaccine composition.
Suitable routes of administration include, without limitation,
subcutaneous, intravenous, nasal, ophthalmic, transdermal,
intramuscular, intradermal, intragastric, perlingual, alveolar,
gingival, intraperitoneal, intravaginal, pulmonary, rectal, and
oral administration. Administration can be by any suitable means,
including injection, topical administration, ingestion, or
inhalation. Single and/or multiple administrations are
contemplated.
[0031] Optionally, B2L protein can be administered using a nucleic
acid molecule encoding the protein. The nucleic acid molecule can
be either DNA, RNA, or a DNA/RNA chimera. Use of DNA-encoded
elicitors of immune responses is discussed, for example, in
McDonnel & Askari, New Engl J. Med. 334, 42-45, 1996; Robinson,
Can. Med. Assoc. J. 152, 1629-32, 1995; Fynan et al., Int. J.
Immunopharmacol. 17, 79-83, 1995; Pardoll & Beckerleg, Immunity
3, 165-69, 1995, and Spooner et al., Gene Therapy 2, 173-80, 1995.
Chimeric RNA/DNA oligonucleotide based gene therapy is discussed in
Lai & Lien, Expert Opin Biol Ther 2001 January; 1(1):41-7.
[0032] A variety of delivery systems, both viral and non-viral, can
be used to administer the B2L-encoding nucleic acid molecule. Such
delivery systems include, but are not limited to, naked plasmid
DNA, viral expression vectors, and nucleic acid molecules in
conjunction with a liposome or a condensing agent. See U.S. Pat.
No. 6,303,372.
[0033] The determination of a therapeutically effective dose of B2L
is well within the capability of those skilled in the art. A
therapeutically effective dose refers to that amount of active
ingredient (i.e., B2L protein or nucleic acid encoding B2L) that
results in an augmentation of the immune response to a
co-administered antigen, compared to that which occurs in the
absence of the therapeutically effective dose.
[0034] The therapeutically effective dose can be estimated
initially either in cell culture assays or in animal models,
usually mice, rats, rabbits, dogs, or pigs. The animal model also
can be used to determine the appropriate concentration range and
route of administration. Such information can then be used to
determine useful doses and routes for administration in humans.
[0035] Therapeutic efficacy and toxicity, e.g., ED50 (the dose
therapeutically effective in 50% of the population) and LD50 (the
dose lethal to 50% of the population), can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals. The dose ratio of toxic to therapeutic effects is the
therapeutic index, and it can be expressed as the ratio,
LD50/ED50.
[0036] Pharmaceutical compositions that exhibit large therapeutic
indices are preferred. The data obtained from cell culture assays
and animal studies are used in formulating a range of dosage for
human use. The dosage contained in such compositions is preferably
within a range of circulating concentrations that include the ED50
with little or no toxicity. The dosage varies within this range
depending upon the dosage form employed, sensitivity of the
patient, and the route of administration.
[0037] Suitable dosages and treatment regimens for administration
of either B2L protein or nucleic acid molecules encoding B2L
include, but are not limited to, daily, twice-or three-times
weekly, weekly, bi-weekly, monthly, bimonthly, or yearly treatments
of about 1, 5, 10, 15, 20, 25, 50, 75, 100, or 200 ug/m.sup.2 (or
approximately 0.05, 0.1, 0.2, 0.25, 0.3, 0.5, 0.75, 1, 2, 5, or 10
mg/kg). Preferred routes of administration include subcutaneous,
intradermal, intramuscular administration. The exact dosage,
treatment regimen, and route of administration, however, will be
determined by the practitioner, in light of factors related to the
subject that requires treatment.
[0038] All patents, patent applications, and references cited in
this disclosure are expressly incorporated herein by reference in
their entireties. The above disclosure generally describes the
present invention. A more complete understanding can be obtained by
reference to the following specific example, which is provided for
purposes of illustration only and is not intended to limit the
scope of the invention.
EXAMPLES
[0039] B2L is an immunostimulatory Parapox protein. Using PCR
amplification of segments of the parapox genome, (D1701 strain), we
identified full-length coding sequences from open reading frames
(ORFs) of Parapox. Specific primers were synthesized to the ORFs
generating individual PCR products. Utilizing linear expression
element (LEE) technology, the PCR products were tested for in vivo
DC recruiting activity. DNA was precipitated on gold beads and the
complexes were introduced by biolistic inoculation into mouse
abdominal skin that had been prepared by treatment with shears and
Nair depilatory. Each mouse was treated with 4-6 shots of the
DNA-gold complexes. The recruitment of dendritic cells to the site
of inoculation was assessed after 4 days. To assess the dendritic
cell recruitment, the mice were euthanized, the location of the
inoculations marked and the abdominal skin was harvested.
Inoculation sites were isolated and trimmed of surrounding tissue.
Skin segments were immersed in cryoprotective medium and quickly
frozen in liquid nitrogen. Thin-sections were cut and stored at
-20.degree. C. for subsequent fixation and staining. The presence
of dendritic cells (FITC-labeled, anti-I-A.sup.d/I-E.sup.d antibody
positive cells with dendritic morphology) was evaluated by
fluorescence microscopy. We identified B2L by its dendritic cell
recruitment activity in this assay, with further observations of
the best humoral immune response occurring at the inoculation
site.
[0040] B2L is an adjuvant for generating a humoral immune response.
Mice were inoculated with a low dose (0.5 .mu.gDNA/ear; 1 .mu.g
total/mouse) of a plasmid encoding human .infin..sub.1-anti-trypsin
(hAAT). When inoculated by itself, the low dose is below the
threshold of antibody induction with a single immunization.
However, when administered in the presence of a genetic adjuvant,
such as a plasmid encoding GM-CSF, an antibody response to HAAT is
observed in most mice. The addition of LEEs encoding B2L enhanced
the generation of an antibody response to hAAT. In fact, the
animals that received the B2L LEEs along with the HAAT antigen
exhibited an earlier antibody response than the mice that received
GM-CSF as an adjuvant. The responses of the 2 groups were similar
with respect to the levels of circulating antibodies to HAAT that
were obtained without a booster inoculation.
[0041] B2L enhances antibody responses to multiple antigens. In
these experiments, B2L was co-inoculated with plasmids encoding
hAAT and hepatitis B surface antigen (HbsAg). The level of each
antigen-encoding plasmid was reduced to 0.2 .mu.g/inoculation with
B2L or control plasmid at 1 .mu.g/inoculation. B2L enhanced the
generation of antibodies in response to both hAAT and HbsAg.
[0042] B2L adjuvants provide a protective immune response for
influenza virus. Mice were immunized twice with plasmid encoding
influenza hemagglutinin (pCMVi-HA). Mice were immunized with 4
levels (0.4-400 ng) of pCMVi-HA plasmid along with 1 .mu.g control
plasmid, pCMVi-mGM-CSF, or pCMVi-B2L. When challenged with
influenza virus, mice that were immunized without adjuvant still
succumbed to the infection. 50% of the mice that received the
highest dose of antigen (400 ng/immunization) without adjuvant died
upon challenge. In contrast, mice that were immunized with this
antigen level in the presence of B2L were fully protected. This
level of protection was also observed with the control adjuvant,
mGM-CSF. In this initial experiment, B2L also enhanced the level of
protection for mice that were immunized with only 40 ng pCMVi-HA.
At this level, B2L exhibited a protective benefit compared to
mGM-CSF.
[0043] PP30 is another Parapox immunostimulatory polypeptide.
Screening the entire dendritic cell recruitment activity and
adjuvant activity, we found a second immunostimulatory Parapox gene
product, designated PP30. SEQ ID NO:6 is a PP30 amino acid
sequence, and SEQ ID NO:5 is a native coding sequence of PP30
derived from Parapox strain D1701. PP30 is also found in
alternative Parapox strains. In general, PP30 may be substituted
for B2L in the compositions and methods disclosed herein, and the
description and examples herein reciting B2L are applicable to PP30
as well, however, the adjuvant activity of PP30 is distinguishable
from B2L as it stimulated an antibody response to hAAT
significantly greater than that to HbsAg. PP30 is a newly
identified gene with no apparent homolog in the NCBI database.
[0044] B2L and PP30 Parapox proteins are immunostimulatory across
Parapox strains. By comparing the sequences of B2L from the D1701
strain to the related gene in the NZ2 strain we identified sequence
differences at both the nucleotide and predicted protein levels.
Therefore, we isolated DNA from the NZ2 strain of Parapox,
amplified the NZ2-B2L gene by PCR and cloned this into pCMVi
plasmid. The efficacy of NZ2-B2L and D1701-B2L was then compared
and showed comparable dendritic cell recruitment activity and
adjuvant activity. Cross-strain immunostimulatory activity was
similarly shown for PP30.
[0045] Direct B2L protein administration enhances specific antibody
response. To demonstrate the effect of the B2L protein on the
generation of a specific antibody response against HBsAg, protein
mixtures without alum were injected into groups of mice:
[0046] Group 1: 1 .mu.g HBsAg protein in PBS ("antigen only"
control)
[0047] Group 2: 1 .mu.g HBsAg protein+3 .mu.l Baypamum.RTM. in PBS
(comparison with whole killed Parapox virus)
[0048] Group 3: 1 .mu.g HBsAg protein+50 ng B2L-H6 in PBS (testing
B2L protein as an adjuvant; note that the protein was expressed as
a 6His fusion protein).
[0049] Group 4: 1 .mu.g HBsAg protein+50 ng SRB4-H6 (comparison
with an irrelevant negative control protein that is also a 6His
fusion protein).
[0050] Group 5: 1 .mu.g HBsAg protein+2.5 .mu.l alum in PBS, mixed
together and left on ice for 30 minutes beforehand (comparison with
alum, the only FDA-approved adjuvant).
[0051] Each mouse was injected with 25 .mu.l into the tibialis
anterior muscle, and blood was collected before injection, and at
each of the time points indicated. Sera were processed and stored
at -20.degree. C. Anti-HBsAg antibody levels were measured by
ELISA. Groups 4 and 5 showed a modest (approximately 2-fold)
increase in antibody elicitation over groups 1 and 2 at multiple
time-points from 30 to 60 days, whereas Group 3 (B2L) showed a much
more dramatic (over a 5-fold) increase over the same controls.
[0052] Direct B2L protein administration stimulates dendritic cell
recruitment. To demonstrate B2L protein promotion of dendritic cell
recruitment, aliquots containing 50 ng of B2L protein were injected
into the skin of a mouse. A control mouse was injected with PBS
alone in a comparable volume. The injected skin was harvested 4
days later. Sections were stained for DC using the anti-MHC II
antibody 2G9, revealing a dramatic B2L protein-enhanced increase in
dendritic cell recruitment.
[0053] B2L has adjuvant activity when coadministered with a tumor
antigen. Tumor peptides frequently are weak immunogens, and immune
responses to them typically require that they be administered
together with an adjuvant. A tumor vaccine consisting of an
emulsion prepared with Montanide ISA 51 containing 100 ug of a
melanoma peptide (TRP-2) and 100 ug of either V5His6-tagged B2L or
and irrelevant protein (hSLC; human secondary lymphoid chemokine)
was administered subcutaneously at the base of the tail three days
after implantation of a melanoma tumor known to express the antigen
included in the vaccine. Tumor growth was monitored until the
nodules reached the volume permitted by an approved animal care and
use protocol. By comparison with animals given the tumor antigen
together with an irrelevant protein, there was a trend toward
reduced tumor growth in mice treated with tumor antigen together
with tagged B2L. The results are consistent with B2L's augmentation
of an immune response to the TRP-2 tumor antigen, which slowed the
growth of the tumor expressing this antigen.
Sequence CWU 1
1
6 1 1137 DNA PARAPOX 1 atgtggccgt tctcctccat ccccctgggc gccgactgcc
gcgtcgtgga gacgctgccc 60 gcagaggtgg cgtctttggc gcagggcaac
atgagcaccc tcgactgctt caccgctatc 120 gccgagtccg cgaagaagtt
cttgtacatc tgcagcttct gctgcaacct gagctccacc 180 aaggagggcg
tcgacgtcaa ggacaagctc tgcacgctcg ccaaggaggg cgtagacgtc 240
acgctgctcg tggacgtgca gagcaaggac aaggacgcgg acgagctgcg cgaggcgggc
300 gtcaactact acaaggtcaa ggtgtccacc aaggagggcg tcggcaacct
tctcggcagc 360 ttctggctct cggacgccgg gcactggtac gtgggaagcg
cctcgctcac gggcgggtcc 420 gtgtccacca tcaagaacct cgggctctac
tccaccaaca agcacctggc ctgggacctc 480 atgaaccgct acaacacctt
ctactccatg atcgtggagc cgaaggtgcc gttcacgcgg 540 ctctgctgcg
ccatcgtcac gcccacggcc acgaacttcc acctcgacca ctccgggggc 600
ggcgtattct tctcggactc gccggagcgc ttcctaggct tctaccgcac gctcgacgag
660 gacctcgtgc tgcaccgcat cgagaacgcc aagaacagca tcgacctctc
gctgctctcg 720 atggtgccgg tgatcaagca cgccagcgcc gtggagtact
ggccgcagat cattgacgcg 780 ctgctgcgcg cggccatcaa ccgcggcgtg
cgcgtgcgcg tgatcattac cgagtggaag 840 aacgcggacc cgctttcggt
ctcggccgcg cgcagcctcg acgactttgg cgtcggcagc 900 gtggacatgt
ccgtgcgcaa gttcgtggta cccggccggg acgacgccgc gaacaacact 960
aagctgctca tcgtggacga caccttcgcg cacctcacgg tcgccaacct cgacggcacg
1020 cactaccgct accacgcctt cgtgagcgtg aacgccgaga agggcgacat
cgtcaaggac 1080 ctgtccgcgg tcttcgagcg ggactggcgc tcggagttct
gcaagccaat aaattaa 1137 2 378 PRT PARAPOX 2 Met Trp Pro Phe Ser Ser
Ile Pro Leu Gly Ala Asp Cys Arg Val Val 1 5 10 15 Glu Thr Leu Pro
Ala Glu Val Ala Ser Leu Ala Gln Gly Asn Met Ser 20 25 30 Thr Leu
Asp Cys Phe Thr Ala Ile Ala Glu Ser Ala Lys Lys Phe Leu 35 40 45
Tyr Ile Cys Ser Phe Cys Cys Asn Leu Ser Ser Thr Lys Glu Gly Val 50
55 60 Asp Val Lys Asp Lys Leu Cys Thr Leu Ala Lys Glu Gly Val Asp
Val 65 70 75 80 Thr Leu Leu Val Asp Val Gln Ser Lys Asp Lys Asp Ala
Asp Glu Leu 85 90 95 Arg Glu Ala Gly Val Asn Tyr Tyr Lys Val Lys
Val Ser Thr Lys Glu 100 105 110 Gly Val Gly Asn Leu Leu Gly Ser Phe
Trp Leu Ser Asp Ala Gly His 115 120 125 Trp Tyr Val Gly Ser Ala Ser
Leu Thr Gly Gly Ser Val Ser Thr Ile 130 135 140 Lys Asn Leu Gly Leu
Tyr Ser Thr Asn Lys His Leu Ala Trp Asp Leu 145 150 155 160 Met Asn
Arg Tyr Asn Thr Phe Tyr Ser Met Ile Val Glu Pro Lys Val 165 170 175
Pro Phe Thr Arg Leu Cys Cys Ala Ile Val Thr Pro Thr Ala Thr Asn 180
185 190 Phe His Leu Asp His Ser Gly Gly Gly Val Phe Phe Ser Asp Ser
Pro 195 200 205 Glu Arg Phe Leu Gly Phe Tyr Arg Thr Leu Asp Glu Asp
Leu Val Leu 210 215 220 His Arg Ile Glu Asn Ala Lys Asn Ser Ile Asp
Leu Ser Leu Leu Ser 225 230 235 240 Met Val Pro Val Ile Lys His Ala
Ser Ala Val Glu Tyr Trp Pro Gln 245 250 255 Ile Ile Asp Ala Leu Leu
Arg Ala Ala Ile Asn Arg Gly Val Arg Val 260 265 270 Arg Val Ile Ile
Thr Glu Trp Lys Asn Ala Asp Pro Leu Ser Val Ser 275 280 285 Ala Ala
Arg Ser Leu Asp Asp Phe Gly Val Gly Ser Val Asp Met Ser 290 295 300
Val Arg Lys Phe Val Val Pro Gly Arg Asp Asp Ala Ala Asn Asn Thr 305
310 315 320 Lys Leu Leu Ile Val Asp Asp Thr Phe Ala His Leu Thr Val
Ala Asn 325 330 335 Leu Asp Gly Thr His Tyr Arg Tyr His Ala Phe Val
Ser Val Asn Ala 340 345 350 Glu Lys Gly Asp Ile Val Lys Asp Leu Ser
Ala Val Phe Glu Arg Asp 355 360 365 Trp Arg Ser Glu Phe Cys Lys Pro
Ile Asn 370 375 3 1137 DNA PARAPOX 3 atgtggccgt tctcctccat
ccccgtgggc gcccaatgcc gcgtcttgga aacgctgccc 60 gcagaggtgg
cgtccctggc gcagggcaac atgagcaccc tcgactgctt caccgccatc 120
gccgagtccg cgaagaaatt tttgtacatc tgcagcttct gctgcaacct gagctccacc
180 aaggagggcg tcgacgtcaa agacaagctc tgcacgctcg ccaaggaagg
cgttgacgtc 240 acgctgctcg tggacgtgca gagcaaggac aaggacgcgg
acgaactgcg cgcggcgggc 300 gtcaactact acaaggtcaa agtgtccacg
cgggaaggcg tcggcaacct tctcggcagc 360 ttctggctct cggacgccgg
gcactggtac gtgggcagcg cctcgctcac gggcgggtcc 420 gtgtccacca
tcaagaacct cgggctctac tccaccaaca agcacctggc ctgggacctc 480
atgaaccgct acaacacctt ctactccatg atcgtggagc cgaaggtgcc gttcacgcgg
540 ctctgctgcg ccgtcgtcac gcccacggcc acgaacttcc acctcaacca
ctccgggggc 600 ggcgtattct tctcggactc gccggagcgc ttcctaggct
tctaccgcac gctcgacgag 660 gacctcgtgc tgcaccgcat cgagaacgcc
aagaacagca tcgacctctc gctgctctcg 720 atggtgccgg tgatcaagca
cgccggcgcc gtggagtact ggccgcggat catagacgcg 780 ctgctgcgcg
cggccatcaa ccgcggcgtg cgcgtgcgcg tgatcatcac cgagtggaag 840
aacgcggacc cgctgtcggt ctcggccgcg cgcagcctcg acgactttgg cgtcggtagc
900 gtggacatgt ccgtgcgcaa gttcgtggta cccggccggg acgacgctgc
gaacaacacc 960 aagctgctta tcgtggacga caccttcgcg cacctcacgg
tcgccaacct cgacggcacg 1020 cactaccgct accacgcctt cgtgagcgtg
aacgccgaga agggcgacat cgtcaaggac 1080 ctgtccgcgg tcttcgagcg
ggactggcgc tcggagtttt gcaagccaat aaattaa 1137 4 378 PRT PARAPOX 4
Met Trp Pro Phe Ser Ser Ile Pro Leu Gly Ala Gln Cys Arg Val Leu 1 5
10 15 Glu Thr Leu Pro Ala Glu Val Ala Ser Leu Ala Gln Gly Asn Met
Ser 20 25 30 Thr Leu Asp Cys Phe Thr Ala Ile Ala Glu Ser Ala Lys
Lys Phe Leu 35 40 45 Tyr Ile Cys Ser Phe Cys Cys Asn Leu Ser Ser
Thr Lys Glu Gly Val 50 55 60 Asp Val Lys Asp Lys Leu Cys Thr Leu
Ala Lys Glu Gly Val Asp Val 65 70 75 80 Thr Leu Leu Val Asp Val Gln
Ser Lys Asp Lys Asp Ala Asp Glu Leu 85 90 95 Arg Ala Ala Gly Val
Asn Tyr Tyr Lys Val Lys Val Ser Thr Arg Glu 100 105 110 Gly Val Gly
Asn Leu Leu Gly Ser Phe Trp Leu Ser Asp Ala Gly His 115 120 125 Trp
Tyr Val Gly Ser Ala Ser Leu Thr Gly Gly Ser Val Ser Thr Ile 130 135
140 Lys Asn Leu Gly Leu Tyr Ser Thr Asn Lys His Leu Ala Trp Asp Leu
145 150 155 160 Met Asn Arg Tyr Asn Thr Phe Tyr Ser Met Ile Val Glu
Pro Lys Val 165 170 175 Pro Phe Thr Arg Leu Cys Cys Ala Val Val Thr
Pro Thr Ala Thr Asn 180 185 190 Phe His Leu Asn His Ser Gly Gly Gly
Val Phe Phe Ser Asp Ser Pro 195 200 205 Glu Arg Phe Leu Gly Phe Tyr
Arg Thr Leu Asp Glu Asp Leu Val Leu 210 215 220 His Arg Ile Glu Asn
Ala Lys Asn Ser Ile Asp Leu Ser Leu Leu Ser 225 230 235 240 Met Val
Pro Val Ile Lys His Ala Gly Ala Val Glu Tyr Trp Pro Arg 245 250 255
Ile Ile Asp Ala Leu Leu Arg Ala Ala Ile Asn Arg Gly Val Arg Val 260
265 270 Arg Val Ile Ile Thr Glu Trp Lys Asn Ala Asp Pro Leu Ser Val
Ser 275 280 285 Ala Ala Arg Ser Leu Asp Asp Phe Gly Val Gly Ser Val
Asp Met Ser 290 295 300 Val Arg Lys Phe Val Val Pro Gly Arg Asp Asp
Ala Ala Asn Asn Thr 305 310 315 320 Lys Leu Leu Ile Val Asp Asp Thr
Phe Ala His Leu Thr Val Ala Asn 325 330 335 Leu Asp Gly Thr His Tyr
Arg Tyr His Ala Phe Val Ser Val Asn Ala 340 345 350 Glu Lys Gly Asp
Ile Val Lys Asp Leu Ser Ala Val Phe Glu Arg Asp 355 360 365 Trp Arg
Ser Glu Phe Cys Lys Pro Ile Asn 370 375 5 408 DNA PARAPOX 5
atggacgcgg tgtccgcgct ctgcgtgagc cctcgcggca gccgccgcca tgttcgtggc
60 gctgcagctc tgggccgtct acgagaacta cgacaacatc cgcgagttca
acgcggcgaa 120 cgcggcgctg gagttcgcgc gcacggcggg cggcccgcgc
gtggaccggc gcgtgttcga 180 ccccaacgac gaggccttcg acgtgcgcaa
gaagtggcgc tgcgtgctct tcaagggcgc 240 ggcggtggcg gcctcggagt
tcgggtttcg ttcgcacgac ggcgtctcgc ccgcgcgctt 300 cgcgagcgtg
ggaggcgtgt gcggacgaga tcttcacggc cgcgagcgac ttccgcgtgt 360
tcaacccgtg tttgccgcca aatcagggca gcgaggcctg cctttttc 408 6 136 PRT
PARAPOX 6 Met Asp Ala Val Ser Ala Leu Cys Val Ser Pro Arg Gly Ser
Arg Arg 1 5 10 15 His Val Arg Gly Ala Ala Ala Leu Gly Arg Leu Arg
Glu Leu Arg Gln 20 25 30 His Pro Arg Val Gln Arg Gly Glu Arg Gly
Ala Gly Val Arg Ala His 35 40 45 Gly Gly Arg Pro Ala Arg Gly Pro
Ala Arg Val Arg Pro Gln Arg Arg 50 55 60 Gly Leu Arg Arg Ala Gln
Glu Val Ala Leu Arg Ala Leu Gln Gly Arg 65 70 75 80 Gly Gly Gly Gly
Leu Gly Val Arg Val Ser Phe Ala Arg Arg Arg Leu 85 90 95 Ala Arg
Ala Leu Arg Glu Arg Gly Arg Arg Val Arg Thr Arg Ser Ser 100 105 110
Arg Pro Arg Ala Thr Ser Ala Cys Ser Thr Arg Val Cys Arg Gln Ile 115
120 125 Arg Ala Ala Arg Pro Ala Phe Phe 130 135
* * * * *