U.S. patent application number 14/318330 was filed with the patent office on 2017-04-27 for allergy inhibitor compositions and kits and methods of using the same.
The applicant listed for this patent is Hsien-Jue Chu, Huali Jin, Youmin Kang, Terry Kaleung Ng, Bin Wang. Invention is credited to Hsien-Jue Chu, Huali Jin, Youmin Kang, Terry Kaleung Ng, Bin Wang.
Application Number | 20170112918 14/318330 |
Document ID | / |
Family ID | 36935235 |
Filed Date | 2017-04-27 |
United States Patent
Application |
20170112918 |
Kind Code |
A9 |
Wang; Bin ; et al. |
April 27, 2017 |
Allergy Inhibitor Compositions And Kits And Methods Of Using The
Same
Abstract
Compositions, methods, and kits for inhibiting an allergic
response against an allergenic protein are disclosed. Compositions,
methods and kits for inhibiting an allergic response against a flea
allergenic protein; a feline allergenic protein; a canine
allergenic protein; a dust mite allergenic protein; a peanut
allergenic protein; a Japanese cedar allergenic protein; and a
blomia tropicalis allergenic protein are disclosed.
Inventors: |
Wang; Bin; (Beijing, CN)
; Jin; Huali; (Beijing, CN) ; Kang; Youmin;
(Beijing, CN) ; Chu; Hsien-Jue; (Bonner Springs,
KS) ; Ng; Terry Kaleung; (Fort Dodge, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wang; Bin
Jin; Huali
Kang; Youmin
Chu; Hsien-Jue
Ng; Terry Kaleung |
Beijing
Beijing
Beijing
Bonner Springs
Fort Dodge |
KS
IA |
CN
CN
CN
US
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20150086593 A1 |
March 26, 2015 |
|
|
Family ID: |
36935235 |
Appl. No.: |
14/318330 |
Filed: |
June 27, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13721449 |
Dec 20, 2012 |
8795675 |
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14318330 |
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11644435 |
Dec 22, 2006 |
8349333 |
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13721449 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/0003 20130101;
A61P 17/04 20180101; A61P 17/02 20180101; A61P 37/08 20180101; A61P
37/00 20180101; A61K 39/0005 20130101; A61P 11/02 20180101; A61K
2039/57 20130101; A61K 2039/53 20130101; A61P 27/14 20180101; A61P
11/06 20180101; A61K 39/0008 20130101; A61K 39/35 20130101 |
International
Class: |
A61K 39/35 20060101
A61K039/35 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 23, 2005 |
CN |
200510132381.X |
Claims
1.-31. (canceled)
32. A therapeutic composition for inhibiting an allergic response,
the composition comprising: (a) an eukaryotic expression vector
comprising a nucleotide sequence encoding an allergenic protein
that comprises an antigen epitope; and (b) an allergenic protein
that comprises an antigenic epitope, wherein the allergenic protein
is selected from the group consisting of: feline allergenic
protein, canine allergenic protein, dust mite allergenic protein,
Japanese cedar allergenic protein, Blomia tropicalis allergenic
protein, and flea salivary allergenic protein.
33. The therapeutic composition of claim 32, wherein the nucleotide
sequence encodes an amino acid sequence selected from the group
consisting of: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID
NO:21, SEQ ID NO:22, and SEQ ID NO:23.
34. The therapeutic composition of claim 32, wherein the nucleotide
sequence comprises a nucleic acid sequence selected from the group
consisting of: SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, SEQ ID NO:24, and SEQ ID NO:25.
35. The therapeutic composition of claim 32, wherein the nucleotide
sequence is operably linked to a promoter selected from the group
consisting of: RSV promoter, CMV promoter, and SV40 promoter.
36. The therapeutic composition of claim 32, wherein a ratio of the
eukaryotic expression vector by weight to the allergenic protein by
weight is between 1:5 and 5:1.
37. The therapeutic composition of claim 36, wherein the ratio of
the eukaryotic expression vector by weight to the allergenic
protein by weight is 1:1.
38. The therapeutic composition of claim 32, wherein a molar ratio
of the eukaryotic expression vector to the allergenic protein is
between 1:100,000 to 20:100,000.
39. The therapeutic composition of claim 7, wherein the molar ratio
of the eukaryotic expression vector to the allergenic protein is
15:100,000.
40. A method of inhibiting an allergenic reaction in a subject in
need thereof, the method comprising administering the therapeutic
composition of claim 32 to the subject.
41. The method of claim 40, wherein the eukaryotic expression
vector and the allergenic protein are administered together.
42. The method of claim 40, wherein the eukaryotic expression
vector and the allergenic protein are administered separately.
43. A method of inducing CD4.sup.+CD25.sup.- Tr cells in a subject
in need thereof, the method comprising administering the
therapeutic composition of claim 32 to the subject.
44. A kit for inhibiting an allergenic response in a subject in
need thereof, the kit comprising: (a) a first container comprising
an eukaryotic expression vector comprising a nucleotide sequence
encoding an allergenic protein that comprises an antigenic epitope;
(b) a second container comprising an allergenic protein that
comprises an antigenic epitope, wherein the allergenic protein is
selected from the group consisting of: feline allergenic protein,
canine allergenic protein, dust mite allergenic protein, Japanese
cedar allergenic protein, Blomia tropicalis allergenic protein, and
flea salivary allergenic protein.
45. The kit of claim 44, wherein the nucleotide sequence encodes an
amino acid sequence selected from the group consisting of: SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, and
SEQ ID NO:23.
46. The kit of claim 44, wherein the nucleotide sequence comprises
a nucleic acid sequence selected from the group consisting of: SEQ
ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ
ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,
SEQ ID NO:24, and SEQ ID NO:25.
47. The kit of claim 44, wherein the nucleotide sequence is
operably linked to a promoter selected from the group consisting
of: RSV promoter, CMV promoter, and SV40 promoter.
48. The kit of claim 44, wherein a ratio of the eukaryotic
expression vector by weight to the allergenic protein by weight is
between 1:5 and 5:1.
49. The kit of claim 48, wherein the ratio of the eukaryotic
expression vector by weight to the allergenic protein by weight is
1:1.
50. The kit of claim 44, wherein a molar ratio of the eukaryotic
expression vector to the allergenic protein is between 1:100,000 to
20:100,000.
51. The kit of claim 50, wherein the molar ratio of the eukaryotic
expression vector to the allergenic protein is 15:100,000.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn.120
as a continuation of U.S. Ser. No. 13/721,449, filed Dec. 20, 2012,
allowed, which is a continuation of U.S. Ser. No. 11/644,435 filed
Dec. 22, 2006, which issued as U.S. Pat. No. 8,349,333 on Jan. 8,
2013, which claims priority to Chinese application number 2005
10132381.X filed Dec. 23, 2005, each of which is incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and kits which
are useful to prevent and inhibit allergic reactions against
allergens, and to methods of using such compositions and kits. The
present invention provides compositions, kits and methods for
preventing and inhibiting flea allergy dermatitis, allergic
reactions to cat and canine fur or danders, dust mites, peanuts,
Japanese cedar pollen and blomia tropicalis allergen.
BACKGROUND OF THE INVENTION
[0003] Allergic reactions to various allergens represent
significant health concerns, particularly in instances in which the
allergic reaction induces severe reactions and/or allergen induced
immediate hypersensitivity (AIH).
[0004] Allergy is considered as the consequence of persistent T
cell activation driving pathogenic inflammation against host dermis
by specific allergens. Several approaches are used to ameliorate
AIH and these include nonspecific immunosuppressive drugs or
monoclonal antibodies targeted to T or B cells (A. J. Van
Oosterhout et al., Am. J. Respir. Cell Mol. Biol. 17, 386 (Sep. 1,
1997); P. Proksch et al, J Immunol 174, 7075 (Jun. 1, 2005)).
However, this situation is compromised as long term treated
recipients can become generally compromised in their ability to
fight infections. Redirecting immunity from Th2 type to Th1 type
has also been demonstrated with limited success (S. Jilek, C.
Barbey, F. Spertini, B. Corthesy, J Immunol 166, 3612 (Mar. 1,
2001)). A recent discovery of T regulatory cells, including the
naturally occurring thymus derived CD4+CD25.sup.+ Treg cells (I. M.
de Kleer et al, J Immunol 111, 6435 (May 15, 2004); D. Lundsgaard,
T. L. Holm, L. Hornum, H. Markholst, Diabetes 54, 1040 (Apr. 1,
2005); M. J. McGeachy, L. A. Stephens, S. M. Anderton, J Immunol
175, 3025 (Sep. 1, 2005); I. Bellinghausen, B. Klostermann, J.
Knop, J. Saloga, J Allergy Clin Immunol 111, 862 (Apr. 1, 2003); E.
M. Ling et al, Lancet 363, 608 (Feb. 21, 2004); and J. Kearley, J.
E. Barker, D. S. Robinson, C. M. Lloyd, J. Exp. Med., jem.20051166
(Nov. 28, 2005)), mucosal induced Th3 cells and antigen induced
CD4.sup.+CD25'' Tr cells have been proposed to be use as
immuno-regulators or suppressors or auto-reactive pathogenesis (H.
Fukaura et al, J. Clin. Invest. 98, 70 (Jul. 1, 1996)). Various
approaches have been explored to induce T regulatory cells to
constrain the auto-reactive T cells. Preferentially, induction of
antigen specific T regulatory cells targeted to allergy, asthma and
autoimmune disease antigens are considered a promising strategy.
Several lines of evidence have indicated that induction of antigen
specific regulatory T cell 1 (TO) is possible via utility of
immatured DCs, suboptimal immunogens or partial blocking the
co-stimulatory molecules in DCs (A. Kumanogoh et al., J Immunol
166, 353 (Jan. 1, 2001); M. K. Levings et al, Blood 105, 1162 (Feb.
1, 2005); and S. K. Seo et al, Nat Med 10, 1088 (Oct. 1, 2004)).
All these approaches are done either in vitro or in experimental
conditions. Induction of Tr cells that can inhibit antigen specific
T cells' function in vivo by co-inoculating antigen-matched DNA and
protein antigens as co-administered vaccines (H. Jin et al,
Virology 337, 1 83 (Jun. 20, 2005)).
[0005] The chief characteristic of the non-host flea is that it is
a hematophagic parasite that may be found in the body of any
mammalian or avian species of animal. Ctenocephalides felis is a
parasite that occurs mainly in cats and dogs, while Ctenocephalides
canis is limited to domestic dogs and feral dogs. Flea allergy
dermatitis (FAD) is the most frequently seen skin ailment in cats
and dogs. FAD results when a flea parasite bites and its saliva
serves as an irritant and elicits an allergic reaction. The
location of the bite appears red, swollen, irritated and itching.
Often the animal will scratch at the bite with its paws, causing
the wound to turn into a skin ulceration and eliciting further
bacterial and fungal infections. This poses a great danger for the
dog or cat and at present no effective pharmacotherapeutic or
preventive methods exist for this disease.
[0006] In general, flea allergen refers to the various differently
sized proteins from flea antigens that cause an allergic reaction.
In some parts of the literature it is referred to as feline flea
saliva allergenic protein FSA1 or Cte f 1. GeneBank AF102502, which
is incorporated herein by reference, discloses the nucleotide
sequences (SEQ ID NO:1) encoding the FSA1 or Cte f 1 protein
derived from the flea salivary gland of the Ctenocephalides felis.
The 653 nucleotide sequence includes coding sequences 1-531 which
include coding sequences for the signal peptide (1-54) and mature
protein sequence (55-528). GeneBank AAD17905, which is incorporated
herein by reference, discloses the amino sequences (SEQ ID NO:2) of
the FSA1 or Cte f 1 protein derived from the flea salivary gland of
the Ctenocephalides felis. including the signal peptide (1-18) and
mature protein sequences (19-176).
[0007] The chief feline allergenic protein is Fel dI. GeneBank
M74953, which is incorporated herein by reference, discloses the
amino acid of and nucleotide sequences (SEQ ID NO:3) encoding the
Fel dI protein derived from the major allergen of the domestic cat.
It possesses the secondary B secretion peptide sequence. This Fel d
I sequence is 416 bp mRNA including the 5' untranslated region made
up of sequences 1-25 and the coding sequence being sequences 26-292
encoding 88 amino acids (SEQ ID NO:4; GeneBank AAC41617, which is
incorporated herein by reference). The signal peptide is encoded by
26-79 and the mature protein is encoded by 80-289. The 3'
untranslated region is 293-416. GeneBank M74952, which is
incorporated herein by reference, discloses the amino acid of and
nucleotide sequences (SEQ ID NO:5) encoding the Fel dI protein
derived from the major allergen of the domestic cat. This Fel dI
sequence is 410 bp mRNA including the 5' untranslated region made
up of sequences 1-7 and the coding sequence being sequences 8-286
encoding 92 amino acids (SEQ ID NO:6; GeneBank AAC37318, which is
incorporated herein by reference). The signal peptide is encoded by
8-73 and the mature protein is encoded by 74-283. The 3'
untranslated region is 287-410.
[0008] The chief canine allergenic proteins are the salivary lipid
promoters Can f1 and Can f2. GeneBank AF027177, which is
incorporated herein by reference, discloses the amino acid of and
nucleotide sequences (SEQ ID NO:7) encoding the Can f1 protein
derived from the salivary lipocalin proteins of the major allergen
of the domestic dog. This Can f1 sequence is 525 bp mRNA encoding
174 amino acids (SEQ ID NO:8; GeneBank AAC48794, which is
incorporated herein by reference).
[0009] GeneBank AF027178, which is incorporated herein by
reference, discloses the amino acid of and nucleotide sequences
(SEQ ID NO:9) encoding the Can f2 protein derived from the salivary
lipocalin proteins of the major allergen of the domestic dog. This
Can f2 sequence is 791 bp mRNA including a coding sequence of
195-737 encoding 180 amino acids (SEQ ID NO: 10; GeneBank AAC48795,
which is incorporated herein by reference).
[0010] GeneBank Ul 1695, which is incorporated herein by reference,
discloses the amino acid of and nucleotide sequences (SEQ ID NO:11)
encoding the dust mite allergy source protein antigen Der P 1. This
Der P 1 sequence is 1099 bp mRNA including a coding sequence of
50-1012 encoding 180 amino acids (SEQ ID NO: 12; GeneBank AAB60125,
which is incorporated herein by reference). The coding sequence
includes coding sequences 50-109 which encode a signal peptide and
coding sequences 344-1009 which encodes the mature peptide.
GeneBank AAB60125 discloses a signal peptide that includes amino
acids 1-20 and a mature protein that includes amino acids
99-320.
[0011] GeneBank L77197, which is incorporated herein by reference,
discloses the amino acid of and nucleotide sequences (SEQ ID NO:
13) encoding the peanut allergy source protein antigen Ara h II.
This Ara h II sequence is 717 bp sequence encoding 110 amino acids
and including a polyA signal 562-567.
[0012] GeneBank AF059616, which is incorporated herein by
reference, discloses the amino acid of and nucleotide sequences
(SEQ ID NO:14) encoding the peanut allergy source protein antigen
Ara h II. This Ara h 5 sequence is 743 bp sequence including a
coding sequence of 17-412. GeneBank AAD55587, which is incorporated
herein by reference, discloses the 131 amino acid protein (SEQ ID
NO: 15).
[0013] GeneBank AB081309, which is incorporated herein by
reference, discloses the amino acid of and nucleotide sequences
(SEQ ID NO: 16) encoding the Japanese cedar (cryptomeria japonicd)
allergy source antigen Cry j 1.1. This Cryj 1.1 sequence is 1295 bp
sequence including a coding sequence of 62-1186 in which a signal
peptide is encoded by 62-124 and the mature protein is encoded by
125-1183 and a polyA site at 1295. GeneBank BAB86286, which is
incorporated herein by reference, discloses the 374 amino acid
protein (SEQ ID NO: 17) including a signal peptide of amino acids
1-21 and a mature protein of amino acids 22-374.
[0014] GeneBank AB081310, which is incorporated herein by
reference, discloses the amino acid of and nucleotide sequences
(SEQ ID NO: 18) encoding the Japanese cedar (cryptomeria japonicci)
allergy source antigen Cry j 1.2. This Cry j 1.2 sequence is 1313
bp sequence including a coding sequence of 46-1170 in which a
signal peptide is encoded by 46-108 and the mature protein is
encoded by 109-1167 and a polyA site at 1313. GeneBank BAB86287,
which is incorporated herein by reference, discloses the 374 amino
acid protein (SEQ ID NO: 19) including a signal peptide of amino
acids 1-21 and a mature protein of amino acids 22-374.
[0015] GeneBank U59102, which is incorporated herein by reference,
discloses the amino acid of and nucleotide sequences (SEQ ID NO:20)
encoding the blomia tropicalis allergy source protein antigen Blo t
5. This Blo t 5 sequence is 537 bp sequence including a coding
sequence of 33-437. GeneBank AAD10850, which is incorporated herein
by reference, discloses the 134 amino acid protein (SEQ ID
NO:21).
[0016] There remains a need for compositions and methods of
preventing and inhibiting the allergic reactions induced by these
allergens.
SUMMARY OF THE INVENTION
[0017] The present invention relates to compositions for preventing
and inhibiting an allergic response against an allergenic protein.
The compositions comprise: [0018] (a) an eukaryotic cell expression
vector containing nucleotide sequences encoding an allergenic
protein or a polypeptide that comprises an antigenic epitope of
said allergenic protein; and, [0019] (b) an allergenic protein or a
polypeptide that comprises an antigenic epitope of said allergenic
protein.
[0020] The present invention provides compositions for preventing
and inhibiting an allergic response against an allergenic protein
selected from the group consisting of: a flea allergenic protein; a
feline allergenic protein; a canine allergenic protein; a dust mite
allergenic protein; a peanut allergenic protein; a Japanese cedar
allergenic protein; and a blomia tropicalis allergenic protein.
[0021] The present invention further relates to kits for preventing
and inhibiting an allergic response against an allergenic protein.
The kits comprise: [0022] (a) a first container comprising a
eukaryotic cell expression vector containing nucleotide sequences
encoding an allergenic protein or a polypeptide that comprises an
antigenic epitope of said allergenic protein; and [0023] (b) a
second container an allergenic protein or a polypeptide that
comprises an antigenic epitope of said allergenic protein.
[0024] The present invention provides kits for preventing and
inhibiting an allergic response against an allergenic protein
selected from the group consisting of: a flea allergenic protein; a
feline allergenic protein; a canine allergenic protein; a dust mite
allergenic protein; a peanut allergenic protein; a Japanese cedar
allergenic protein; and a blomia tropicalis allergenic protein.
[0025] The present invention further relates to methods of
preventing and inhibiting an allergic reaction to an allergenic
protein in an individual. The methods comprise the step or steps of
administering to the individual [0026] (a) an eukaryotic cell
expression vector containing nucleotide sequences encoding an
allergenic protein or a polypeptide that comprises an antigenic
epitope of said allergenic protein; and [0027] (b) an allergenic
protein or a polypeptide that comprises an antigenic epitope of
said allergenic protein.
[0028] The present invention provides of preventing and inhibiting
an allergic reaction to an allergenic protein in an individual
wherein said allergenic protein is selected from the group
consisting of: a flea allergenic protein; a dust mite allergenic
protein; a peanut allergenic protein; a Japanese cedar allergenic
protein; and a blomia tropicalis allergenic protein.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1A, FIG. 1B, and FIG. 1C refer to a Flea allergy model
in mice. C57b/6 mice were primed twice biweekly with flea antigens
or saline as a negative control, challenged with the flea antigens,
or PBS as the negative control, histamine as the positive control
intradermally. In FIG. 1A, the local reactions after the skin test
were measured at 30 min after the challenge. FIG. 1B shows
anti-flea antigens of IgE production. In FIG. 1C, CD4+ T cell
proliferation responses stimulated by flea antigens in vitro, were
tested in mice. Results are representative of at least three
experiments. * P<0.05, compared with naive control groups as
indicated.
[0030] FIG. 2A, FIG. 2B, FIG. 2C, and FIG. 2D show that
co-immunization of DNA and protein suppresses the development of
immediate hyper-sensitivity reaction. FIG. 2A shows data from skin
tests of mice after co-immunization with F or pcDF100+F. FIG. 2B
shows that dose dependent skin responses in mice after
co-immunization with pcDF100+F is displayed. FIG. 2C shows
anti-flea antigen levels of IgE and IgG1 after induction and
treatment. FIG. 2D shows CD4+ T cell proliferation responses
stimulated by flea antigen-specific in vitro. Results are
representative of at least three experiments. * P<0.05, compared
with V+F and F vaccination groups as indicated.
[0031] FIG. 3A, FIG. 3B, and FIG. 3C show that CD4.sup.+CD25'' T
cells are responsible for the observed suppression. In FIG. 3A,
5.times.10.sup.5 of CD3.sup.+ T cells were isolated from the
spleens of flea antigen immunized mice and added to 96-well-plates.
At the same time, 1.times.10.sup.5 of splenocytes from naive, F,
V+F and pcDF100+F immunized mice were also added to the same plate.
Similarly, 1.times.10.sup.5 non-T cells or T cells, purified
CD8.sup.+, CD4.sup.+ or CD4.sup.+CD25'' T cells were isolated from
the spleens of V+F or pcDF100+F immunized mice. These Co-cultures
were stimulated with flea antigen (50 ug/ml) in the presence of
1.times.10.sup.5 bone marrow derived DCs for 48 h in vitro.
Proliferation was examined by MTS-PMS (Promega) according to
manufactors instructions and stimulation index (SI) was determined
by the formula: counts of flea-antigen stimulated/counts of
non-stimulated cultures). In FIG. 3B, 1.times.10.sup.6 of
splenocytes from naive, F, V+F and pcDF100+F immunized mice were
adoptively transferred into naive C57 mice. Similarly,
1.times.10.sup.6 non-T cells or T cells, 1.times.10.sup.6 purified
CD8.sup.+, CD4.sup.+, 5.times.10.sup.5 CD4.sup.+CD25'' and
CD4.sup.+CD25.sup.+T cells were isolated from spleens of pcDF100+F,
V+F, F immunized or naive control mice and were adoptively
transferred into syngeneic flea-antigen primed mice and skin test
responses were examined. FIG. 3C shows that co-administration of
DNA and protein induce antigen-specific suppression.
I.times.10.sup.6CD4.sup.+CD25 T cells were isolated from spleens of
pcDF100+F, V+F immunized or naive control mice and were adoptively
transferred into naive mice, which were then immunized with
specific Flea antigen or non-specific OVA protein 24 hours after
transfer, then CD4.sup.+ T cells were isolated and their
proliferation was analyzed. Results shown in the figure are
representative of two experiments. * P<0.05 compared with V+F
and F transfers as indicated.
[0032] FIG. 4A, FIG. 4B, and FIG. 4C show that DCs from pcDF100+F
co-immunized mice induce Trcells in vitro. FIG. 4A shows pcDF100+F
co-immunization restricted MLR stimulatory activities on DCs. 48 h
after immunization, DCs isolated from spleen of mice were used to
stimulate T cell proliferation in MLR. T cell proliferation was
measured by CFSE activity. Results are representative of one of two
respective experiments. * P<0.05 compared with V+F and F
vaccination groups as indicated. FIG. 4B shows data from DCs
isolated from spleens of pcDF100+F.sub.7 V+F, F immunized or naive
control mice co-cultured with naive CD4.sup.+ T cells. The T cells
were restimulated once for two days for 3 cycles, using fresh DCs,
and were then analyzed after each stimulation cycle for IL-10, IL-4
and IFN-y positive cell numbers, and for the ability to regulated
MLR stimulatory activities. In FIG. 4C, MLR was performed with APC
from C57 mice and T cells from Balb/c mice. T cell proliferation
was measured by CFSE. Results are representative of two individual
experiments. * P<0.05 compared with V+F and F vaccination groups
as indicated.
[0033] FIG. 5A, FIG. 5B, and FIG. 5C show dermatological scores
data.
[0034] FIG. 6 shows results of skin tests before
co-immunization.
[0035] FIG. 7 shows results of skin tests after
co-immunization.
[0036] FIG. 8 shows dermatological scores data after
co-immunization.
[0037] FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, and FIG. 9F
show data demonstrating therapeutic effects of co-immunization on
the FAD cats.
[0038] FIG. 10A, FIG. 10B, and FIG. 10C show data demonstrating
therapeutic effects of co-immunization on the FAD cats.
[0039] FIG. 11 shows a map of plasmid pVAX1-K-FSA1.
DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0040] The present invention provides compositions, kits and
methods which prevent and inhibit allergic reactions, and allergen
induced immediate hypersensitivity. The present invention provides
compositions, kits and methods which prevent and inhibit flea
allergy dermatitis, compositions, kits and methods which prevent
and inhibit feline allergy, compositions, kits and methods which
prevent and inhibit canine allergy, compositions, kits and methods
which prevent and inhibit mite allergy, compositions, kits and
methods which prevent and inhibit peanut allergy, compositions,
kits and methods which prevent and inhibit Japanese cedar allergy
and compositions, kits and methods which prevent and inhibit blomia
tropicalis allergy.
[0041] The compositions of the invention comprise an allergenic
protein or a peptide or protein which comprises an antigenic
epitope of the allergenic protein and an expression vector which
encodes an allergenic protein or a peptide or protein which
comprises an antigenic epitope of the allergenic protein.
[0042] The kits of the invention comprise a container that
comprises an allergenic protein or a peptide or protein which
comprises an antigenic epitope of the allergenic protein and
container that comprises an expression vector which encodes an
allergenic protein or a peptide or protein which comprises an
antigenic epitope of the allergenic protein.
[0043] The methods of the invention comprise administering the
compositions of the invention and/or the components of a kit of the
invention in combination to an individual who has or is susceptible
to allergic reactions, or allergen induced immediate
hypersensitivity.
[0044] The allergenic protein or a peptide or protein which
comprises an antigenic epitope of the allergenic protein present in
the composition or kit and used in the method, and allergenic
protein or a peptide or protein which comprises an antigenic
epitope of the allergenic protein encoded by the expression vector
present in the composition or kit and used in the method have amino
acid sequence overlap such that they share epitopes, i.e at least
one epitope of the allergenic protein or a peptide or protein which
comprises an antigenic epitope of the allergenic protein present in
the composition or kit and used in the method is the same as at
least one epitope of the allergenic protein or a peptide or protein
which comprises an antigenic epitope of the allergenic protein
encoded by the expression vector present in the composition or kit
and used in the method. In some embodiments, the allergenic protein
or a peptide or protein which comprises an antigenic epitope of the
allergenic protein present in the composition or kit and used in
the method is the same as the allergenic protein or a peptide or
protein which comprises an antigenic epitope of the allergenic
protein encoded by the expression vector present in the composition
or kit and used in the method. In some embodiments, the allergenic
protein or a peptide or protein which comprises an antigenic
epitope of the allergenic protein present in the composition or kit
and used in the method is a fragment of the allergenic protein or a
peptide or protein which comprises an antigenic epitope of the
allergenic protein encoded by the expression vector present in the
composition or kit and used in the method. In some embodiments, the
allergenic protein or a peptide or protein which comprises an
antigenic epitope of the allergenic protein encoded by the
expression vector present in the composition or kit and used in the
method is a fragment of the allergenic protein or a peptide or
protein which comprises an antigenic epitope of the allergenic
protein present in the composition or kit and used in the method.
In some embodiments, the allergenic protein or a peptide or protein
which comprises an antigenic epitope of the allergenic protein
present in the composition or kit and used in the method is a
fragment of the allergenic protein or a peptide or protein which
comprises an antigenic epitope of the allergenic protein encoded by
the expression vector present in the composition or kit and used in
the method. In some embodiments, one or both of 1) the allergenic
protein or a peptide or protein which comprises an antigenic
epitope of the allergenic protein present in the composition or kit
and used in the method and 2) the allergenic protein or a peptide
or protein which comprises an antigenic epitope of the allergenic
protein encoded by the expression vector present in the composition
or kit and used in the method is identical to a naturally occurring
protein which is an allergen. In some embodiments, both of 1) the
allergenic protein or a peptide or protein which comprises an
antigenic epitope of the allergenic protein present in the
composition or kit and used in the method and 2) the allergenic
protein or a peptide or protein which comprises an antigenic
epitope of the allergenic protein encoded by the expression vector
present in the composition or kit and used in the method are
identical to a naturally occurring protein which is an allergen. In
some embodiments, one or both of 1) the allergenic protein or a
peptide or protein which comprises an antigenic epitope of the
allergenic protein present in the composition or kit and used in
the method and 2) the allergenic protein or a peptide or protein
which comprises an antigenic epitope of the allergenic protein
encoded by the expression vector present in the composition or kit
and used in the method is identical to a fragment of a naturally
occurring protein which is an allergen. In some embodiments, both
of 1) the allergenic protein or a peptide or protein which
comprises an antigenic epitope of the allergenic protein present in
the composition or kit and used in the method and 2) the allergenic
protein or a peptide or protein which comprises an antigenic
epitope of the allergenic protein encoded by the expression vector
present in the composition or kit and used in the method are
identical to a fragment of a naturally occurring protein which is
an allergen. In some embodiments, the allergenic protein or a
peptide or protein which comprises an antigenic epitope of the
allergenic protein present in the composition or kit and used in
the method is identical to a fragment of a naturally occurring
protein which is an allergen and the allergenic protein or a
peptide or protein which comprises an antigenic epitope of the
allergenic protein encoded by the expression vector present in the
composition or kit and used in the method is identical to a
naturally occurring protein which is an allergen. In some
embodiments, the allergenic protein or a peptide or protein which
comprises an antigenic epitope of the allergenic protein present in
the composition or kit and used in the method is identical to a
naturally occurring protein which is an allergen and the allergenic
protein or a peptide or protein which comprises an antigenic
epitope of the allergenic protein encoded by the expression vector
present in the composition or kit and used in the method is
identical to a fragment of naturally occurring protein which is an
allergen.
[0045] In some embodiments, the composition or kit includes an
allergenic protein such as a protein from a pathogen, food,
environmental factor or irritant. In some embodiments, the
composition or kit includes a peptide or protein which includes an
antigenic epitope of an allergenic protein such as a protein from a
pathogen, food, environmental factors or irritant. Similarly, in
some embodiments, the composition or kit includes an expression
vector which encodes an allergenic protein such as a protein from a
pathogen, food or irritant and in some embodiments, the composition
or kit includes an expression vector which encodes a peptide or
protein which includes an antigenic epitope of an allergenic
protein such as a protein from a pathogen, food, environmental
factor or irritant.
[0046] In some embodiments, an allergenic protein or peptide or
protein which includes an antigenic epitope of an allergenic
protein that is encoded by the expression vector is identical to
the allergenic protein or peptide or protein which includes an
antigenic epitope of an allergenic protein included in the
composition or kit. In some embodiments, an allergenic protein or
peptide or protein which includes an antigenic epitope of an
allergenic protein that is encoded by the expression vector is
different from the allergenic protein or peptide or protein which
includes an antigenic epitope of an allergenic protein included in
the composition or kit. In some embodiments, the peptide or protein
included in the composition is the allergenic protein. In some
embodiments, the peptide or protein included in the composition is
a fragment of the allergenic protein. In some embodiments, the
peptide or protein encoded by the expression vector is the
allergenic protein. In some embodiments, the peptide or protein
encoded by the expression vector is a fragment of the allergenic
protein. According to the invention, the methods comprise
administering the compositions in amounts sufficient to suppress
the allergic reaction against the allergenic protein when the
individual is subsequently exposed to such protein.
[0047] In some embodiments, the present invention provides
inhibitors for flea allergy dermatitis. The flea allergy dermatitis
inhibitor of the present invention comprises a eukaryotic cell
expression vector containing flea salivary allergenic protein (such
as felis salivary antigen 1 (FSA1 or Cte f1)) or a peptide or
protein that comprises an antigenic epitope of such allergenic
protein, in combination with a flea salivary allergenic protein
(such as felis salivary antigen 1 (FSA1 or Cte f1)) or a peptide or
protein that comprises an antigenic epitope of such allergenic
protein.
[0048] In some embodiments, the present invention provides
inhibitors for feline allergy. The feline allergy inhibitor of the
present invention comprises a eukaryotic cell expression vector
containing feline allergenic protein (such as Fel dI) or a peptide
or protein that comprises an antigenic epitope of such allergenic
protein, in combination with a feline allergenic protein (such as
Fel dI) or a peptide or protein that comprises an antigenic epitope
of such allergenic protein.
[0049] In some embodiments, the present invention provides
inhibitors for canine allergy. The canine allergy inhibitor of the
present invention comprises a eukaryotic cell expression vector
containing canine allergenic protein (such as Can f1 or Can f2) or
a peptide or protein that comprises an antigenic epitope of such
allergenic protein, in combination with a canine allergenic protein
(such as Can f1 or Can f2) or a peptide or protein that comprises
an antigenic epitope of such allergenic protein.
[0050] In some embodiments, the present invention provides
inhibitors for dust mite allergy. The dust mite allergy inhibitor
of the present invention comprises a eukaryotic cell expression
vector containing a dust mite allergy allergenic protein (such as
Der PI or Der F1) or a peptide or protein that comprises an
antigenic epitope of such allergenic protein, in combination with a
mite allergy allergenic protein (such as Der PI or Der F1) or a
peptide or protein that comprises an antigenic epitope of such
allergenic protein.
[0051] In some embodiments, the present invention provides
inhibitors for peanut allergy. The peanut allergy inhibitor of the
present invention comprises a eukaryotic cell expression vector
containing a peanut allergy allergenic protein (such as Ara HII or
Ara H5) or a peptide or protein that comprises an antigenic epitope
of such allergenic protein, in combination with a peanut allergy
allergenic protein (such as Ara HII or Ara H5) or a peptide or
protein that comprises an antigenic epitope of such allergenic
protein.
[0052] In some embodiments, the present invention provides
inhibitors for Japanese cedar allergy. The Japanese cedar allergy
inhibitor of the present invention comprises a eukaryotic cell
expression vector containing a Japanese cedar allergy allergenic
protein (such as Cry j 1.1 or Cry j 1.2) or a peptide or protein
that comprises an antigenic epitope of such allergenic protein, in
combination with a Japanese cedar allergy allergenic protein (such
as Cry j 1.1 or Cry j 1.2) or a peptide or protein that comprises
an antigenic epitope of such allergenic protein.
[0053] In some embodiments, the present invention provides
inhibitors for blomia tropicalis allergy. The blomia tropicalis
allergy inhibitor of the present invention comprises a eukaryotic
cell expression vector containing a blomia tropicalis allergy
allergenic protein (such as Blo t5) or a peptide or protein that
comprises an antigenic epitope of such allergenic protein, in
combination with a blomia tropicalis allergy allergenic protein
(such as Blo t5) or a peptide or protein that comprises an
antigenic epitope of such allergenic protein.
[0054] The allergenic protein may be expressed in Escherichia coli
or eukaryotic cells (for example, yeast or CHO cells), for example,
molecular cloning methodology is used to incorporate the allergenic
protein coding sequence into the corresponding expression vector,
causing the protein product to be expressed through the Escherichia
coli, yeast or CHO cell systems. Purification is then used to
obtain the allergenic protein. Similarly, peptides or proteins may
be designed which include antigenic epitopes of allergenic
proteins. Nucleic acid sequences encoding such peptides or proteins
can be incorporated into expression vectors and produced in host
cells where they express the peptide or protein which is then
purified or peptides may be synthesized. Alternatively, the
allergenic protein may be purified from natural sources.
[0055] The eukaryotic cell expression vector included in the
compositions or kits of the invention may be an expression vector
composed of a plasmid expression vector, a viral expression vector
or bacteriophage expression vector. Plasmid DNA and chromosome DNA
fragment-formed expression vector and other expression vectors are
well known and commonly used in the field of genetic engineering.
In some embodiments, the plasmid vector pVAX1 (Invitrogen) is used.
In some embodiments, the plasmid vector provax which has the CMV
promoter, an hCG leader and bovine growth hormone poly A is used.
In some embodiments, the plasmid vector is a pcDNA3 plasmid
(Invitrogen) which comprises a human cytomegalovirus
immediate-early (CMV) promoter, bovine growth hormone
polyadenylation signal (BGH polyA), T7 sequence, ColE1 origin of
replication, and the JE virus signal sequence.
[0056] In the eukaryotic cell expression vectors, the coding
sequence for the allergenic protein or peptide or protein that
comprises an antigenic epitope of such allergenic protein is
operably linked to regulatory sequences required for eukaryotic
expression. Examples of suitable promoters include an RSV (Rous
sarcoma virus) promoter, a CMV (cytomegalovirus) promoter such as
the CMV immediate early promoter, an SV40 virus promoter, Mouse
Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus
(HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney
virus, ALV, Epstein Barr Virus (EBV), as well as promoters from
human genes such as human Actin, human Myosin, human Hemoglobin,
human muscle creatine and human metallothionein. Examples of
polyadenylation signals useful to practice the present invention,
include but are not limited to SV40 polyadenylation signals and LTR
polyadenylation signals. In addition to the regulatory elements
required for DNA expression, other elements may also be included in
the DNA molecule. Such additional elements include enhancers. The
enhancer may be selected from the group including but not limited
to: human Actin, human Myosin, human Hemoglobin, human muscle
creatine and viral enhancers such as those from CMV, RSV and
EBV.
[0057] In some embodiments, the proportion of eukaryotic cell
expression vector to allergenic protein or peptide or protein that
comprises an antigenic epitope of such allergenic protein is
1:5-5:1; the preferred option is: 1:1 (the mole ratio is
1-20:100,000; the preferred molar ration is 15:100,000).
[0058] In some embodiments, the inhibitor composition or
combination of kit components is introduced into the organism
intramuscularly, intracutaneously/intradermally, transdermally,
subcutaneously, intravenously and through mucosal tissue by means
of injection, nebulizer/aerosol/spraying, nose drops, eye drops,
orally, sublingual, buccal, vaginal, penetration, absorption,
physical or chemical means; or it may be introduced into the
organism through other physical mixture or package. The kit
components do not have to be delivered together, nor do they have
to be delivered at the same site or by the same route of
administration.
[0059] The pharmaceutical composition may be introduced by various
means including, for example, the needle injection, needleless
injector, gene gun, electroporation, and microprojectile
bombardment.
[0060] The composition and kit components may be formulated by one
having ordinary skill in the art with compositions selected
depending upon the chosen mode of administration. Suitable
pharmaceutical carriers are described in the most recent edition of
Remington's Pharmaceutical Sciences, A. Osol, a standard reference
text in this field.
[0061] For parenteral administration, formulations may be provided
as a solution, suspension, emulsion or lyophilized powder in
association with a pharmaceutically acceptable parenteral vehicle.
Examples of such vehicles are water, saline and dextrose solution.
Liposomes and nonaqueous vehicles such as fixed oils may also be
used. The vehicle or lyophilized powder may contain additives that
maintain isotonicity (e.g., sodium chloride, mannitol) and chemical
stability (e.g., buffers and preservatives). The formulation is
sterilized by commonly used techniques. Injectable compositions may
be sterile and pyrogen free.
[0062] The dosage administered varies depending upon factors such
as: pharmacodynamic characteristics; its mode and route of
administration; age, health, and weight of the recipient; nature
and extent of symptoms; kind of concurrent treatment; and frequency
of treatment. In some embodiments, the amount of composition used
or the amount of combination of kit components is generally 1250
ug/kg body weight/administration; with the composition or kit
components administered once every 1-30 days, preferably once every
7-14 days. In some embodiments, a single dose is administered. In
some embodiments, multiple doses are administered. In some
embodiments, a total of 2-3 administrations are administered.
[0063] In some embodiments, the compositions or kits are
administered to individuals who are suffering from an allergic
reaction. In some embodiments, the compositions or kits are
administered to individuals who are not suffering from an allergic
reaction but who have been exposed to the allergen or likely to
have been exposed to the allergen. In some embodiments, the
compositions or kits are administered to individuals who are not
suffering from an allergic reaction but who are known to be
allergic to the allergen, ie. who have previously had allergic
responses to the allergen.
[0064] The methods of the present invention are useful in the
fields of both human and veterinary medicine. Accordingly, the
present invention relates to treatment and prevention of allergic
reactions in mammals, birds and fish. The methods of the present
invention can be particularly useful for mammalian species
including human, bovine, ovine, porcine, equine, canine and feline
species.
[0065] The Examples set out below include representative examples
of aspects of the present invention. The Examples are not meant to
limit the scope of the invention but rather serve exemplary
purposes. In addition, various aspects of the invention can be
summarized by the following description. However, this description
is not meant to limit the scope of the invention but rather to
highlight various aspects of the invention. One having ordinary
skill in the art can readily appreciate additional aspects and
embodiments of the invention.
EXAMPLES
Example 1
Polypeptide Synthesis of Flea Salivary Allergenic Peptides and
Construction of a Eukaryotic Cell Expression Vectors for Expression
of the Same
[0066] The described flea salivary allergenic protein (FSA)
possesses the amino acid residue sequence as described in SEQ ID
NO:2.
[0067] The synthesized polypeptide that comprises the amino acid
residues described in SEQ ID NO: 22 is named pep66. The synthesized
polypeptide that comprises amino acids described in SEQ ID NO: 23
is named pep 100.
[0068] Nucleotide sequences that encode pep66 have the nucleotide
sequence described in SEQ ID NO 24 and are named FAD66.
[0069] Nucleotide sequences that encode pep 100 have the nucleotide
sequence described in SEQ ID NO 25 and are named FAD 100.
[0070] Eukaryotic vectors that encode pep66 and pep 100 comprise at
least one nucleotide sequence described in SEQ ID NO 24 or SEQ ID
NO: 25 and are named pcDF66 or pcDF100, respectively.
[0071] Flea Salivary Allergenic Protein was purchased from the
Greer Laboratory Company (Lenoir, N.C., United States) and was
formulated by the systematic flea cultivation methods described by
Lee, et al., in Parasite Immunology 19:13-19, 1997. At the end of
one adult year, female fleas were obtained from isolated salivary
glands of infected animals. Salivary gland cells were suspended in
SDS-reduction buffer and agitated in an oscillator for 30 seconds.
Cells were pulverized and crude protein was stored at -20.degree.
C.
[0072] The described eukaryotic cell expression vectors comprising
nucleotide sequences that encode FSA epitopes pep66 and pep 100 are
named pcDF66 or pcDF100, respectively. 1. Synthesis of FSA
polypeptides pep66, pep 100, and their encoded genes
[0073] We verified the amino acid sequences of MHC Class II
epitopes of FSA and the chemically synthesized peptides pep66 and
pep 100 using Epitlot software. Sequences of the newly synthesized
genes and protein products are below:
[0074] Peptide 66-80: QEKEKCMKFCKKVCK (SEQ ID NO: 22), called
pep66;
[0075] Peptide 100-114: GPDWKVSKECKDPNN (SEQ ID NO: 23), called pep
100.
[0076] Nucleotide sequences that encode pep66 and pep 100 have been
named FAD66 and FAD 100, respectively. The nucleotide sequences
comprise the following sequences:
[0077] FAD66: CAAGAGAAAG AAAAATGTAT GAAATTTTGC AAAAAAGTTT GCAAA
(SEQ ID NO: 24
[0078] FAD 100: GGTCCTGATT GGAAAGTAAG CAAAGAATGC AAAGATCCCA ATAAC
(SEQ ID NO: 25). 2. Construction of FAD66 and FAD 100 expression
vectors
[0079] Expression vector pGFP (Clontech, Mountain View, Calif.,
U.S.A.) was purchased as a template. We conducted Polymerase chain
reaction (PCR) amplification of FSA nucleotide sequences to label
the FSA nucleotide sequence with the Green Fluorescent Protein
(GFP) gene. Primer extension was completed by use of the P66F and
PR primers as well as the PI OOF and PR primers. Sequences for the
primers used in cloning method are as follows: P66F: 5'-AAGCTTGCCA
TGCAAGAGAA AGAAAAATGT ATGAAATTTT GCAAAAAAGT TTGCAAAGGTACC GCCATGG
TGAGCAAGGG CGAGGA-3' (SEQ ID NO: 26) (the 13.sup.th site-57.sup.th
site basic group from the 5' terminal end of the amplification
product said sequence is FAD66; the 58th site-63rd site basic group
from the 5' terminal end of the amplification product is the Kpn 1
recognition site; the first through sixth basic nucleotide from the
5' terminal end of the amplification product is the Hind III
recognition site); PR: 5'-TTA GGTACCTTAC TTGTAC AGCTCGTCCAT-3' (SEQ
ID NO: 27) (the 4.sup.th site-9.sup.th site basic group from the 5'
terminal end of the amplification product in said sequence is the
Kpn I recognition site), PI OOF: 5'-AAGCTTGCCA TG GGTCCTGA
TTGGAAAGTA AGCAAAGAAT GCAAAGATCC CAATAACGGT ACC
GCCATGGTGAGCAAGGGCGAGGA-3' (SEQ ID NO: 28) (the 13.sup.th
site-57.sup.th site basic group from the 5' terminal end of the
amplification product in said sequence is FAD66; the 58.sup.th
site-63.sup.rd site basic group from the 5' terminal end of the
amplification product is the Kpn I recognition site; the 1.sup.st
site-6.sup.th site basic group from the 5' terminal end of the
amplification product in said sequence is the Hind III recognition
site), PR: 5'-TTA GGTACCTTAC TTGTAC AGCTCGTCCAT-3' (SEQ ID NO: 27)
(the 4.sup.th site-9.sup.th site basic group from the 5' terminal
end of the amplification product is the Kpn I recognition site).
PCR product and eukaryotic expression vector pcDNA3 (Invitrogen
Corp., Carlsbad, Calif., U.S.A.) were digested using BamHX and Hind
III. The amplification product was ligated into the plasmid using
T4 DNA ligase, and then transformed into Escherichia coli Top 10.
After E. coli were grown in incubators, plasmid DNA was extracted
and restriction digestion was performed using Kpn I to obtain a
positive clones. Positive clones included plasmid pcDF66-GFP
containing FAD66 and GFP genes and plasmid pcDF100-GFP containing
FAD 100 and GFP genes. After using pcDF66-GFP and pcDF100-GFP for
Kpn I digestion, low melting point agarose gel electrophoresis was
used to recover large fragments, and then self-binding is
conducted. Finally, the product is transformed into Escherichia
coli Top 10, the plasmid is extracted and restriction endonuclease
Kpn/digestion assay is used to obtain a positive clone. Obtained
clones included FAD66 expression vector pcDF66 and FAD 100
expression vector pcDF100.
[0080] Normal simian kidney cells (CV1 cells) (purchased from the
Institute of Cell Biology, Shanghai) were cultured in DMEM
containing 10% fetal calf serum under 5% CO2, and 37.degree. C.
Transfections of pcDF66-GFP and pcDF100-GFP were performed on the
CV1 cells in a 35 mm culture dish with 2.5.times.10.sup.5 cells
per/ml and 2 ml per dish. Purification of the plasmids was
performed in accordance with the methodology described in the
Guidebook for Molecular Cloning Experimentation (3.sup.rd edition)
(Chinese translation) (translated by Huang Peitang et al., Science
Publishing Company, published September 2002). A positive ion
liposome medium (Lipofectamine.TM. 2000, Invitrogen) was used to
transfect and culture the CV1 cells according to the manufacturer's
instructions (Invitrogen, CA, USA). After 24 hours of incubation,
the cell culture is observed under fluorescence microscopy showing
pcDF100-GFP and pcDF66-GFP was expressed in eukaryotic cells. The
results demonstrate that pcDF100-GFP and pcDF66-GFP also may be
expressed in eukaryotic cells.
Example 2
Flea Allergy Dermatitis Inhibitor as Therapy for Flea Allergy
Dermatitis
[0081] Experiments in which Kunming white, BALB/c and C57BL/6 mice
are immunized with a vector comprising FSA protein and nucleotide
sequences that encode FSA proteins, or proteins thereof,
demonstrate that immunization is an effective therapy for flea
allergy dermatitis. Useful vectors for immunization can comprise a
eukaryotic cell expression vector further comprising a nucleotide
sequences encoding FSA protein (for example, pcDF66 or pcDF100),
and either FSA synthesized peptides (pep66 or pep 100) or FSA
protein (flea salivary allergenic protein). The immunization
efficacy is superior to that of an immunization vector merely
comprising a eukaryotic cell expression vector that included
nucleotide sequences that encodes either FSA peptides or FSA
protein. Inhibition studies demonstrate that the DNA sequences
encoding different epitopes have different efficacies. For
instance, FAD 100 appears to have a stronger therapeutic effect in
suppressing Flea Allergy Dermatitis. Results in each strain of
mouse were similar, indicating that immunosuppressive activity of
the immunization is not limited to MHC genetic backgrounds.
Therefore, we can directly deduce from the above results that
through use of a FAD Inhibitor comprising a eukaryotic cell
expression vector which further comprises a nucleotide sequence
that encodes FSA protein and either a FSA protein or a FSA peptide,
it is possible to effectively inhibit flea allergy dermatitis.
[0082] T-cell proliferation experiments and related cytokine
expansion experiments demonstrate that a FAD Inhibitor, comprising
a eukaryotic cell expression vector that encodes FSA protein and an
FSA protein or FSA peptide, inhibit antigen-specific T-cell
proliferation thereby suppressing an allergic reaction.
Immunosuppression may be induced through IL-10, thus inhibiting
IL-5, IL-13 and other cytokine expression levels. The FAD Inhibitor
in the present invention may effectively prevent and/or treat flea
allergy dermatitis, especially those cases caused by
Ctenocephalides felis. 1. Kunming white mice experiments.
[0083] Three Hundred Sixty (360) female Kunming white mice were
divided into a total of 12 groups of equal numbers. Each mouse in
the first group was immunized with 100 microliters of 0.9% NaCl
aqueous solution containing 100 micrograms of pcDF66. Each mouse in
the second group was immunized with 100 microliters of 0.9% NaCl
aqueous solution containing 100 micrograms pep66. Each mouse in the
third group was immunized with 100 microliters of 0.9% NaCl aqueous
solution containing 50 micrograms of pcDF66 and 50 micrograms of
pep66. Each mouse in the fourth group was immunized with 100
microliters of 0.9% NaCl aqueous solution containing 100 micrograms
of pcDF100. Each mouse in the fifth group was immunized with 100
microliters of 0.9% NaCl aqueous solution containing 100 micrograms
of pep 100. Each mouse in the sixth group was immunized with 100
microliters of 0.9% NaCl aqueous solution containing 50 micrograms
of pcDF100 and 50 micrograms of pep100. Each mouse in the seventh
group was immunized with 100 microliters of 0.9% NaCl aqueous
solution containing 50 micrograms of pcDF66 and 50 micrograms of
pep 100. Each mouse in the eighth group was immunized with 100
microliters of 0.9% NaCl aqueous solution containing 50 micrograms
of pcDF100 and 50 micrograms of pep66. Each mouse in the ninth
group was immunized with 100 microliters of 0.9% NaCl aqueous
solution containing 100 micrograms of inactivated flea antigen
(purchased from the Greer Lab Company, North Carolina, United
States). Each mouse in the tenth group was immunized with 100
microliters of 0.9% NaCl aqueous solution containing 50 micrograms
of inactivated flea antigen (purchased from the Greer Lab Company,
North Carolina, United States) and 50 micrograms of pcDF66. Each
mouse in the eleventh group was immunized with 100 microliters of
0.9% NaCl aqueous solution containing 50 micrograms of inactivated
flea antigen (purchased from the Greer Lab Company, North Carolina,
United States) and 50 micrograms of pcDF100. Each mouse in the
twelfth group was immunized with 100 microliters of 0.9% NaCl
aqueous solution containing 100 micrograms of pcDNA3 to serve as a
control. Fourteen days after the first immunization, a booster
immunization was administered in the same dosage amount. Seven days
after the booster immunization, skin tests were conducted using the
following methods.
[0084] Hair was removed from the ventral murine chest cavity and an
intracutaneous injection of 30 .PHI.l of inoculation of 1 ug/ul FSA
protein was injected into 10 subjects. At the same time a histamine
solution (with a concentration of 0.01% histamine) and PBS was
injected in equal amounts to serve as positive controls and
negative controls. There were 10 subjects for each control group.
Twenty minutes after each injection, we measured blister diameters
in micrometers. The t test results indicated that pcDF100 and pep
100 compound immunization (the sixth group) demonstrated a notable
difference (P<0.05) when compared to pcDF100 single immunization
(the fourth group) or pep 100 single immunization (the fifth
group). There was a notable difference (P<0.05) between pcDF66
and pep66 compound immunization (the third group) and pcDF66 single
immunization (the first group), while there was no notable
difference with pep66 single immunization (the second group). Flea
antigen and pcDF66 (the tenth group) or pcDF100 (the eleventh
group) compound immunizations were notably different (P<0.05)
than the results generated by flea antigen alone (the ninth group),
The tenth and eleventh groups did not display any notable
difference as compared to pcDF66 (the first group) or pcDF100 (the
fourth group) immunizations. There was no difference in blister
diameters measured in the sixth and seventh groups as compared to
the expression vector or epitope polypeptide single immunity groups
(Groups 1, 2, 3 and 4).
[0085] Based on the preceding skin test results, we can conclude
that a eukaryotic cell expression vector comprising FSA protein or
a nucleotide sequence encoding FSA peptide and said FSA peptide, or
a eukaryotic cell expression vector comprising FSA protein or a
nucleotide sequence that encodes FSA peptides and FSA protein
reduces skin allergies in an antigen-specific way. These results
indicate that reduction of allergic reactions on the skin may be
induced through immunosuppression. 2. BALB/c and C57B/6 mice skin
test results
[0086] In order to further verify the results obtained above with
Kunming white mice, two pure strains of mice (BALB/c and C57B/6)
were tested to determine whether immunosuppression of FAD was MHC
restricted. Experimental methodologies were the same as those
methodologies performed in the Kunming white mice. The t test
results indicate that pcDF100 and pep 100 immunization (the sixth
group) had no notable difference (P<0.05) as compared to pcDF100
single immunization (the fourth group) or pep 100 single
immunization (the fifth group). Results from the flea antigen and
pcDF100 compound immunization (the eleventh group) demonstrated a
very significant difference (PO.01) compared to the results
generated by immunizations of flea antigen (the ninth group) or
pcDF100 (the fourth group). There was no significant difference
among immunizations of Flea antigen and pcDF66 immunization (the
tenth group), flea antigen immunization alone (the ninth group), or
pcDF66 immunization alone (the first group). There was no
significant difference among pcDF100 and pep66 immunization (the
eighth group), pcDF100 immunization alone (the fourth group), or
pep66 immunization alone (the second group).
[0087] The preceding results indicate that anti-allergic
immunosuppression is antigen-specific. For example, immunization
with pcDF100 and pep 100 was more effective in as compared to
immunization with pcDF100 or pep100 alone. Immunization with the
flea antigen and pcDF100 were more effective than any immunization
vectors that included only one component.
[0088] Differences also exist in the level of immunosuppression
among those groups that effectively treated FAD. For example, the
flea antigen and pcDF100 compound immunity group had more of an
effective immunosuppressive effect than compared any of the single
immunity groups. The t test results indicate that pcDF100 and pep
100 compound immunity (the sixth group) are significantly different
(P<0.05) when compared to results of immunization using pcDF100
alone (the fourth group) or pep100 alone (the fifth group). There
are significant differences (P<0.05) in the immunosuppressive
effect of the immunizations performed with pcDF100 and pep66
compound immunity (the eighth group) as compared to pcDF100 alone
(the fourth group) or pep66 alone (the second group). There was an
extremely significant difference (P<0.01) in immunizations using
Flea antigen and pcDF100 (the eleventh group) as compared to flea
antigen (the ninth group) or pcDF100 alone (the fourth group).
[0089] We interpret our results to conclude that anti-allergic
immunosuppression is antigen-specific. For example, immunization
was more effective in the pcDF100 and pep100 compound immunity
group as compared to the pcDF100 single immunity group or the
pep100 single immunity group. In addition, pep66 and pep100
peptides may have cross-reactivity. For example, immunization was
more effective with pcDF100 and pep66 immunization as compared to
pcDF100 single immunity group or the pep66 single immunity group.
Immunization using the flea antigen and pcDF66 compound immunity
did not produce clear immunosuppression. These results are
consistent with those obtained in the BALB/c group. Although there
are slight differences in the effectiveness of the immunization
among each strain of mice studied, experimental results of the
three different strains of mice same conclusion: immunization
vectors comprising eukaryotic cell expression vectors further
comprising FSA protein or nucleotide sequences that encode FSA
peptides and FSA peptides; or eukaryotic cell expression vectors
comprising FSA protein or nucleotide sequences that encode FSA
peptides and FSA protein may mount effective anti-allergic
immunosuppression.
Example 3
T-Cell Proliferation in Immunized Mice
[0090] Three Hundred Sixty (360) BALB/c mice and three hundred
sixty C57B/6 mice were each divided into 12 groups of 30 mice per
group. Immunization was performed in accordance with the
methodology described in Example 2. At seven days after the booster
immunization, splenic T-cells were isolated and T-cell expansion
activity was tested. The specific methodology was: under aseptic
conditions, splenic cells were taken from mice and used to form a
single-cell suspension fluid. A hemolytic solution was used to
remove red blood cells, which were then washed three times using
PBS fluid. The cells were centrifuged and a cell count taken. Cell
concentration was adjusted to 1.times.10.sup.6 cells/ml, and each
cell suspension from each animal was divided into four experimental
groups and plated into a 96-well culture plate. To one group, 100
ul Con-A (mitogen) was added to a final concentration of 5 fig/ml.
Specific antigen (flea antigen) was added to serve as a stimulant
to a final concentration of 5 ng/ml to the second group. No
stimulant was added for a negative control group, and 100 ul BSA
was added to a final concentration of 2 u.g/ml to serve as another
unrelated antigen. After being incubated at 37.degree. C. in a CO2
culture for 48 h, 100 u.l MTS was added to each well at a final
concentration of 5 mg/ml. After 4 hours of incubation, an enzyme
labeler was used to read the OD value at 492 nm and calculate the
stimulation index (SI=tested OD+non-stimulated OD). T-cell
proliferation results for the BALB/c mice indicated that eukaryotic
cell expression vectors comprising a nucleotide sequence that
encodes FSA peptide flea salivary and said FSA peptide generate
notable antigen-specific immunosuppression of FAD. For example,
immunization was more effective from the vectors comprising pcDF100
and pep100 (the sixth group) and the vectors comprising flea
antigen and pcDF100 (the eleventh group) as compared to
immunization with vectors comprising single components. In
addition, there was no clear immunosuppression demonstrated by
immunization vectors comprising the pcDF66 and pep66 group (the
third group), the flea antigen and pcDF66 group (the tenth group)
as compared to the corresponding single immunity groups. Results
using vectors comprising the pcDF100 and pep 100 compound immunity
group and the flea antigen and pcDF100 compound immunity group are
consistent with the immunosuppression effect shown by the skin
tests. There was an extremely significant difference (P<0.01) in
the immunosuppressive effect of the immunization vectors comprising
the pcDF100 and pep66 (the eighth group) as compared to the
immunosuppressive effect seen in the corresponding single immunity
group.
[0091] C57B/6 T-cell proliferation results indicate that eukaryotic
cell expression vectors containing nucleotide sequences that encode
FSA peptide and said FSA peptide may produce clear antigen-specific
immunosuppression. For example, there is a clear difference (PO.05)
in the effect of immunization of pcDF66 and pep66 compound immunity
(the third group) as compared to the corresponding single immunity
groups (the first group and the second group). Additionally, there
was an extremely significant difference (P<0.01) in the effect
of immunization in the pcDF100 and pep100 compound immunity group
(the sixth group) as compared to the corresponding single immunity
groups (the fourth group and the fifth group). The immunization of
mice using vector comprising the flea antigen and pcDF66 (the tenth
group) is clearly more effective (P<0.05) than the corresponding
single immunity groups (the ninth group and the first group). There
was an extremely significant difference (P<0.01) in the effect
of the immunization of the flea antigen and pcDF100 compound
immunity group (the eleventh group) as compared to the
corresponding single immunity groups (the ninth group and the
fourth group). In addition, cross-reactivity exists between the two
epitopes. For example, there is a clear difference (PO.05) in
T-cell proliferation profiles with the pcDF66 and pep100 compound
immunity group (the seventh group) and the corresponding single
immunity groups (the first group and the fifth group). There is
also a clear difference (P<0.05) in T-cell proliferation
profiles of the pcDF100 and pep66 compound immunity group (the
eighth group) and the corresponding single immunity groups (the
fourth group and the second group). The T-cell proliferation
profiles of in the pcDF100 and pep100 compound immunity group, the
pcDF100 and pep66 compound immunity group and the flea antigen and
pcDF100 compound immunity group results were all consistent with
the skin test results.
Example 4
Changes in the Cytokine Levels of Immunized Mice
[0092] There were 360 Kunming white mice, 360 BALB/c mice, and 360
C57B/6 mice, and each set of subjects strain was divided into 12
groups of 30 mice. Immunization was performed in accordance with
the method described in Example 2. Seven days after the booster
immunization, the spleen was excised and total RNA (TRIZOL, Dingguo
Biological Company) isolated. Reverse transcription for cDNA was
performed in accordance with the Dalianbao Biological Company's RNA
RT-PCR operating handbook. Briefly 1 jag of purified total RNA was
placed in a 250 uL centrifuge tube. Then the following reagents
were added in sequence: 4 ul MgCl.sub.2, 2 ul 10* buffer solution,
8.5 ul DEPC water, 2 ul dNTP mixture, 0.5 ul RNase inhibitor, 0.5
ul M-MLV reverse transcriptase (Promega), 0.5 uL Oligo (dT)i2
primer. The response conditions were 42.degree. C. for 30 min,
99.degree. C. for 5 min and 5.degree. C. for 5 minutes. The gene
family hypoxanthine phosphoribosyltransferase (HPRT) was used as
the internal source expression standard. The various groups' cDNA
concentrations were adjusted to make all sample concentrations
consistent. Then 2 .PHI.l of cDNA were used to conduct PCR
amplification assessing the expression levels of the three cytokine
genes: IFN-y, IL-4, and IL-10. The reaction's required primer and
PCR reaction conditions are as indicated in Table 1. (Because the
gene family HPRT has fixed expression in vivo, it is used as the
template for the control's internal source expression
standard).
TABLE-US-00001 TABLE 1 HPRT, IFN-y, IL-4 and IL-10 Primer Sequence
and PCR Reaction Specifications. Target gene Primer Response
conditions HPRT 5' GTTGGATACAGGCCAGACTTTGTTG (SEQ ID 94.degree. C.
30 sec, 60.degree. C. 30 sec NO: 29) 3' GAGGGTAGGCTGGCCTATGGCT (SEQ
and 72.degree. C. 40 sec ID NO: 30) IFN-Y 5' CATTGAAAGCCTAGAAAGTCTG
(SEQ ID 94.degree. C. 30 sec, 58.degree. C. 30 sec NO: 31) 3'
CTCATGGAATGCATCCTTTTTCG (SEQ and 72.degree. C. 40 sec IDNO: 32)
IL-4 5' GAAAGAGACCTTGACACAGCTG (SEQ 94.degree. C. 30 sec,
54.degree. C. 30 sec IDNO: 33) 3' GAACTCTTGCAGGTAATCCAGG (SEQ and
72.degree. C. 40 sec ID NO: 34) IL-10 5' CCAGTTTACCTGGTAGAAGTGATG
(SEQ ID 94.degree. C. 30 sec, 56.degree. C. 30 sec NO: 35) and
72.degree. C. 40 sec 3'TGTCTAGGTCCTGGAGTCCAGCAGACTCAA
(SEQIDNO.-36)
[0093] Bio-Rad Image software (Quantity One 4.2.0) was used to
analyze images taken of the electrophoresis gels of the PCR
products. Expression profile results obtained were generally
consistent each strain of mouse. IL-IO expression levels in the
pcDF66 and pep66 compound immunity (Group 3) were higher than
pcDF66 single immunity (Group 1) and the pep66 single immunity
(Group 2). IL-10 expression levels in the pcDF100 and pep 100
compound immunity (Group 6) were higher than pcDF100 single
immunity group (Group 4) or pep 100 single immunity group (Group
5). IL-10 expression levels in the flea antigen and pcDF66 compound
immunity group (Group 10) were higher than flea antigen (Group 9)
or pcDF66 single immunity groups (Group 1). IL-10 expression levels
in the flea antigen and pcDF66 compound immunity group (Group 11)
were higher than flea antigen (Group 9) or pcDF100 single immunity
groups (Group 4). There was no clear difference in the 1L-4 and
IFN-y expression levels among the various groups. The results
suggest that immunization conducted with eukaryotic cell expression
vector comprising nucleotide sequences that encode FSA and said FSA
protein or said FSA peptide compound enhanced IL-10 expression
levels.
[0094] In addition, expression profiles were generated on IL-5 and
IL-13 in the three types of mice. Results indicated that for the
eukaryotic cell expression vectors comprising nucleotide sequences
that encode FSA peptides and said FSA protein or peptide (Group 3,
Group 6, Group 10 and Group 11), IL-5 and IL-13 levels were clearly
lower than those of the respective single immunity groups.
Example 5
Detection of Blood IgE Levels in Immunized Mice
[0095] Two groups of 360 BALB/c and 360 C57B/6 mice were each
divided into 12 groups of 30 mice each and immunization was
performed as described in Example 2. Blood was taken intravenously
from the eye socket prior to immunization and 14 days after the
booster immunization. The blood was separated by centrifugation and
IgE levels were assessed using ELISA. The coated antigen used on
the ELISA plates was flea antigen purchased from Greer Laboratory
(Lenoir, N.C., United States). The first component on the ELISA
plate was separated blood sera and the second component used for
binding was the sheep anti-mice IgE antibody labeled with
horseradish peroxidase antioxidant enzyme. The substrate was added
to the system after antibody was bound to the antigens and the
enzyme labeler was used to read the OD values at 492 nm. The IgE
production in Kunming white mice, BALB/c mice, C57B/6 mice after
immunization are generally consistent for all three groups of mice.
Except for the flea antigen single immunity group (the ninth
group), IgE levels for the groups were relatively low. The IgE
levels produced after immunization with the flea antigen single
immunity group (the ninth group) were the highest levels across all
sets of mice. IgE levels were greatly reduced in the group
immunized with flea antigen and pcDF66 (the tenth group) and the
group immunized with the flea antigen and pcDF100 (the 11 group) as
compared to the flea antigen single immunity group (the ninth
group). This indicates that a eukaryotic cell expression vector
comprising nucleotide sequences that encode FSA peptides and said
FSA protein reduces IgE levels after immunization.
Example 6
Feline Allergenic Protein Antigen Fel d. I.I (Fel d I with the
Minor B Leader) Encoded Genetic Clone and Eukaryotic Expression
Assay
[0096] 1. The following sequences were used as Fel d I.1 primers.
Primer were artificially synthesized:
[0097] (a) Fel d I.1 PI 5' primer: AAGCTTGGATGTTAGACGC (SEQ ID NO
37)
[0098] (b) Fel d I.1 P2 3' primer: GGTACCTTAACACAGAGGAC (SEQ ID NO
38)
[0099] 2. Fel d I.1 expression vector construction
[0100] Fel d I.1 cDNA was used as a template for PCR amplification
of the Fel d I.1 gene using Fel d 1.1 PI and Fel d I.1 P2. The
primers are further described below:
[0101] (a) Fel d I.1 PI 5'-AAGCTTGGATGTTAGACGC-3' (SEQ ID NO 37)
(the 1.sup.st site-6.sup.th site basic group from the 5' terminal
end of the primer in this sequence is the Hind\\\recognition site;
the 9.sup.th site-11.sup.th site is the original initiator
code);
[0102] (b) Fel d I.1 P2 5'-GGTACCTTAACACAGAGGAC-3' (SEQ ID NO 38)
(the 1.sup.st site-6.sup.th site basic group from the 5' terminal
end of the. primer in this sequence is the Kpn I recognition site;
the 7.sup.th site-9.sup.th site is the original termination
code).
[0103] Kpn I and Hind III, respectively, were used for digesting
the PCR product and eukaryotic expression vector pVAX1 (Invitrogen
Corp.). Digested nucleotide fragments were then ligated using T4
DNA ligase. The resultant plasmid product was transformed into
Escherichia coli Top 10, grown for maximum copy number, and then
the plasmid isolated using methods known by those of ordinary skill
in the art. We performed a restriction digestion using the Kpn/and
Hind III endonucleases. Selected plasmid clones named pVAX1-Fel d
I.1 comprised the pVax plasmids sequence and the Fel d I.1 gene.
Through sequential analysis (Augct Co. Ltd., Beijing China),
further analysis was performed to obtain the final Fel d I.1 PI
expression vector, pFeld 1.1.
[0104] 3. pFel d I.1 Eukaryotic Expression
[0105] Normal simian kidney cells (CV1 cells, purchased from the
Shanghai Cell Institute) were cultured in DMEM containing 10% fetal
calf serum under 5% CO.sub.2 and 37.degree. C. conditions.
2.5.times.10.sup.5 cells per/ml were pipeted in a 35 mm culture
dishes in a 2 mL volume. Transfections were performed using
standard methods. Briefly, purification of the plasmids was
performed in accordance with the methodology in the Guidebook for
Molecular Cloning Experimentation (third edition, Chinese
translation) (translated by Huang Peitang et al., Science
Publishing Company, published September 2002). A positive ion
liposome medium (Lipofectamine.TM. 2000, Invitrogen) was used to
transfect the cultured CV1 cells according to the manufacturer's
instructions (Invitrogen, CA, USA). After 24 hours of transfection,
the cells were collected and an RNA extraction reagent (TRIZOL,
Dingguo Biological Company) was used to isolate total cellular RNA.
In order to prevent contamination, total extracted RNA was
separately loaded into several EP tubes. A micropipette was used to
carefully suction 2 ul of total RNA extracted and RT-PCR reagent
used to expand total cellular cDNA. After the specimen was added,
reverse transcription was performed at 42.degree. C. for 30-60
minutes, denaturation at 99.degree. C. for 5 minutes, and The
reaction tube was set aside at 5.degree. C. for 5 minutes prior to
extraction for use. Using the isolated cDNA as a template for gene
amplification PCR was performed using Feld1.1P1 and FeldI.1P2 as
the primers. The PCR products were subject to low melting point
agarose gel analysis to detect Feld1.1 positive bands. The results
demonstrate that Fel dI.1 is expressed in eukaryotic cells.
Example 7
Feline Allergenic Protein Antigen Fel d. 1.2 Genetic Clone and
Eukaryotic Expression Assay
[0106] 1. The following sequences were used as Fel d I.2 primers.
Primers were artificially synthesized:
[0107] (a) Fel d I.2 P1 5' primer: AAGCTTGGATGAAGGGGGCTC (SEQ ID
NO:39)
[0108] (b) Fel d I.2 P2 3' primer: GGTACCTTAACACAGAGGAC (SEQ ID
NO:40)
[0109] 2. Fel d I.2 expression vector construction
[0110] Fel d I.2 cDNA was used as a template for PCR amplification
using Fel d I.2 PI and Fel d I.2 P2 primers to generate copies of
the Fel d I.2 gene. The primers are further described below: (a)
Fel d I.2 PI 5'-AAGCTTGGATGAAGGGGGCTC-3' (SEQ ID NO:39) (the
1.sup.st site-6.sup.th site basic group from the 5' terminal end of
the primer is the Hind III recognition site; the 9.sup.th
site-11.sup.th site from the terminal end of the primer is the
original initiator code); (b) Fel d I.2 P2
5'-GGTACCTTAACACAGAGGAC-3' (SEQ ID NO:40) (the 1.sup.st
site-6.sup.th site basic group from the 5' terminal end of the
primer is the Kpn I recognition site, the 7.sup.th site-9* site
from the terminal end of the primer is the original termination
code).
[0111] Kpn I and Hind III in succession were used for digesting the
PCR product and eukaryotic expression vector, pVAX1 (Invitrogen
Corp.). The digested plasmid and PCR product were ligated using T4
DNA ligase. The product was transformed into Escherichia coli Top
10, the plasmid extracted, and restriction endonuclease Kpn 1 and
Hind 111 digestion assay used to obtain a positive clone, that is,
plasmid pVAX1-Fel d I.2 containing the Can f 1 gene. Through
sequential analysis (Augct Co. Ltd., Beijing China), further assay
correction was performed to obtain the Fel d I.2 expression vector,
pFeld I.2.
[0112] 3. pFeldI.2 Eukaryotic Expression
[0113] The pFeld I.2 expression vector was subjected to the
digestion, transformation, isolation, and ligation protocols
previously described in Example 6, Section 3. Isolated PCR products
were subjected to low melting point agarose gel testing to confirm
Feld1.2 positive bands. The results prove that Feld1.2 is expressed
in eukaryotic cells.
Example 8
Canine Allergenic Protein Antigen Can f 1 Genetic Clone and
Eukaryotic Expression Assay
[0114] 1. Can f 1 Primer Synthesis
[0115] The following sequences were used as Can f 1 primers.
Primers were artificially synthesized:
[0116] Can f 1 PI 5' primer: AAGCTTATGAAGACCCTGCTCCTCAC (SEQ ID NO:
41) Can f 1 P2 3' primer: GGTACCCTACTGTCCTCCTGGAGAGC (SEQ ID NO:
42)
[0117] 2. Can f 1 Expression Vector Construction
[0118] Can f 1 cDNA was used as a template to perform PCR
amplification using Can f 1 PI and Can f 1 P2 primers to generate
copies of the Can f 1 gene. The primers are further described
below: (a) Can f 1 PI 5'-AAGCTTATGAAGACCCTGCTCCTCAC-3' (SEQ ID
NO:41) (the 1.sup.st site-6.sup.th site basic group from the 5'
terminal end of the primer sequence is the Hind III recognition
site; the 7.sup.th site-9.sup.th site from the terminal end of the
primer sequence is the original initiator code); (b) Can f 1 P2
5.sup.r-GGTACCCTACTGTCCTCCTGGAGAGC-3' (SEQ ID NO:42) (the 1.sup.st
site-6.sup.th site basic group from the 5' terminal end of the
primer sequence is the Kpn I recognition site; the 7.sup.th
site-9.sup.th site from the %' terminal end of the sequence is the
original termination code).
[0119] PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to
the digestion, transformation, isolation, and ligation methods as
described in Example 7, Section 2. Positive clones were selected
and analyzed using sequential analysis discussed in Example 7,
Section 2 to obtain the Can f 1 expression vector, pCanf1.
[0120] 3. pCanf1 Eukaryotic Expression
[0121] The pCanf1 expression vector was subjected to transfection,
total RNA isolation, and RT-PCR protocols as previously described
in Example 6, Section 3. Isolated PCR products were subjected to
low melting point agarose gel testing to confirm pCanf1 positive
bands. The results prove that pCanf1 is expressed in eukaryotic
cells.
Example 9
Canine Allergenic Protein Antigen Can f 2 Genetic Clone and
Eukaryotic Expression Assay
[0122] 1. Can f 2 Primer Synthesis
[0123] The following sequences were used as Can f 2 primers.
Primers were artificially synthesized:
[0124] Can f 2 PI 5' primer: AAGCTT ATGCAGCTCCTACTGCTG (SEQ ID NO:
43) Can f 2 P2 3' primer: GGTACCCTAGTCTCTGGAACCC (SEQ ID NO:
44)
[0125] 2. Can f 2 Expression Vector Construction
[0126] Can f 2 CDNA cDNA was used as a template to perform PCR
amplification using Can f 2 PI and Can f 2 P2 primers to generate
copies of the Can f 2 gene. The primers are further described
below: (a) Can f 2 PI 5'-AAGCTT ATGCAGCTCCTACTGCTG-3' (SEQ ID
NO:43) (the 1.sup.st site-6.sup.th site basic group from the 5'
terminal end of the primer sequence is the Hind III recognition
site; the 7.sup.th site-9.sup.th site is the original initiator
code); (b) Can f 2 P2 5' GGTACCCTAGTCTCTGGAACCC-3' (SEQ ID NO:44)
(the 1.sup.st site-6.sup.th site basic group from the 5' terminal
end of this primer sequence is the Kpn I recognition site, the
1.sup.x site-9.sup.l site is the original termination code).
[0127] PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to
the digestion, transformation, isolation, and ligation methods as
described in Example 7, Section 2. Positive clones were selected
and analyzed using sequential analysis discussed in Example 7,
Section 2 to obtain the Can f 2 expression vector, pCanf2.
[0128] 3. pCanf2 Eukaryotic Expression
[0129] The pCanf2 expression vector was subjected to transfection,
total RNA isolation, and RT-PCR protocols as previously described
in Example 6, Section 3. Isolated PCR products were subjected to
low melting point agarose gel testing to confirm pCanf2 positive
bands. The results prove that pCanf2 is expressed in eukaryotic
cells.
Example 10
Dust Mite Allergenic Protein Antigen Der p 1 Genetic Clone and
Eukaryotic Expression Assay
[0130] 1. Der p 1 Primer Synthesis
[0131] The following sequences were used as Der p 1 primers.
Primers were artificially synthesized:
[0132] Der p 1 PI 5' primer: AAGCTTAACATGAAAATTGTTTTGG (SEQ ID NO:
45) Der p 1 P2 3' primer: GGTACCGTTTAGAGAATGACAACAT (SEQ ID NO:
46)
[0133] 2. Der p 1 Expression Vector Construction
[0134] Der p 1 cDNA was used as a template to perform PCR
amplification using Der pi PI and Der p 1 P2 primers to generate
copies of the Der p 1 gene. The primers are further described
below:
[0135] (a) Der p 1 PI 5'-AAGCTTAACATGAAAATTGGTTTTGG-3' (SEQ ID
NO:45) (the 1.sup.st site-6.sup.th site basic group from the 5'
terminal end of the primer sequence is the Hind III recognition
site, the 10.sup.th site-12.sup.th site is the original initiator
code);
[0136] (b) Der p 1 P2 5'-GGTACCGTTTAGAGAATGACAACAT-3' (SEQ ID
NO:46) (the 1.sup.st site-6.sup.th site basic group from the 5'
terminal end of the primer sequence is the Kpn I recognition site,
the 9.sup.th site-11.sup.th site is the original termination
code).
[0137] PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to
the digestion, transformation, isolation, and ligation methods as
described in Example 7, Section 2. Positive clones were selected
and analyzed using sequential analysis discussed in Example 7,
Section 2 to obtain the Der p 1 expression vector, pDerp1.
[0138] 3. Der p 1 Eukaryotic Expression
[0139] The pDerp1 expression vector was subjected to transfection,
total RNA isolation, and RT-PCR protocols as previously described
in Example 6, Section 3. Isolated PCR products were subjected to
low melting point agarose gel testing to confirm pDerp1 positive
bands. The results prove that pDerp1 is expressed in eukaryotic
cells.
Example 11
Peanut Allergenic Protein Antigen Ara h II Genetic Clone and
Eukaryotic Expression Assay
[0140] 1. Ara h II Primer Synthesis
[0141] The following sequences were used as Ara h II primers.
Primers were artificially synthesized:
[0142] Ara h II PI 5' primer: AAGCTTCTCATGCAGAAGAT (SEQ ID NO: 47)
Ara h II P2 3' primer: GGTACCTTAGTAT CTGTCTC (SEQ ID NO: 48)
[0143] 2. Ara h II Expression Vector Construction
[0144] Ara h II cDNA was used as a template to perform PCR
amplification using Ara h II PI and Ara h II P2 primers to generate
copies of the Ara h II gene. The primers are further described
below: (a) Ara h II PI 5'-AAGCTTCTCATGCAGAAGAT-3.sup.f (SEQ ID
NO:47) (the 1.sup.st site-6.sup.th site basic group from the 5'
terminal end of the primer sequence is the HindlU recognition site,
the 10.sup.th site-12.sup.th site is the original initiator code),
Ara h II P2 5'-GGTACCTTAGTATCTGTCTC-3' (SEQ ID NO:48) (the 1.sup.st
site-6.sup.th site basic group from the 5' terminal end of the
primer sequence is the Kpn I recognition site, the 7.sup.th
site-9.sup.th site from the 5' terminal end of the primer sequence
is the original termination code).
[0145] PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to
the digestion, transformation, isolation, and ligation methods as
described in Example 7, Section 2. Positive clones were selected
and analyzed using sequential analysis discussed in Example 7,
Section 2 to obtain the Ara h II expression vector, pArahII.
[0146] 3. Ara h II Eukaryotic Expression
[0147] The pArahII expression vector was subjected to transfection,
total RNA isolation, and RT-PCR protocols as previously described
in Example 6, Section 3. Isolated PCR products were subjected to
low melting point agarose gel testing to confirm pArahII positive
bands. The results prove that pArahII is expressed in eukaryotic
cells.
Example 12
Peanut Allergenic Protein Antigen Ara h 5 Genetic Clone and
Eukaryotic Expression Assay
[0148] 1. Ara h 5 Primer Synthesis
[0149] The following sequences were used as Ara h 5 primers.
Primers were artificially synthesized:
[0150] Ara h 5 PI 5' primer: AAGCTTATGTCGTGGCAAAC (SEQ ID NO: 49)
Ara h 5 P2 3' primer: GGTACCTAAAGACCCGTATC (SEQ ID NO: 50)
[0151] 2. Ara h 5 Expression Vector Construction
[0152] Ara h 5 cDNA was used as a template to perform PCR
amplification using Ara 5 PI and Ara h 5 P2 primers to generate
copies of the Ara h 5 gene. The primers are further described
below: (a) Ara h 5 PI 5'-AAGCTTATGTCGTGGCAAAC-3' (SEQ ID NO:49)
(the 1.sup.st site-b 6.sup.th site basic group from the 5' terminal
end of the primer sequence is the Hind III recognition site; the
7.sup.th site-9.sup.th site is the original initiator code); (b)
Ara h 5 P2 5'-GGTACCTAAAGACCCGTATC-3' (SEQ ID NO:50) (the 1.sup.st
site-6.sup.th site basic group from the 5' terminal end of the
primer sequence is the Kpn I recognition site; the 7* site-9.sup.l
site from the 5' terminal end of the primer sequence is the
original termination code).
[0153] PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to
the digestion, transformation, isolation, and ligation methods as
described in Example 7, Section 2. Positive clones were selected
and analyzed using sequential analysis discussed in Example 7,
Section 2 to obtain the Ara h 5 expression vector, pArah5.
[0154] 3. pArah5 Eukaryotic Expression
[0155] The pArah5 expression vector was subjected to transfection,
total RNA isolation, and RT-PCR protocols as previously described
in Example 6, Section 3. Isolated PCR products were subjected to
low melting point agarose gel testing to confirm pArah5 positive
bands. The results prove that pArah5 is expressed in eukaryotic
cells.
Example 13
Japanese Cedar (Cryptomeria japonica) Pollen Allergenic Protein
Antigen Cry j 1.1 Genetic Clone and Eukaryotic Expression Assay
[0156] 1. Cry j 1.1 Primer Synthesis
[0157] The following sequences were used as Cry j 1.1 primers.
Primers were artificially synthesized
[0158] Cry j 1. 1 PI 5' primer: AAGCTTATGGATTCCCCTTGCTTAT (SEQ ID
NO:51) Cry j 1. 1 P2 3' primer: GGTACCATCAACAACGTTTAGAG (SEQ ID NO:
52)
[0159] 2. Cry j 1.1 Expression Vector Construction
[0160] Cry j 1.1 cDNA was used as a template to perform PCR
amplification using Cry j 1.1 PI and Cry j 1.1 P2 primers to
generate copies of the Cry j 1.1 gene. The primers are further
described below:
[0161] (a) Cry j 1.1 PI 5'-AAGCTTATGGATTCCCCTTGCTTAT-3' (SEQ
IDN0:51) (the 1.sup.st site-6.sup.th site basic group from the 5'
terminal end of the primer sequence is the Hind III recognition
site; the 7.sup.th site-9.sup.th site is the original initiator
code);
[0162] (b) Cry j 1.1 P2 5'-GGTACCATCAACAACGTTTAGAG-3' (SEQ ID
NO:52) (the 1.sup.st site-6.sup.th site basic group from the 5'
terminal end of the primer sequence is the Kpn I recognition site;
the 7 site-9 site from the 5' terminal end of the primer sequence
is the original termination code).
[0163] PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to
the digestion, transformation, isolation, and ligation methods as
described in Example 7, Section 2. Positive clones were selected
and analyzed using sequential analysis discussed in Example 7,
Section 2 to obtain the Cry j 1.1 expression vector, pCryj1.1.
[0164] 3. pCryj1.1 Eukaryotic Expression
[0165] The pCryj1.1 expression vector was subjected to
transfection, total RNA isolation, and RT-PCR protocols as
previously described in Example 6, Section 3. Isolated PCR products
were subjected to low melting point agarose gel testing to confirm
pCryj1.1 positive bands. The results prove that Cry j 1.1 may be
expressed in eukaryotic cells.
Example 14
Japanese Cedar (Cryptomeria japonica) Pollen Allergenic Protein
Antigen Cry j 1.2 Genetic Clone and Eukaryotic Expression Assay
[0166] 1. Cry j 1.2 Primer Synthesis
[0167] The following sequences were used as Cry j 1.2 primers.
Primers were artificially synthesized
[0168] Cry j 1.2 PI 5' primer: AAGCTTATGGATTCCCCTTGCTTAG (SEQ ID
NO: 53) Cryj 1.2 P2 3' primer: GGTACCTCAACAACGTTTAGAGAGAG (SEQ
IDNO:54)
[0169] 2. Cry j 1.2 Expression Vector Construction
[0170] Cryj 1.2 cDNA was used as a template to perform PCR
amplification using Cryj 1.2 PI and Cryj 1.2 P2 primers to generate
copies of the Cryj 1.1 gene. The primers are further described
below:
[0171] (a) Cry j 1.2 PI 5'-AAGCTTATGGATTCCCCTTGCTTAG-3' (SEQ ID
NO:53) (the 1.sup.st site-6.sup.th site basic group from the 5'
terminal end of the primer sequence is the Hind III recognition
site; the 7.sup.th site-9.sup.th site from the 5' terminal end of
the primer sequence is the original initiator code);
[0172] (b) Cry j 1.2 P2 5'-GGTACCTCAACAACGTTTAGAGAGAG-3' (SEQ ID
NO:54) (the 1.sup.st site-6.sup.th site basic group from the 5'
terminal end of the primer sequence is the Kpn I recognition site;
the 7.sup.th site-9.sup.th site from the 5' terminal end of the
primer sequence is the original termination code).
[0173] PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to
the digestion, transformation, isolation, and ligation methods as
described in Example 7, Section 2. Positive clones were selected
and analyzed using sequential analysis discussed in Example 7,
Section 2.to obtain the Cry j 1.2 expression vector, pCryj1.2.
[0174] 3. pCryj1.2 Eukaryotic Expression
[0175] The pCryj1.2 expression vector was subjected to the
transfection, total RNA isolation, and RT-PCR protocols previously
described in Example 6, Section 3. Isolated PCR products were
subjected to low melting point agarose gel testing to confirm
pCryj1.2 positive bands. The results prove that Cry j 1.2 may be
expressed in eukaryotic cells.
Example 15
Blomia tropicalis Allergenic Protein Antigen Blo t 5 Genetic Clone
and Eukaryotic Expression Assay
[0176] 1. Blo t 5 Primer Synthesis
[0177] The following sequences were used as Blo t 5 primers.
Primers were artificially synthesized Blo t 5 PI 5' primer:
AAGCTTACAATGAAGTTCGC (SEQ ID NO: 55) Blo t 5 P2 3' primer:
GGTACCAATTTTTATTGGGT (SEQ ID NO: 56)
[0178] 2. Blo t 5 Expression Vector Construction
[0179] Blo t 5 cDNA was used as a template to perform PCR
amplification using Blo t 5 PI and Blo t 5 P2 primers to generate
copies of the Blo t 5 gene. The primers are further described
below:
[0180] (a) Blo t 5 PI 5'-AAGCTTACAATGAAGTTCGC-3' (SEQ ID NO:55)
(the 1.sup.st site-6.sup.th site basic group from the 5' terminal
end of the primer sequence is the Hind III recognition site; the
10.sup.th-12.sup.th site from the 5' terminal end of the primer
sequence is the original initiator code);
[0181] (b) Blo t 5 P2 5'-GGTACCAATTTTTATTGGGT-3' (SEQ ID NO:56)
(the 1.sup.st site-6.sup.th site basic group from the 5' terminal
end of the primer sequence is the Kpn I recognition site; the
12.sup.th site-14.sup.th site from the 5' terminal end of the
primer sequence is the original termination code).
[0182] PCR fragments and pVAX1 (Invitrogen, Inc.) were subjected to
the digestion, transformation, isolation, and ligation methods as
described in Example 7, Section 2. Positive clones were selected
and analyzed using sequential analysis discussed in Example 7,
Section 2 to obtain the Blo t 5 expression vector, pBlot5.
[0183] 3. pBlotSEukaryotic Expression
[0184] The pBlot5 expression vector was subjected to transfection,
total RNA isolation, and RT-PCR protocols as previously described
in Example 6, Section 3. Isolated PCR products were subjected to
low melting point agarose gel testing to confirm pBlot5 positive
bands. The results prove that pBlot5 may be expressed in eukaryotic
cells.
Example 16
Induction of Adaptive T Regulatory Cells that Suppress the Allergic
Response: Conversion of T Regulatory Cells by Suboptimal DCs that
are Induced by Co-Immunization of DNA and Protein Vaccines
[0185] Total flea extracts induce immediate intradermal allergic
reactions in mice, therefore this system can be used to evaluate
immunotherapeutic methods aimed at amelioration of AIH. Using this
rodent model of AIH induced by flea allergens, co-immunization of
DNA and protein vaccines encoding the flea salivary specific
antigen (FSA1) ameliorates experimental AIH, including antigen
induced wheel formation, elevated T cell proliferation,
infiltration of lymphocytes and mast cells to the site of
challenge. The amelioration of AIH was directly related to the
induction of a specific population of flea antigenic specific T
cells exhibiting a CD4.sup.+/CD257FoxP3.sup.+ phenotype, a
characteristic of Tr. These Tr also express IL-10, IFN-y and the
transcriptional factor T-bet after antigen stimulation. These Tr
are driven by MHC-II.sup.+/CD40.sup.low DC populations that are
induced by the co-immunization of DNA and protein vaccines. These
studies identify important cellular players in the control of AIH.
Exploitation of these cellular regulations and their induction will
provide novel direction to develop therapies for allergic and
related disorders.
Methods
[0186] Histology Analysis.
[0187] On day 14 following the last immunization, skin samples from
mice were collected and fixed in 4% of paraformaldehyde, embedded
in paraffin blocks from every group of mice. Four to
five-micrometer sections were cut and placed on sylan-coated glass
slides prior to rehydrated in xylene and washed with decreasing
concentrations of alcohol solutions. The endogenous peroxidase
activity was blocked by 3% hydrogen peroxide at room temperature
for 10 min and the antigen retrieval was accomplished by boiling
the slides in 0.01 M citrate buffer (pH 6.0). Lastly, slides were
stained with hematoxylin and eosin (H&E) or toluidine blue for
mast cells and analyzed under a light microscope for histology
changes.
[0188] Skin Test.
[0189] On day 14 after the last immunization, the mice were
challenged with 1 Hg/ul of flea-saliva-antigen on nonlesional
lateral thorax skin intradermally, PBS is used as a negative
control and histamine is used as a positive control. The diameter
of the skin reaction was measured within 20 min after challenge by
using a calibrated micrometer. Reaction was considered as a
positive when the injection site was larger than half the size of
the sums of diameters injected comparing the positive and negative
control challenges.
[0190] T Cell Recall Responses.
[0191] The T cells isolated from immunized mice on day 14 were
cultured at 5.times.10.sup.4 cells/well in triplicate in 96-well
plates containing RPMI-10/5% FCS and then stimulated with 20 ug/ml
of Flea-saliva-antigen for 48 h. Following the stimulation, cell
proliferation was assessed by a colorimetric reaction after the
addition of 20 pi of an MTS-PMS (Pormaga, USA) solution for 2-4 hrs
and its color density was read at 570 nm by plate reader (Magellan,
Tecan Austria GmbH).
[0192] Measurement of Flea Antigen-Specific Antibodies.
[0193] Serum concentration of anti-flea IgG1, IgG2a, IgG2b, IgM and
IgE isotypes were measured using flea antigen coated plates by
ELISA and detection with specific horseradish peroxidase-conjugated
rat anti-IgG1, IgG2a, IgG2b, IgM and IgE antibodies
(SouthernBiotech, Birmingham, USA), absorbance (450 nm) was
measured using an ELISA plate reader (Magellan, Tecan Austria
GmbH).
[0194] RT-PCR.
[0195] Total RNA was isolated from spleen and skin tissue 14 d
after immunization using TRIzol reagent (Promega). cDNA was
synthesized and PCR was performed in a 50 pi reaction mixture with
5 u, 1 cDNA and 1.0 M of each of the following primers: HPRT, IL-2,
IFN-y, IL-4, IL-5, IL-13, IL-10 and 1L-12Q8). For Gata3 and T-bet
analysis, CD4.sup.+CD25'' T cells were isolated and total RNA was
prepared. RT-PCR was done as described using specific primer
sequences as follows for Gata3, 5'-GGAGGCATCCAGACCCGAAAC-3.sup.1
(forward) (SEQ ID NO: 57) and 5'-ACCATGGCGGTGACC-ATGC-3.sup.1
(reverse) (SEQ ID NO: 58); for T-bet,
5'-TGAAGCCCACACTCCTACCC-3.sup.1 (forward) (SEQ ID NO: 59); and
5'-GCGGCATTTTCTCAGTTGGG-3.sup.1 (reverse) (SEQ ID NO: 60).
[0196] Isolation of CD4.sup.+CD25'' T Cells and Adoptive
Transfer.
[0197] Single splenocyte suspensions were prepared from mouse
spleen and CD4.sup.+CD25'' T cells were isolated and purified by
using the MagCellect Mouse CD4.sup.+CD25.sup.+ Regulatory T Cell
Isolation Kit according to the manufacturer's protocol (R&D
Systems, Inc., USA). The purity of the selected cell populations
was 96-98%. The purified cells (1.times.10.sup.6 per mouse) were
adoptively transferred intravenously into C571BL6 mice.
[0198] CFSE Labeling and Co-Culture of Cells.
[0199] Naive CD4.sup.+ T cells isolated from C57/BL6 mice were
labeled with CFSE (Molecular Probes). For assay of regulatory
activity, 1.times.104 pcDF100+F induced regulatory or control T
cells were co-cultured with 4.times.104 purified and CFSE labeled
naive CD4.sup.+ T cells in the presence of flea antigen (100
u.g/ml) and 1.times.104 bone marrow derived DCs. For some cultures,
Tr cells were co-cultured with anti-IL-10, anti-IFN-y or an isotype
control antibody at 100 g/ml. After 72 h, cells were collected and
labeled then CFSE+ cells were selected for analysis by flow
cytometry.
[0200] Flow Cytometry.
[0201] CD4.sup.+CD25'' T cells were isolated and incubated on ice
with PE-conjugated antibodies to CD44, CD69, CD62L (eBioscience,
CA, USA). Flow cytometry of cytokine production and FoxP3
expression in T cells were performed, single cell suspensions were
prepared from the animals spleens and Fc receptors were blocked
with excess anti-Fc (BD PharMingen, USA). Cells were washed with
ice-cold PBS. For intracellular cytokine staining, T cells were
stimulated overnight with Con A (Sigma-Aldrich) in the presence of
anti-CD28 mAb (BD PharMingen, USA). Collected cells were fixed with
4% paraformaldehyde and permeabilized with 0.1% saponin
(Sigma-Aldrich). For staining of surface of CD4 or cytoplasmic
IL-10, IL-4, IFN-y or FoxP3, the appropriate concentrations of
phycoerythrin-labeled antibodies (eBioscience, CA, USA) were added
to premeabilized cells for 30 min on ice followed by washing twice
with cold PBS. Samples were processed and screened using
FACSCalibur and data were analyzed with Cell Questpro software
(BD).
Results
[0202] A flea salivary allergen, FSA1, has been identified and
implicated as one of the causes for allergy dermatitis observed in
cats and dogs. The degree of skin reaction or intradermal test
(TDT) to assess the immediate intradermal flea-antigen reactivity
can be achieved by flea allergy challenge intradermally. As
expected, administration of mice with flea antigen induced
significant skin reactions (FIG. 1A), induced mast cells
activation, induced coincident IgE production (FIG. 1B), and
induced strong CD4+ T cell proliferation responses (FIG. 1C) in C57
mice when compared with naive control animals after intradermal
challenge. This data demonstrates the utility of the flea antigen
allergic model for evaluation of novel therapeutic strategies
against AIH.
[0203] This model was used to examine the ability of the
co-inoculation strategy to protect animals from the immediate
hypersensitivity after flea allergen challenge. C57/BL6 mice were
pre-sensitized with various test vaccines and animals were
intradermal challenged with the flea extracts, or with histamine as
the positive or using PBS as the negative control. The immediate
hypersensitivity reaction was blocked in group immunized 14 days
earlier of co-inoculation of a plasmid DNA, pcDF100, encoding an
epitope of FSA1 (aa100-1 14) mixed with the total flea proteins
(designated as pcDF100+F) (FIG. 2A). To determine if the inhibition
observed was due to the plasmid backbone or rather was related to a
DNA construct encoding another region of FSA1. Robust inflammation
of the challenged sites were observed in the mice immunized with
either vector control plus flea proteins (designated as V+F), or a
DNA construct encoding another region of FSA1 at aa66-80, pD66,
again mixed with the flea proteins (designated as pcDF66+F, FIG.
2A). We also examined the influence of host immune balance toward
Th1 type on the inhibition of allergic reaction, the pcDF100 primed
and the flea protein boosted animals (designated as
pcDF100.fwdarw.F; FIG. 2A). The severe reaction following challenge
was observed in mice primed with pcDF100 and boosted with F (FIG.
2A), suggesting that induction of a strong immune response worsens
the allergic reaction.
[0204] Histological analysis revealed infiltrations by leukocytes
and mast cells in the skin lesions of mice immunized with F or V+F
at the challenge sites; whereas, mice immunized with pcDF100+F
showed normal intradermal structure which was free of inflammatory
cells.
[0205] We next analyzed if the blocking of the immune reaction
induced by the co-inoculation was a dose dependent. pcDF100 at dose
of 25, 50, 100 and 200 (ig was co-immunized with 100 ug of flea
proteins, respectively. Dosage at 50 jig of pcDF100 showed
significant inhibition of the intradermal reaction which reached
maximal inhibition at 100 (ig. A dosage of 25 ug exhibited only a
minimal effect on lesion formation, as the animals developed severe
reactions similar to those observed in animals inoculated with
either F or V+F (FIG. 2B) or positive controls.
[0206] High levels of IL-4, IL-5 and IL-13 are the characteristics
of allergic reactions and these immune modulators are implicated in
allergy severity. Different profiles of the cytokines associated
with the co-inoculated regimens were examined. Mice co-inoculated
with F or V+F produced higher level of mRNA expressions for IL-4,
IL-5 and IL-13; whereas, mice co-immunized with pcDF100+F produced
relative low levels of these cytokines, suggesting that an
anti-inflammatory immune regulatory function was derived from the
co-inoculation of pcDF100+F. No significant differences were found
in the levels of IL-2 or IFN-y among the co-inoculated regimens,
but IFN-y was slightly higher in mice primed with pcDF100 and
boosted by F. This result again suggests that the induced
inhibition of the allergic reaction by co-immunization is not due
to an un-balanced Th2 to Th1 response by the allergenic-specific
T-helper cells.
[0207] As flea antigen triggered IgE-mediated allergy is well
characterized, the ability of co-inoculation of pcDF100+F to
inhibit anti-flea induced IgE production was examined. The levels
of anti-flea IgE and IgG in sera were measured on days 14, 28 and
42 and were reduced slightly in mice co-immunized with pcDF100+F
compared with groups immunized with V+F or F alone (FIG. 2C),
suggesting that the co-inoculation does not influence the IgE
production.
[0208] Proliferative CD4.sup.+ T cells are known to be involved in
the development of immediate hypersensitivity. I isolated CD4.sup.+
T cells from the spleen of mice co-immunized with F, V+F or
pcDF100+F was examined on day 14 after the second immunization for
their recall proliferative responses to flea antigens in vitro.
Immunizations of F and V+F resulted in strong proliferation of
CD4.sup.+ T cells; whereas the CD4.sup.+ T cells isolated from
pcDF100+F immunized mice showed little, if any, proliferation in
response to the flea antigen stimulation (FIG. 2D). These results
suggest that the inhibition of hypersensitivity observed by
co-immunization of pcDF100+F is related to the non-responsive
antigen specific CD4.sup.+ T cells.
[0209] The results indicate that the co-immunization with pcDF100
and flea proteins antigen induces an inhibition of the allergic
reaction via down-regulating the levels of inflammatory cytokines
and CD4.sup.+ cells induced by flea intradermal challenge. This
suggests that the prevention of allergy is likely antigen specific
since the antigen mismatched combinations did not produce the same
effect.
[0210] To test if antigen-specific Tr cells have been induced by
the co-immunization of flea DNA and protein vaccines, splenocytes
were collected from co-immunized C57B/6 mice and mixed with the
flea-antigen-specific effector T cells of syngeneic mice to exam
their ability to inhibit recall proliferative responses in vitro.
As shown in FIG. 3A, splenocytes from mice co-immunized with
pcDF100+F significantly inhibited T cell recall immune responses.
In contrast, splenocytes from mice immunized with F or V+F as well
as from naive mice failed to inhibit the antigen specific T cell
proliferative responses (FIG. 3A). This indicates that cells within
the splenocyte population likely generated during the
co-immunization of animals can suppress this antigen specific T
cell proliferation. Purified non-T cells, T cells or subsets of T
cells from the spleen of mice co-immunized with pcDF100+F were
identified and tested individually for suppression of recall
proliferation. Significant inhibition was observed from reactions
of either purified T cells, purified CD4.sup.+ cells, or purified
CD4.sup.+CD25'' cells from the mice co-immunized with pcDF100+F,
but not from other subsets of cells (FIG. 3A). However, the
inhibitions from CD4.sup.+CD25.sup.+ cells are in general thought
to be independent of antigen sensitization, whereas the inhibition
of CD4.sup.+CD25'' cells of this reaction is in an antigen
dependent manner (FIG. 3A).
[0211] To further examine this issue in vivo, adoptive transfer was
utilized. Antigen naive syngeneic recipient mice were adoptively
transferred with either total splenocytes, T, CD4.sup.+ or
CD8.sup.+ cells isolated from C57B/6 mice co-immunized with
pcDF100+F, F or V+F, respectively. All recipient mice were next
challenged intradermally by flea extract to induce the
hypersensitivity. Splenocytes, T and CD4.sup.+ T cells from mice
immunized with the pcDF100+F, but not with the F or V+F, were all
able to suppress the development of the immediate hypersensitivity
reaction (FIG. 3B). In contrast, CD8.sup.+ T cells isolated from
all three experimental groups and naive control mice did not
suppress this reaction. Both in vitro and in vivo results indicate
that CD4.sup.+CD25'' Tr cells can mediate this suppression.
[0212] To investigate the observed role of CD4.sup.+CD25'Tr antigen
specificity, the CD4.sup.+C25'' Trcells taken from C57B/6 mice
co-immunized with pcDF100+F were adoptively transferred into the
syngeneic recipient mice that were subsequently immunized twice at
a biweekly interval with flea-antigen or OVA in Freunds' complete
adjuvant (FCA). On day 14 after the last immunization, T cells were
isolated and tested for their ability to proliferate to either flea
antigen or OVA in vitro. T cells from recipient mice immunized with
flea antigen did not respond the flea antigen stimulation in vitro;
whereas, the T cells from recipient mice immunized with OVA-FCA
responded well with OVA stimulation, but not to the flea antigens
stimulation in vitro. As the controls, the naive mice immunized
with flea antigen respond well to the flea antigen stimulation, but
not to OVA stimulation in vitro and vice versa (FIG. 3C). This
result indicates that adoptive transferred CD4.sup.+C25* Tr cells
only inhibit the flea antigen specific T cell priming and
proliferation in vivo; while the responses from the irrelevant
antigen specific T cells were not affected.
[0213] Taken together, these data demonstrate that CD4.sup.+CD25''
Tr cells were induced by the co-immunization of DNA and protein
vaccines. These appear to be unique CD4+ Tr cells as they function
in an antigen-specific manner.
[0214] To determine if Tr cells induced by co-inoculation express
certain types of cytokines and unique markers associated with Tr
cells as previously documented (J. D. Fontenot, M. A. Gavin, A. Y.
Rudensky, Nat Immunol 4, 330 (Apr. 1, 2003); M. G. Roncarolo, R.
Bacchetta, C. Bordignon, S. Narula, M. K. Levings, Immunol Rev 182,
68 (Aug. 1, 2001); and P. Stock et al, Nat Immunol 5, 1149 (Nov. 1,
2004)), CD4.sup.+CD25'' cells were isolated from the mice immunized
with F, V+F or pcDF100+F on days 1, 3, 7 and 14 post immunization
and T cell profiles were followed by intracellular staining with
specific fluorescent labeled antibodies. After co-immunization, on
days 1, 3, 7 and 14, CD4.sup.+CD25'' T cells were isolated from F,
V+F and pcDF100+F immunized mice and intracellular cytokine
production for IL-10, IFN-y and IL-4 expression was assessed by
flow cytometry. CD4.sup.+CD25'' Tr cells isolated from F, V+F,
pcDF100+F and naive mice as controls were analyzed for expression
of CD69, CD44 and CD62L, and for their expression of FoxP3. Tr
cells express T-bet but not gata-3. On day 14 after immunization,
total RNA was extracted from CD4.sup.+CD25'' T cells from all three
groups and RT-PCR was used to test the expression of HPRT, T-bet
and gata-3. Over the course of 14 days, the CD4.sup.+CD25'' T cells
isolated from mice co-immunized with pcDF100+F expressed high
levels of IL-10, IFN-y, FoxP3, and a minimal amount of IL-4. In
contrast, CD4.sup.+CD25'' T cells produced a higher level of IL-4
and no expressions of FoxP3, IL-10, or IFN-y from the mice
immunized with F or V+F. Since the transcriptional factor Foxp3,
has been demonstrated to be a hallmark of the Tr cells, the
co-vaccination induced CD4.sup.+CD25''Tr cells can be categorized
into the regulatory class of T cells but they have a unique
phenotype. T cell activation markers are expressed equally high
including CD44 and CD69 and low for CD62L among the immunized
groups, suggesting that the induced CD4.sup.+CD25' Tr cells are
fully activated by the immunization.
[0215] To analyze the Th phenotype for the induced CD4.sup.+CD25''
Tr cells based on the observed cytokine expression patterns as
described above, the expression of both T-bet and gata-3 genes of
the CD4.sup.+CD25'' T cells from mice immunized with F, V+F or
pcDF100+F were analyzed by the RT-PCR method. The results show that
the CD4.sup.+CD25'' T cells from the pcDF100+F immunized mice, but
not from the F or V+F immunized mice, expressed higher level of
T-bet, a hallmark for Th1 cells. In the contrast, the
CD4.sup.+CD25'' T cells from the F and V+F immunized mice expressed
higher levels of gata-3, a characteristic Th2 cells.
[0216] Collectively, these data demonstrate that CD4.sup.+CD25'' T
cell induced by the co-immunization of DNA and protein vaccines has
as an adaptive Th1 phenotypic Tr cell which can suppress the
antigen-specific CD4.sup.+ T cell proliferative function.
[0217] Since antigen presenting cell (APC) activates T cells to
promote adaptive immunity, the induction of CD4.sup.+CD25'' Tr cell
is apparently through specific APC activation by the co-inoculation
of DNA+protein vaccines. To investigate this question, a similar
experiments described as above was set up to assess dendritic cell
(DC) function and phenotype and their influence on naive T cells.
The effects of co-inoculation on the maturation of DC was analyzed.
Expression of costimulatory molecules on DCs from co-immunized mice
was examined. Splenocytes were isolated and stained for expression
of surface markers on DCs by gating on CD11 positive cells 48 h
after co-immunization of pcDF100+F, V+F or F. Co-immunization of
pcDF100+F did not affect maturation of DCs as DCs isolated from the
spleen 48 h after the co-immunization expressed high and similar
levels of CD80, CD86, MHC II, IL-12, IL-6, IL-la/f3, IFN-a/8 and
TNF-a which are the characteristics of matured DCs; whereas these
molecules on the immature DCs from naive control mice remain
expressed at relatively low levels, suggesting the co-inoculation
enables the immature DC be induced to mature. However, we observed
that the level of CD40 expression was dramatically reduced in
pcDF100+F co-immunized mice compared to all other groups,
suggesting a unique phenotype of DC may be involved in the
induction of the observed Tr. DCs from mice immunized with V+F or F
were observed to have the ability to activate heterogeneous T cells
to proliferate; whereas the Immature DCs of naive mice have no such
ability as expected (FIG. 4A). Interestingly, DCs from mice
co-immunized with pcDF100+F had a restricted capacity to activate T
cells to proliferate (FIG. 4B), suggesting that an alternative
mechanism of Tr induction is induced in the co-immunization group.
To explore if the DC of co-immunized mice are able to convert naive
T cells to a Tr phenotype, DCs obtained from mice after their being
co-immunized with pcDF100+F were co-cultured with syngeneic naive
CD4.sup.+ T cells in vitro and subsequently characterized the
resulting CD4.sup.+ T cells by FACS. These T cells upregulated
higher levels of CD44, and CD69, but lower levels of CD62L,
suggesting that the DC again has the capacity to activate T cells.
Similar results were obtained from analysis of DCs from V+F or F
co-immunized mice. Furthermore, the activated CD4.sup.+T cells in
the pcDF100+F group produced significant higher level of IL-10 and
IFN-y, but reduced IL-4 (FIG. 4B). However, activated T cells from
V+F or F immunized groups produce significant level of IL-4, but
little IL-10 and IFN-y (FIG. 4B). The cytokine production were
analyzed after three rounds of re-stimulation with fresh DCs
isolated from the D+F immunized mice. These studies demonstrated an
increase in the production of IL-10 in T-cells, which is one of
characteristic of regulatory T cells. To further characterize their
regulatory function, IL-10.sup.+ T cells were isolated after DC
stimulation and subjected to MLR to see if they block responder T
cell proliferation in the MLR. As shown, the proliferation of
responder T cells were inhibited by the presence of IL-10.sup.+ T
cells, but not from T cells isolated from co-culture with DCs of
mice immunized with V+F and F, or the control animals (FIG. 4C).
These data demonstrate that DCs from the D+F co-immunized mice
develop large populations of T regulatory cells in vitro.
[0218] To demonstrate the same conversion in vivo, DCs collected
from BALB/c mice after co-immunization with pcDFlOO+F were
co-transferred with syngenic naive CD4.sup.+T cells into nude mice
(nu/nu) and subsequently analyzed these transferred T cells by FACS
for intracellular staining for IL-lO and T reg markers. DCs from
pcDFlOO+F co-immunized mice induced Tr cells in vivo. DCs isolated
from spleens of pcDFlOO+F, V+F, F immunized or naive control mice
were co-adoptive-transfer with naive CD4.sup.+CD25.sup.-T cells.
The T cells were analyzed for IL-10, IFN-.gamma., IL-4, FoxP3 and
CD25 on day 3 and 7. Co-transferred T cells expressed more IL-10,
FoxP3 and IFN-.gamma., but little IL-4. However, co-transferred T
cells with DCs from V+F or F immunized mice expressed higher levels
of IL-4, but little IL-10 and IFN-.gamma., supporting the in vitro
data. Specific IL-10 cytokine production was elevated after two
rounds of re-stimulation with the DCs freshly isolated from D+F
immunized mice. These data demonstrate that DCs from D+F
co-immunized mice drive likely naive T cells into Tr cells in vivo.
Consistent with previous results, such conversion was only made
within the CD4.sup.+cD25'' population since the frequency of
CD25.sup.+ was not observed to be influenced testing vivo.
[0219] Finally, experiments were performed to identify what
molecules in the interaction between DC and T cells play a role in
the development of Tr. Since the Tr is within the activated T cell
compartment as demonstrated above, the signals should include
classic activation pathway signals. Important among these is the
up-regulation of major histocompatibility complex (MHC) class II
and co-stimulatory molecules (CD80/CD86), which provide the two
requisite signals for naive T cell activation. To study this issue,
DCs were isolated from spleen of mice 48 h after pcDF100+F
co-immunization and co-cultured with naive CD4.sup.+ T cells from
naive syngeneic mice in the presence or absence of reagents to
block signaling molecules including anti-CD80, anti-CD86, anti-CD40
and anti-MHC-II. After seven days of re-stimulation, T cells were
isolated and added into MLR system to examine their regulatory
functions. The results showed that both anti-CD40 and anti-MHC II
mAbs can partially reverse the suppressive effects on MLR by the Tr
cells; whereas mAbs against CD80, CD86 and had no ability to block
the induction of the suppression phenotype. These results
demonstrate that both CD40 and MHC II signals play roles in the
induction of Tr in the flea allergen Induced immediate
hypersensitivity model.
[0220] Adaptive Trl cells have been observed and induced by DCs
processing a suboptimal immunogen or subimmunogen to inhibit normal
T cells' function mediated via secretion of IL-10 or TGF-b or both.
It has been further demonstrated that immature DCs drive the
differentiation of IL-10-producing Trl cells with producing IL-10,
TGF-R, and IFN-y, but they do not produce IL-4 or IL-2, they are
hyporesponsive to antigenspecific and polyclonal activation. In
additional evidence has shown that suboptimal activation of DC by a
minute antigen stimulation can induces the Trl conversion. This
stimulation of DCs lacks the co-stimulation signaling and thus
presents a tolerance signal to T cells.
[0221] Co-inoculation of DNA encoding a flea antigen with flea
protein induces adaptive Tr cells which inhibit the allergic
reaction induced by flea allergen challenge. The cells exhibit a
phenotype of CD4.sup.+CD25''Foxp3.sup.+ and suppress in vivo and in
vitro antigen specific as well as MLR immune responses through the
production of IL-10 and IFN-y. MHC-ll.sup.+/CD40.sup.low DC
populations are induced by such co-immunization and in turn to
convert naive T cells into Tr cells.
Example 17
Testing the Prevention and/or Therapeutic Approaches Against FAD
Allergen Through Co-Immunization with Vaccines in a Feline FAD
Model Establishing the Feline Model and Testing Co-Immunization
[0222] There are more than 15 kinds of allergen in the flea saliva.
Among them, an 18 kDa protein, flea saliva antigen 1 (FSA1), has
been determined a main allergen which can cause FAD. This gene has
been cloned and expressed it in the E. coli system. In addition,
the pVAX-FSA1 eukaryotic expressing construct has also been
prepared.
[0223] A cat FAD model was established and subsequently used to
demonstrate that the therapeutic effect of co-immunization of
FSA1+pVAX-FSA1 on the established FAD in cats.
Determination of Number of Fleas, Duration of a Cycle
Animal and Parasite
[0224] Ten pathogen-free cats were purchased from North China
Pharmaceutical Group (Shijiazhuang, Hebei) and housed animal
facility at the Center for Disease Control and Prevention of China
(CDC) in the course of experiment. All cats were over one year old,
which is an important factor since the younger animals may be
tolerable to the flea allergen in this experiment. The cats were
grouped with breed and sex randomly. The sterile fleas were
supplied by the CDC.
[0225] To determine relationship of infestation and FAD symptom, 10
cats were separated into three groups, including an experimental
group with six cats and two control groups with two cats each. One
control group was treated nitenpyram, and another was not treated
with the nitenpyram. Each experimental cat was lived separated on
day 0 and infestated with 100 fleas. After two days, all cats in
this group were given the nitenpyram to remove the flea from their
bodies. Two of the control cats were also given this medicine on
day 0. This challenge cycle was repeated every other week for 7
times
Immunization Scheme for the FAD Induced or Control Cats with FSA1
and pVAX-FSA1 as Co-Immunogens
[0226] The 6 FAD cats were separated into three groups. Two of them
were co-immunized with 400 u.g FSA1 i.p. and 400 u,g pVAX-FSA1
plasmid at double lateral abdomen subcutaneously. Two were
co-immunized with 400 u.g FSA1 i.p. and 400 u.g pVAX vector
subcutaneously. The last two were not immunized and used as the
positive control group. The 4 un-FAD cats were separated into two
groups. Two were co-immunized with 400 u.g FSA1 i.p. and 400( g
pVAX-FSA1 plasmid at double lateral abdomen subcutaneously and the
other two were immunized with 400 u.g FSA1 i.p. and 400 u.g pVAX
vector subcutaneously. The two control cats with nitenpyram were
sent into different groups. The cats were immunized three times: at
days 0, 9, and 16. After that, the cats were challenged for six
cycles as above. During the second immunization, a cycle of
challenge was done at the same time, because we wanted to keep
positive cats at the susceptible state for next therapy. The
program of immunization was as Table 2.
TABLE-US-00002 TABLE 2 Immunization Cat PSA1 + pVAX- PSA1 + pVAX
Treat with No. FSA1 vector nitenpyram prophylactive 1 - + + 2 + - -
4 + - + 5 - + - therapeutic 8 + - + 9 - + + 10 - + + 11 + - +
Positive control 3 - - + 7 - - +
Dermatological Scores.
Method:
[0227] Cats were scored by the dermatological assessments two days
after ceasing each infestation. The assessments were included
erythema, papules, crusts, scale, alopecia, excoriation. The body
of each cat was divided into three portions to assess which part
might have the most severe clinical outcome. According the previous
documented report, three parts were consisted of: (1) back, from
the head to the tail of dorsal surface; (2) double lateral abdomen,
from the scapula to the tail; (3) chest and underside, from
laryngeal to the caudomedial thighs.
Smearing Counts of Peripheral Blood Cells from the Cats
Method:
[0228] The anti-coagulant peripheral blood at 2 ml was collected on
days 0, 2, 16, 30, 44, 58, 62 from the experimental group and day
0, 14, 28, 42, 56, 60 from the control groups. The same samples
were collected after the immunization. A drop of blood was smeared
on a clean glass slide. After the smear was dried, the cells were
stained with Wright-Giemsa stain for 15 min. After a rinse in
deionized water, the smear was gently dried with Kimwipe paper and
dehydrated by 96% and 100% ethanol with each 10 seconds treatment.
The slide was then treated with xylene for 30 min.
[0229] After the staining, nucleus and cytoplasm of blood cell was
distinguished by a blue and pink staining. The percentage of each
type of cells was counted by a total number of 200 cells in a view
under a light microscope.
Therapeutic Approaches:
[0230] The same methods as above were used except that total RNA
was extracted from mononuclear cells which were isolated from
peripheral blood on day 7 post the third immunization from each
group.
Statistical Analysis
[0231] Analysis of variance (ANOVA) was used to detect the
differences which included dermatology scores from each cycle,
scores of various lesions and scores on different sites among three
groups. The differences of skin test and IgE level were determined
also by ANOVA. Statistical analyses of other ones were performed
using the Student's/-test. In these analyses, data were converted
into logarithmic plot. If the P<0.05, indicates a significant
difference.
[0232] Statistical analyses of were also performed using the
Student's/-test. In these analyses, the data was converted into
log. If the PO.05, the data indicated significant differences.
Results:
Observation and Dermatological Scores of the Cats
[0233] Lesions were scored according to their types, locations,
sizes and numbers.
[0234] The total scores were analyzed from the groups, and the
lesions and sites for the experimental group were assessed. The FAD
group registered more scores than in other groups. The scores were
increased at the fourth infestation cycle, and then stayed at the
level of 5.0. The scores in the FAD group were significant higher
than in other control groups (P<0.01). These results support the
conclusion that the cat FAD model was valid and feasible to
evaluate a therapeutic or prophylactic treatment against the
allergic reaction.
[0235] To assess where the lesions occur most, what type, and when
to occur, statistic analysis was done and it was found that papules
and fur loss were the most common factors to contribute to the
dermatology scores in FAD group. The reading of erythema was not a
contributing factor since the erythema reached to the peak on day
44, but it fallen at the end of this experiment, indicating no
persistency. On the other hand, the papules were increased, and
remained at a high level after 44 days and reached to the peak on
the day 86 (P<0.05), suggesting its persistency. Similarly, fur
loss was maintained at high level after day 44 (P<0.05). These
two readouts are indicated as a good onset to reflect FAD. The
scores from other lesions were randomly distributed and
inconsistent in the two control groups, in which no significant
difference of lesions were observed.
[0236] Although, most of dermal lesions tended to be located on the
backs and heads compared other sites on day 44 (P<0.05), the
lesions were extended all over the body at the end of the
experiment. This observation was different from the previous
reports in dog, of which the chest and underside were tended to
have the most lesions. That may owe to the habitual differences of
the cats and dogs.
[0237] To eliminate the interference by the nitenpyram treatment,
any differences between these treated with nitenpyram and the ones
without treatment were assessed. From the dermatology score, no
significant difference was seen in the control groups. Thus, the
nitenpyram treatment did not influence the results obtained from
our experiments (FIG. 5).
Cell Percentage in Peripheral Blood by Blood Smear Analysis
[0238] A comparison of the number of each type of cells in
peripheral blood from the different groups showed that the number
of eosinophils was higher in the experimental group than those in
control groups. Blood smears were performed for each infestation
cycle, but the percent of each type of cells became constant after
the third cycle. As the result from day 58 show in Table 3, the
significant changes occurred in the number of eosinophils among the
three groups. Since the increase of eosinophils is related to
allergic diseases as previously documented, these results indicate
that the cats in the experimental group were more susceptible to
the infestation of fleas. There was no obvious difference between
the two control groups, the nitenpyram seems not to interfere the
cell numbers in the cats' peripheral blood.
TABLE-US-00003 TABLE 3 Cell types and their percentage in
peripheral blood Lympho- Mono- Neutro- Baso- Eosino- cytes cytes
phils phils phils (%) (%) <%) (%) (%) Experimental 23.4 2.6 61.5
1.0 11.5 group Control group 21.8 3.6 70.5 0.7 3.4 with nitenpyram
Control group 23.1 3.6 68.3 0.6 4.4 without nitenpyram
Histopathological Examination
[0239] First, the differences between the normal skins and those
with lesions were analyzed. Skin biopsies were chosen from all
groups on day 58. However, the skin biopsies from one cat may also
display differences in some degree, especially those with lesions.
That was not enough to indicate whether the cats infestated with
fleas were induced to have FAD. For this reason, the skin biopsies
from each group were collected after the IDT with the flea extracts
since IDT with flea provides an antigen specific recalled immune
responses.
Skin Test to Evaluate Whether the Cats were Allergic to the Flea
Extracts.
[0240] The diameter of wheal or bleb was measured 15 min after the
IDT injection. Since each cat had a different level of sensitivity
to the IDT, the results from each individual animal were recorded.
FIG. 6 shows the skin reactions in all groups. An allergic reaction
was considered to have occurred if an IDT value was above the
average of threshold (the sum of saline and histamine is divided by
2), or not occurred if an IDT value was below the average of the
threshold. The results are shown in Table 4. Cats with dermatology
scores at 5.0 or above were identified as the positive for FAD.
TABLE-US-00004 TABLE 4 Comparison of dermatological assessment with
the intradermal skin test (IDT). Control Control group Positive
Positive Positive skin Positive Positive Cat Experimental group
with without Dermatology clinical skin test test with skin test
skin test No. group nitenpyram nitenpyram scores scores with BSA
flea extract with FS with FSA1 I No Yes No 0 - - - - - 2 No No Yes
1 - - - - - 4 No Yes No 1 - - - - - 5 No No Yes 0 - - - - - 3 Yes
No No 7 + - + - + 7 Yes No No 6 + - + - - 8 Yes No No 8 + - + - + 9
Yes No No 2 - - + - + 10 Yes No No 6 + - + - + 11 Yes No No 3 - - +
- +
Skin Test to Determine the Effect of Co-Immunization of FSA1 and
pVAX-FSA1
[0241] The cats with FAD were co-immunized with FSA1+pVAX-FSA1 or
vector+FSA1 as described in experimental design B in Table 2.
Before and after the immunizations, the cats were tested by IDT
with various flea antigens or control antigens. The IDT readings
were recorded on 7 days after the last immunization as summarized
in FIG. 7 and Table 5.
[0242] The results showed that FAD cats co-immunized with the
FSA1+pVAX-FSA1 had less skin-reactions to the flea extracts or flea
specific antigens (such as FSA1 protein) challenges; whereas the
cats immunized with vector+FSA1 had more skin-reactions to the same
challenges.
[0243] Comparing the differences before and after immunization, we
observed that the skin reaction was much smaller after the
immunization than before in the co-immunized group as shown FIG. 7,
suggesting the co-immunization significant decreased sensitivity to
the flea challenge. On contrary, no significant effect was seen in
groups immunized with FSA1 and pVAX vector, indicating that the
cats were remained their allergic status. The status of all cats
was listed in Table 5.
TABLE-US-00005 TABLE 5 FSA1 FSAl + IDT IDT IDT The state for Cat
Positive and pVAX - with with flea with FE before No. control pVAX
FSAl BSA extracts FSA1 immunization I No Yes No - 2 No No Yes - 4
No No Yes - 5 No Yes No - 3. Yes No No - + - + 7 Yes No No - + - +
8 No No Yes - - - + 9 No Yes No - + - + 10 No Yes No - + - + II No
No Yes - - - +
Therapeutic Effects of Co-Immunization on the FAD Cats
[0244] To determine the therapeutic effect of co-immunization on
lesions of the FAD cats, the lesions were recorded over period of
53 days after the first immunization and shown in FIG. 8. The
lesion scores in the co-immunized group were dramatically reduced
from 5.5 to 2 (FIG. 8, square points on line). Whereas, the effects
on the group immunized with FSA1 and pVAX vector was reduced but to
a lesser extent (FIG. 8, triangle points on line). The result
suggested the co-immunization had a therapeutic effect on the
established FAD in cats.
Correlation of the Type of Lesions Affected by the Co-Immunization
on the FAD Cats
[0245] To correlate which type of lesions reduced with the
co-immunization, the scores on each type of lesions in every group
were analyzed and the results are shown in FIG. 9A-9F. Only the
score of the papules were reduced from 4.0 to 0.5 and coincident
with the co-immunization of FSA1+pVAX-FSA1 (FIG. 9B, square points
on line). Other type of lesions in experimental group was remained
unchanged.
Correlation of the Location of Lesions Affected by the
Co-Immunization on the FAD Cats Therapeutic Effects of
Co-Immunization on the FAD Cats
[0246] After analysis the correlation, the data showed that the
scores of double lateral abdomen in therapeutic group were reduced
from 1.5 to 0 (FIG. 10B) after the co-immunization of
FSA1+pVAX-FSA1, but no effect in the other places as shown in FIGS.
10A-10C. The therapeutic group immunized with FSA1 and pVAX vector
showed a lingering response (FIG. 10B, triangle points). The
dermatologic scores of FAD cats immunized with FSA1 and pVAX vector
were reduced from 1.5 to 0.5 after 7 days of the last immunization.
In contrast, those in the co-immunized with FSA1+pVAX-FSA1 were
reduced promptly after its first immunization and remained at low
level thorough (FIG. 10B, square points).
SUMMARY
[0247] These data demonstrate the successful induction of a FAD
model in feline by flea infestations. Physiological or pathogenic
parameters in the FAD feline have been characterized which can be
used as a model to evaluate treatment of immunotherapeutic or
prophylactic approaches.
[0248] The co-immunization of FSA1+pVAX-FSA1 vaccines was
demonstrated to suppress the established FAD in cats. Such
suppression seems to be an antigenic specific, which supports the
results observed in mouse studies.
Example 18
pVAXI-K-FSA1
[0249] Plasmid pVAX1-K-FSA1 comprises the FSA1 coding sequence
linked to a Kozak sequence in plasmid backbone pVAX (Invitrogen).
The sequence of the K-FSA1 insert is SEQ ID NO:61. Nucleotides 1-9
correspond to the Kozak sequence, the open reading from of FSA1
plus missed 8 amino acids begins at nucleotide 10. A map of the
plasmid pVAX1-K-FSA1 is shown in FIG. 11.
Sequence CWU 1
1
611640DNACtenocephalides felis 1atgaattatt gttttttagt atttttagta
tatttagtat ttgcagttaa tggggaagat 60atttggaaag ttaataaaaa atgtacatca
ggtggaaaaa atcaagatag aaaactcgat 120caaataattc aaaaaggcca
acaagttaaa atccaaaata tttgcaaatt aatacgagat 180aaaccacata
caaatcaaga gaaagaaaaa tgtatgaaat tttgcaaaaa agtttgcaaa
240ggttatagag gagcttgtga tggcaatatt tgctactgca gcaggccaag
taatttaggt 300cctgattgga aagtaagcaa agaatgcaaa gatcccaata
acaaagattc tcgtcctacg 360gaaatagttc catatcgaca acaattagca
attccaaata tttgcaaact aaaaaattca 420gagaccaatg aagattccaa
atgcaaaaaa cattgcaaag aaaaatgtcg tggtggaaat 480gatgctggat
gtgatggaaa cttttgttat tgtcgaccaa aaaataaata ataattataa
540taaataaatt gttatagtta ttagttatcc catcacatat tagaaaagtg
gcttataatt 600tatgaacaat ataacacata aattagttgt gtaaaaaaaa
6402176PRTCtenocephalides felis 2Met Asn Tyr Cys Phe Leu Val Phe
Leu Val Tyr Leu Val Phe Ala Val1 5 10 15Asn Gly Glu Asp Ile Trp Lys
Val Asn Lys Lys Cys Thr Ser Gly Gly 20 25 30Lys Asn Gln Asp Arg Lys
Leu Asp Gln Ile Ile Gln Lys Gly Gln Gln 35 40 45Val Lys Ile Gln Asn
Ile Cys Lys Leu Ile Arg Asp Lys Pro His Thr 50 55 60Asn Gln Glu Lys
Glu Lys Cys Met Lys Phe Cys Lys Lys Val Cys Lys65 70 75 80Gly Tyr
Arg Gly Ala Cys Asp Gly Asn Ile Cys Tyr Cys Ser Arg Pro 85 90 95Ser
Asn Leu Gly Pro Asp Trp Lys Val Ser Lys Glu Cys Lys Asp Pro 100 105
110Asn Asn Lys Asp Ser Arg Pro Thr Glu Ile Val Pro Tyr Arg Gln Gln
115 120 125Leu Ala Ile Pro Asn Ile Cys Lys Leu Lys Asn Ser Glu Thr
Asn Glu 130 135 140Asp Ser Lys Cys Lys Lys His Cys Lys Glu Lys Cys
Arg Gly Gly Asn145 150 155 160Asp Ala Gly Cys Asp Gly Asn Phe Cys
Tyr Cys Arg Pro Lys Asn Lys 165 170 1753416DNACtenocephalides felis
3ggcctggcgg tgctcctgga aaaggatgtt agacgcagcc ctcccaccct gccctactgt
60tgcggccaca gcagattgtg aaatttgccc agccgtgaag agggatgttg acctattcct
120gacgggaacc cccgacgaat atgttgagca agtggcacaa tacaaagcac
tacctgtagt 180attggaaaat gccagaatac tgaagaactg cgttgatgca
aaaatgacag aagaggataa 240ggagaatgct ctcagcttgc tggacaaaat
atacacaagt cctctgtgtt aaaggagcca 300tcactgccag gagccctaag
gaagccactg aactgatcac taagtagtct cagcagcctg 360ccatgtccag
gtgtcttact agaggattcc agcaataaaa gccttgcaat tcaaac
416488PRTCtenocephalides felis 4Met Leu Asp Ala Ala Leu Pro Pro Cys
Pro Thr Val Ala Ala Thr Ala1 5 10 15Asp Cys Glu Ile Cys Pro Ala Val
Lys Arg Asp Val Asp Leu Phe Leu 20 25 30Thr Gly Thr Pro Asp Glu Tyr
Val Glu Gln Val Ala Gln Tyr Lys Ala 35 40 45Leu Pro Val Val Leu Glu
Asn Ala Arg Ile Leu Lys Asn Cys Val Asp 50 55 60Ala Lys Met Thr Glu
Glu Asp Lys Glu Asn Ala Leu Ser Leu Leu Asp65 70 75 80Lys Ile Tyr
Thr Ser Pro Leu Cys 855410DNACtenocephalides felis 5ctgcatcatg
aagggggctc gtgttctcgt gcttctctgg gctgccttgc tcttgatctg 60gggtggaaat
tgtgaaattt gcccagccgt gaagagggat gttgacctat tcctgacggg
120aacccccgac gaatatgttg agcaagtggc acaatacaaa gcactacctg
tagtattgga 180aaatgccaga atactgaaga actgcgttga tgcaaaaatg
acagaagagg ataaggagaa 240tgctctcagc ttgctggaca aaatatacac
aagtcctctg tgttaaagga gccatcactg 300ccaggagccc taaggaagcc
actgaactga tcactaagta gtctcagcag cctgccatgt 360ccaggtgtct
tactagagga ttccagcaat aaaagccttg caattcaaac
410692PRTCtenocephalides felis 6Met Lys Gly Ala Arg Val Leu Val Leu
Leu Trp Ala Ala Leu Leu Leu1 5 10 15Ile Trp Gly Gly Asn Cys Glu Ile
Cys Pro Ala Val Lys Arg Asp Val 20 25 30Asp Leu Phe Leu Thr Gly Thr
Pro Asp Glu Tyr Val Glu Gln Val Ala 35 40 45Gln Tyr Lys Ala Leu Pro
Val Val Leu Glu Asn Ala Arg Ile Leu Lys 50 55 60Asn Cys Val Asp Ala
Lys Met Thr Glu Glu Asp Lys Glu Asn Ala Leu65 70 75 80Ser Leu Leu
Asp Lys Ile Tyr Thr Ser Pro Leu Cys 85 907525DNACtenocephalides
canis 7atgaagaccc tgctcctcac catcggcttc agcctcattg cgatcctgca
ggcccaggat 60accccagcct tgggaaagga cactgtggct gtgtcaggga aatggtatct
gaaggccatg 120acagcagacc aggaggtgcc tgagaagcct gactcagtga
ctcccatgat cctcaaagcc 180cagaaggggg gcaacctgga agccaagatc
accatgctga caaatggtca gtgccagaac 240atcacggtgg tcctgcacaa
aacctctgag cctggcaaat acacggcata cgagggccag 300cgtgtcgtgt
tcatccagcc gtccccggtg agggaccact acattctcta ctgcgagggc
360gagctccatg ggaggcagat ccgaatggcc aagcttctgg gaagggatcc
tgagcagagc 420caagaggcct tggaggattt tcgggaattc tcaagagcca
aaggattgaa ccaggagatt 480ttggaactcg cgcagagcga aacctgctct
ccaggaggac agtag 5258174PRTCtenocephalides canis 8Met Lys Thr Leu
Leu Leu Thr Ile Gly Phe Ser Leu Ile Ala Ile Leu1 5 10 15Gln Ala Gln
Asp Thr Pro Ala Leu Gly Lys Asp Thr Val Ala Val Ser 20 25 30Gly Lys
Trp Tyr Leu Lys Ala Met Thr Ala Asp Gln Glu Val Pro Glu 35 40 45Lys
Pro Asp Ser Val Thr Pro Met Ile Leu Lys Ala Gln Lys Gly Gly 50 55
60Asn Leu Glu Ala Lys Ile Thr Met Leu Thr Asn Gly Gln Cys Gln Asn65
70 75 80Ile Thr Val Val Leu His Lys Thr Ser Glu Pro Gly Lys Tyr Thr
Ala 85 90 95Tyr Glu Gly Gln Arg Val Val Phe Ile Gln Pro Ser Pro Val
Arg Asp 100 105 110His Tyr Ile Leu Tyr Cys Glu Gly Glu Leu His Gly
Arg Gln Ile Arg 115 120 125Met Ala Lys Leu Leu Gly Arg Asp Pro Glu
Gln Ser Gln Glu Ala Leu 130 135 140Glu Asp Phe Arg Glu Phe Ser Arg
Ala Lys Gly Leu Asn Gln Glu Ile145 150 155 160Leu Glu Leu Ala Gln
Ser Glu Thr Cys Ser Pro Gly Gly Gln 165 1709791DNACtenocephalides
canis 9agagctggac ccgtgtgtgt gctggccaat gagccctgga gggtccggct
ccagagtacc 60ctcttggcac agggccgagt ccatcgggac agatgaacct agaggactcc
actgccctcc 120catccacggg gccgggtcac cagactctgc aagtctccag
ctgtcgccaa acccagacag 180aaggtgctgt ggacatgcag ctcctactgc
tgaccgtggg cctggcactg atctgtggcc 240tccaggctca ggagggaaac
catgaggagc cccagggagg cctagaggag ctgtctggga 300ggtggcactc
cgttgccctg gcctccaaca agtccgatct gatcaaaccc tgggggcact
360tcagggtttt catccacagc atgagcgcaa aggacggcaa cctgcacggg
gatatcctta 420taccgcagga cggccagtgc gagaaagtct ccctcactgc
gttcaagact gccaccagca 480acaaatttga cctggagtac tggggacaca
atgacctgta cctggcagag gtagacccca 540agagctacct gattctctac
atgatcaacc agtacaacga tgacaccagc ctggtggctc 600acttgatggt
ccgggacctc agcaggcagc aggacttcct gccggcattc gaatctgtat
660gtgaagacat cggtctgcac aaggaccaga ttgtggttct gagcgatgac
gatcgctgcc 720agggttccag agactagggc ctcagccacg cagagagcca
agcagcagga tctcacctgc 780ctgagtacgg t 79110180PRTCtenocephalides
canis 10Met Gln Leu Leu Leu Leu Thr Val Gly Leu Ala Leu Ile Cys Gly
Leu1 5 10 15Gln Ala Gln Glu Gly Asn His Glu Glu Pro Gln Gly Gly Leu
Glu Glu 20 25 30Leu Ser Gly Arg Trp His Ser Val Ala Leu Ala Ser Asn
Lys Ser Asp 35 40 45Leu Ile Lys Pro Trp Gly His Phe Arg Val Phe Ile
His Ser Met Ser 50 55 60Ala Lys Asp Gly Asn Leu His Gly Asp Ile Leu
Ile Pro Gln Asp Gly65 70 75 80Gln Cys Glu Lys Val Ser Leu Thr Ala
Phe Lys Thr Ala Thr Ser Asn 85 90 95Lys Phe Asp Leu Glu Tyr Trp Gly
His Asn Asp Leu Tyr Leu Ala Glu 100 105 110Val Asp Pro Lys Ser Tyr
Leu Ile Leu Tyr Met Ile Asn Gln Tyr Asn 115 120 125Asp Asp Thr Ser
Leu Val Ala His Leu Met Val Arg Asp Leu Ser Arg 130 135 140Gln Gln
Asp Phe Leu Pro Ala Phe Glu Ser Val Cys Glu Asp Ile Gly145 150 155
160Leu His Lys Asp Gln Ile Val Val Leu Ser Asp Asp Asp Arg Cys Gln
165 170 175Gly Ser Arg Asp 180111099DNADermatophagoides
pteronyssinus 11gaattccttt ttttttcttt ctctctctaa aatctaaaat
ccatccaaca tgaaaattgt 60tttggccatc gcctcattgt tggcattgag cgctgtttat
gctcgtccat catcgatcaa 120aacttttgaa gaatacaaaa aagccttcaa
caaaagttat gctaccttcg aagatgaaga 180agctgcccgt aaaaactttt
tggaatcagt aaaatatgtt caatcaaatg gaggtgccat 240caaccatttg
tccgatttgt cgttggatga attcaaaaac cgatttttga tgagtgcaga
300agcttttgaa cacctcaaaa ctcaattcga tttgaatgct gaaactaacg
cctgcagtat 360caatggaaat gctccagctg aaatcgattt gcgacaaatg
cgaactgtca ctcccattcg 420tatgcaagga ggctgtggtt catgttgggc
tttctctggt gttgccgcaa ctgaatcagc 480ttatttggct taccgtaatc
aatcattgga tcttgctgaa caagaattag tcgattgtgc 540ttcccaacac
ggttgtcatg gtgataccat tccacgtggt attgaataca tccaacataa
600tggtgtcgtc caagaaagct actatcgata cgttgcacga gaacaatcat
gccgacgacc 660aaatgcacaa cgtttcggta tctcaaacta ttgccaaatt
tacccaccaa atgtaaacaa 720aattcgtgaa gctttggctc aaacccacag
cgctattgcc gtcattattg gcatcaaaga 780tttagacgca ttccgtcatt
atgatggccg aacaatcatt caacgcgata atggttacca 840accaaactat
cacgctgtca acattgttgg ttacagtaac gcacaaggtg tcgattattg
900gatcgtacga aacagttggg ataccaattg gggtgataat ggttacggtt
attttgctgc 960caacatcgat ttgatgatga ttgaagaata tccatatgtt
gtcattctct aaacaaaaag 1020acaatttctt atatgattgt cactaattta
tttaaaatca aaatttttag aaaatgaata 1080aattcattca caaaaatta
109912320PRTEscherichia coli 12Met Lys Ile Val Leu Ala Ile Ala Ser
Leu Leu Ala Leu Ser Ala Val1 5 10 15Tyr Ala Arg Pro Ser Ser Ile Lys
Thr Phe Glu Glu Tyr Lys Lys Ala 20 25 30Phe Asn Lys Ser Tyr Ala Thr
Phe Glu Asp Glu Glu Ala Ala Arg Lys 35 40 45Asn Phe Leu Glu Ser Val
Lys Tyr Val Gln Ser Asn Gly Gly Ala Ile 50 55 60Asn His Leu Ser Asp
Leu Ser Leu Asp Glu Phe Lys Asn Arg Phe Leu65 70 75 80Met Ser Ala
Glu Ala Phe Glu His Leu Lys Thr Gln Phe Asp Leu Asn 85 90 95Ala Glu
Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala Pro Ala Glu Ile 100 105
110Asp Leu Arg Gln Met Arg Thr Val Thr Pro Ile Arg Met Gln Gly Gly
115 120 125Cys Gly Ser Cys Trp Ala Phe Ser Gly Val Ala Ala Thr Glu
Ser Ala 130 135 140Tyr Leu Ala Tyr Arg Asn Gln Ser Leu Asp Leu Ala
Glu Gln Glu Leu145 150 155 160Val Asp Cys Ala Ser Gln His Gly Cys
His Gly Asp Thr Ile Pro Arg 165 170 175Gly Ile Glu Tyr Ile Gln His
Asn Gly Val Val Gln Glu Ser Tyr Tyr 180 185 190Arg Tyr Val Ala Arg
Glu Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg 195 200 205Phe Gly Ile
Ser Asn Tyr Cys Gln Ile Tyr Pro Pro Asn Val Asn Lys 210 215 220Ile
Arg Glu Ala Leu Ala Gln Thr His Ser Ala Ile Ala Val Ile Ile225 230
235 240Gly Ile Lys Asp Leu Asp Ala Phe Arg His Tyr Asp Gly Arg Thr
Ile 245 250 255Ile Gln Arg Asp Asn Gly Tyr Gln Pro Asn Tyr His Ala
Val Asn Ile 260 265 270Val Gly Tyr Ser Asn Ala Gln Gly Val Asp Tyr
Trp Ile Val Arg Asn 275 280 285Ser Trp Asp Thr Asn Trp Gly Asp Asn
Gly Tyr Gly Tyr Phe Ala Ala 290 295 300Asn Ile Asp Leu Met Met Ile
Glu Glu Tyr Pro Tyr Val Val Ile Leu305 310 315 32013717DNAArachis
hypogaea 13gctcaccata ctagtagccc tcgccctttt cctcctcgct gcccacgcat
ctgcgaggca 60gcagtgggaa ctccaaggag acagaagatg ccagagccag ctcgagaggg
cgaacctgag 120gccctgcgag caacatctca tgcagaagat ccaacgtgac
gaggattcat atgaacggga 180cccgtacagc cctagtcagg atccgtacag
ccctagtcca tatgatcgga gaggcgctgg 240atcctctcag caccaagaga
ggtgttgcaa tgagctgaac gagtttgaga acaaccaaag 300gtgcatgtgc
gaggcattgc aacagatcat ggagaaccag agcgataggt tgcaggggag
360gcaacaggag caacagttca agagggagct caggaacttg cctcaacagt
gcggccttag 420ggcaccacag cgttgcgact tggacgtcga aagtggcggc
agagacagat actaaacacc 480tatctcaaaa aaagaaaaga aaagaaaaga
aaatagctta tatataagct attatctatg 540gttatgttta gttttggtaa
taataaagat catcactata tgaatgtgtt gatcgtgtta 600actaaggcaa
gcttaggtta tatgagcacc tttagagtgc ttttatggcg ttgtctatgt
660tttgttgctg cagagttgta accatcttga aataatataa aaagatcatg ttttgtt
71714766DNAArachis hypogaea 14agaaagagaa gacaagatgt cgtggcaaac
ctacgtcgat aaccaccttc tctgcgaaat 60tgaaggcgac cacctctcct ccgccgcaat
cctcggccaa gacggcggtg tttgggctca 120gagctctcat ttccctcagt
tcaagcctga ggaaattact gctatcatga acgactttgc 180tgagcctgga
tcgctcgccc ctaccgggtt gtacctcggt ggcaccaaat acatggttat
240ccaaggtgaa cccggagcta tcattccagg gaagaagggt cctggtggtg
ttaccattga 300gaagacgaat caggcgttaa tcatcggaat ctacgataag
ccaatgactc cggggcagtg 360caacatgatt gttgaaaggc tgggtgatta
tctcattgat acgggtcttt aagtcctctt 420tgttatttct tgttatctgc
ttgcttattt cactggctcc tatacgaggc ttcgcatcga 480tgtgccaaga
gaatgctcga ttgtagtgta ataatattaa ttgatgggta ttcaaaagtc
540atgggatctg cgtctaggga agaagttatg gtgcttgaga agtgaatgat
aactatcatc 600tctgttgttg tgctttttag cgggtatctg tatacaattt
acaagtggtt ttaatgctgt 660gggcataaat gggcattaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaa 76615131PRTArachis hypogaea 15Met Ser
Trp Gln Thr Tyr Val Asp Asn His Leu Leu Cys Glu Ile Glu1 5 10 15Gly
Asp His Leu Ser Ser Ala Ala Ile Leu Gly Gln Asp Gly Gly Val 20 25
30Trp Ala Gln Ser Ser His Phe Pro Gln Phe Lys Pro Glu Glu Ile Thr
35 40 45Ala Ile Met Asn Asp Phe Ala Glu Pro Gly Ser Leu Ala Pro Thr
Gly 50 55 60Leu Tyr Leu Gly Gly Thr Lys Tyr Met Val Ile Gln Gly Glu
Pro Gly65 70 75 80Ala Ile Ile Pro Gly Lys Lys Gly Pro Gly Gly Val
Thr Ile Glu Lys 85 90 95Thr Asn Gln Ala Leu Ile Ile Gly Ile Tyr Asp
Lys Pro Met Thr Pro 100 105 110Gly Gln Cys Asn Met Ile Val Glu Arg
Leu Gly Asp Tyr Leu Ile Asp 115 120 125Thr Gly Leu
130161295DNACryptomeria japonica 16aatctgctca taatcatagc atagccgtat
agaaagaaat tctacactct gctaccaaaa 60aatggattcc ccttgcttag tagcattact
ggttttctct tttgtaattg gatcttgctt 120ttctgataat cccatagaca
gctgctggag aggagactca aactgggcac aaaacagaat 180gaagctcgca
gattgtgcag tgggcttcgg aagctccacc atgggaggca agggaggaga
240tctttatacg gtcacgaact cagatgacga ccctgtgaat cctgcaccag
gaactctgcg 300ctatggagca acccgagata ggcccctgtg gataattttc
agtgggaata tgaatataaa 360gctcaaaatg cctatgtaca ttgctgggta
taagactttt gatggcaggg gagcacaagt 420ttatattggc aatggcggtc
cctgtgtgtt tatcaagaga gttagcaatg ttatcataca 480cggtttgtat
ctgtacggct gtagtactag tgttttgggg aatgttttga taaacgagag
540ttttggggtg gagcctgttc atcctcagga tggcgatgct cttactctgc
gcactgctac 600aaatatttgg attgatcata attctttctc caattcttct
gatggtctgg tcgatgtcac 660tcttacttcg actggagtta ctatttcaaa
caatcttttt ttcaaccatc ataaagtgat 720gttgttaggg catgatgatg
catatagtga tgacaaatcc atgaaggtga cagtggcgtt 780caatcaattt
ggacctaact gtggacaaag aatgcccagg gcacgatatg gacttgtaca
840tgttgcaaac aataattatg acccatggac tatatatgca attggtggga
gttcaaatcc 900aaccattcta agtgaaggga atagtttcac tgcaccaaat
gagagctaca agaagcaagt 960aaccatacgt attggatgca aaacatcatc
atcttgttca aattgggtgt ggcaatctac 1020acaagatgtt ttttataatg
gagcttattt tgtatcatca gggaaatatg aagggggtaa 1080tatatacaca
aagaaagaag ctttcaatgt tgagaatggg aatgcaactc ctcaattgac
1140aaaaaatgct ggggttttaa catgctctct ctctaaacgt tgttgatgat
gcatatattc 1200tagcatgttg tactatctaa attaacatca acaagaaata
tatcatgatg tatattgttg 1260tattgatgtc aaaataaaaa tgttctttta ctatt
129517374PRTCryptomeria japonica 17Met Asp Ser Pro Cys Leu Val Ala
Leu Leu Val Phe Ser Phe Val Ile1 5 10 15Gly Ser Cys Phe Ser Asp Asn
Pro Ile Asp Ser Cys Trp Arg Gly Asp 20 25 30Ser Asn Trp Ala Gln Asn
Arg Met Lys Leu Ala Asp Cys Ala Val Gly 35 40 45Phe Gly Ser Ser Thr
Met Gly Gly Lys Gly Gly Asp Leu Tyr Thr Val 50 55 60Thr Asn Ser Asp
Asp Asp Pro Val Asn Pro Ala Pro Gly Thr Leu Arg65 70 75 80Tyr Gly
Ala Thr Arg Asp Arg Pro Leu Trp Ile Ile Phe Ser Gly Asn 85 90 95Met
Asn Ile Lys Leu Lys Met Pro Met Tyr Ile Ala Gly Tyr Lys Thr 100 105
110Phe Asp Gly
Arg Gly Ala Gln Val Tyr Ile Gly Asn Gly Gly Pro Cys 115 120 125Val
Phe Ile Lys Arg Val Ser Asn Val Ile Ile His Gly Leu Tyr Leu 130 135
140Tyr Gly Cys Ser Thr Ser Val Leu Gly Asn Val Leu Ile Asn Glu
Ser145 150 155 160Phe Gly Val Glu Pro Val His Pro Gln Asp Gly Asp
Ala Leu Thr Leu 165 170 175Arg Thr Ala Thr Asn Ile Trp Ile Asp His
Asn Ser Phe Ser Asn Ser 180 185 190Ser Asp Gly Leu Val Asp Val Thr
Leu Thr Ser Thr Gly Val Thr Ile 195 200 205Ser Asn Asn Leu Phe Phe
Asn His His Lys Val Met Leu Leu Gly His 210 215 220Asp Asp Ala Tyr
Ser Asp Asp Lys Ser Met Lys Val Thr Val Ala Phe225 230 235 240Asn
Gln Phe Gly Pro Asn Cys Gly Gln Arg Met Pro Arg Ala Arg Tyr 245 250
255Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Trp Thr Ile Tyr
260 265 270Ala Ile Gly Gly Ser Ser Asn Pro Thr Ile Leu Ser Glu Gly
Asn Ser 275 280 285Phe Thr Ala Pro Asn Glu Ser Tyr Lys Lys Gln Val
Thr Ile Arg Ile 290 295 300Gly Cys Lys Thr Ser Ser Ser Cys Ser Asn
Trp Val Trp Gln Ser Thr305 310 315 320Gln Asp Val Phe Tyr Asn Gly
Ala Tyr Phe Val Ser Ser Gly Lys Tyr 325 330 335Glu Gly Gly Asn Ile
Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu Asn 340 345 350Gly Asn Ala
Thr Pro Gln Leu Thr Lys Asn Ala Gly Val Leu Thr Cys 355 360 365Ser
Leu Ser Lys Arg Cys 370181313DNACryptomeria japonica 18tagcatagcc
gtatagaaag aaattctaca ctctgctacc aaaaaatgga ttccccttgc 60ttagtagcat
tactggtttt ctcttttgta attggatctt gcttttctga taatcccata
120gacagctgct ggagaggaga ctcaaactgg gcacaaaaca gaatgaagct
cgcagattgt 180gcagtgggct tcggaagctc caccatggga ggcaagggag
gagatcttta tacggtcacg 240aactcagatg acgaccctgt gaatcctgca
ccaggaactc tgcgctatgg agcaacccga 300gataggcccc tgtggataat
tttcagtggg aatatgaata taaagctcaa aatgcctatg 360tacattgctg
ggtataagac ttttgatggc aggggagcac aagtttatat tggcaatggc
420ggtccctgtg tgtttatcaa gagagttagc aatgttatca tacacggttt
gtatctgtac 480ggctgtagta ctagtgtttt ggggaatgtt ttgataaacg
agagttttgg ggtggagcct 540gttcatcctc aggatggcga tgctcttact
ctgcgcactg ctacaaatat ttggattgat 600cataattctt tctccaattc
ttctgatggt ctggtcgatg tcactcttac ttcgactgga 660gttactattt
caaacaatct ttttttcaac catcataaag tgatgttgtt agggcatgat
720gatgcatata gtgatgacaa atccatgaag gtgacagtgg cgttcaatca
atttggacct 780aactgtggac aaagaatgcc cagggcacga tatggacttg
tacatgttgc aaacaataat 840tatgacccat ggactatata tgcaattggt
gggagttcaa atccaaccat tctaagtgaa 900gggaatagtt tcactgcacc
aaatgagagc tacaagaagc aagtaaccat acgtattgga 960tgcaaaacat
catcatcttg ttcaaattgg gtgtggcaat ctacacaaga tgttttttat
1020aatggagctt attttgtatc atcagggaaa tatgaagggg gtaatatata
cacaaagaaa 1080gaagctttca atgttgagaa tgggaatgca actcctcaat
tgacaaaaaa tgctggggtt 1140ttaacatgct ctctctctaa acgttgttga
tgatgcatat attctagcat gttgtactat 1200ctaaattaac atcaacaaga
aatatatcat gatgtatatt gttgtattga tgtcaaaata 1260aaaatgtatc
ttttactatt tatcaacatg ttatctttga tgtgcaagtt aat
131319374PRTCryptomeria japonica 19Met Asp Ser Pro Cys Leu Val Ala
Leu Leu Val Phe Ser Phe Val Ile1 5 10 15Gly Ser Cys Phe Ser Asp Asn
Pro Ile Asp Ser Cys Trp Arg Gly Asp 20 25 30Ser Asn Trp Ala Gln Asn
Arg Met Lys Leu Ala Asp Cys Ala Val Gly 35 40 45Phe Gly Ser Ser Thr
Met Gly Gly Lys Gly Gly Asp Leu Tyr Thr Val 50 55 60Thr Asn Ser Asp
Asp Asp Pro Val Asn Pro Ala Pro Gly Thr Leu Arg65 70 75 80Tyr Gly
Ala Thr Arg Asp Arg Pro Leu Trp Ile Ile Phe Ser Gly Asn 85 90 95Met
Asn Ile Lys Leu Lys Met Pro Met Tyr Ile Ala Gly Tyr Lys Thr 100 105
110Phe Asp Gly Arg Gly Ala Gln Val Tyr Ile Gly Asn Gly Gly Pro Cys
115 120 125Val Phe Ile Lys Arg Val Ser Asn Val Ile Ile His Gly Leu
Tyr Leu 130 135 140Tyr Gly Cys Ser Thr Ser Val Leu Gly Asn Val Leu
Ile Asn Glu Ser145 150 155 160Phe Gly Val Glu Pro Val His Pro Gln
Asp Gly Asp Ala Leu Thr Leu 165 170 175Arg Thr Ala Thr Asn Ile Trp
Ile Asp His Asn Ser Phe Ser Asn Ser 180 185 190Ser Asp Gly Leu Val
Asp Val Thr Leu Thr Ser Thr Gly Val Thr Ile 195 200 205Ser Asn Asn
Leu Phe Phe Asn His His Lys Val Met Leu Leu Gly His 210 215 220Asp
Asp Ala Tyr Ser Asp Asp Lys Ser Met Lys Val Thr Val Ala Phe225 230
235 240Asn Gln Phe Gly Pro Asn Cys Gly Gln Arg Met Pro Arg Ala Arg
Tyr 245 250 255Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Trp
Thr Ile Tyr 260 265 270Ala Ile Gly Gly Ser Ser Asn Pro Thr Ile Leu
Ser Glu Gly Asn Ser 275 280 285Phe Thr Ala Pro Asn Glu Ser Tyr Lys
Lys Gln Val Thr Ile Arg Ile 290 295 300Gly Cys Lys Thr Ser Ser Ser
Cys Ser Asn Trp Val Trp Gln Ser Thr305 310 315 320Gln Asp Val Phe
Tyr Asn Gly Ala Tyr Phe Val Ser Ser Gly Lys Tyr 325 330 335Glu Gly
Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu Asn 340 345
350Gly Asn Ala Thr Pro Gln Leu Thr Lys Asn Ala Gly Val Leu Thr Cys
355 360 365Ser Leu Ser Lys Arg Cys 37020537DNABlomia tropicalis
20aaaacactca caatccacaa actcaaacaa caatgaagtt cgccatcgtt cttattgcct
60gctttgccgc ttcggttttg gctcaagagc acaagccaaa gaaggatgat ttccgaaacg
120aattcgatca cttgttgatc gaacaggcaa accatgctat cgaaaaggga
gaacatcaat 180tgctttactt gcaacaccaa ctcgacgaat tgaatgaaaa
caagagcaag gaattgcaag 240agaaaatcat tcgagaactt gatgttgttt
gcgccatgat cgaaggagcc caaggagctt 300tggaacgtga attgaagcga
actgatctta acattttgga acgattcaac tacgaagagg 360ctcaaactct
cagcaagatc ttgcttaagg atttgaagga aaccgaacaa aaagtgaagg
420atattcaaac ccaataaaaa tttagaattg tacaatttta catttttgat
atgattaaat 480gtcaataaat gttcaataaa taaattcaat ttttaactat
aaaaaaaaaa aaaaaaa 53721134PRTBlomia tropicalis 21Met Lys Phe Ala
Ile Val Leu Ile Ala Cys Phe Ala Ala Ser Val Leu1 5 10 15Ala Gln Glu
His Lys Pro Lys Lys Asp Asp Phe Arg Asn Glu Phe Asp 20 25 30His Leu
Leu Ile Glu Gln Ala Asn His Ala Ile Glu Lys Gly Glu His 35 40 45Gln
Leu Leu Tyr Leu Gln His Gln Leu Asp Glu Leu Asn Glu Asn Lys 50 55
60Ser Lys Glu Leu Gln Glu Lys Ile Ile Arg Glu Leu Asp Val Val Cys65
70 75 80Ala Met Ile Glu Gly Ala Gln Gly Ala Leu Glu Arg Glu Leu Lys
Arg 85 90 95Thr Asp Leu Asn Ile Leu Glu Arg Phe Asn Tyr Glu Glu Ala
Gln Thr 100 105 110Leu Ser Lys Ile Leu Leu Lys Asp Leu Lys Glu Thr
Glu Gln Lys Val 115 120 125Lys Asp Ile Gln Thr Gln
1302215PRTArtificial Sequenceartificial pep66 peptide; engineered
de novo 22Gln Glu Lys Glu Lys Cys Met Lys Phe Cys Lys Lys Val Cys
Lys1 5 10 152315PRTArtificial Sequenceartificial peptide named
pep100; engineered de novo 23Gly Pro Asp Trp Lys Val Ser Lys Glu
Cys Lys Asp Pro Asn Asn1 5 10 152445DNAArtificial
Sequenceartificial primer sequence named FAD66 primer; engineered
de novo 24caagagaaag aaaaatgtat gaaattttgc aaaaaagttt gcaaa
452545DNAArtificial Sequenceartificial primer sequence named FAD100
primer; engineered de novo 25ggtcctgatt ggaaagtaag caaagaatgc
aaagatccca ataac 452686DNAArtificial Sequenceartificial primer
sequence named P66F Primer; engineered de novo 26aagcttgcca
tgcaagagaa agaaaaatgt atgaaatttt gcaaaaaagt ttgcaaaggt 60accgccatgg
tgagcaaggg cgagga 862730DNAArtificial Sequenceartificial primer
sequence named PR Primer; engineered de novo 27ttaggtacct
tacttgtaca gctcgtccat 302886DNAArtificial Sequenceartificial primer
sequence named P100F primer; engineered de novo 28aagcttgcca
tgggtcctga ttggaaagta agcaaagaat gcaaagatcc caataacggt 60accgccatgg
tgagcaaggg cgagga 862925DNAArtificial Sequenceartificial primer
sequence named HPRT 5' Primer; engineered de novo 29gttggataca
ggccagactt tgttg 253022DNAArtificial Sequenceartificial primer
sequence named HPRT 3' primer; engineered de novo 30gagggtaggc
tggcctatgg ct 223122DNAArtificial Sequenceartificial primer
sequence named IFN-gamma 5' primer; engineered de novo 31cattgaaagc
ctagaaagtc tg 223223DNAArtificial Sequenceartificial primer
sequence named IFN-gamma 3' primer; engineered de novo 32ctcatggaat
gcatcctttt tcg 233322DNAArtificial Sequenceartificial primer
sequence named IL-4 5' primer; engineered de novo 33gaaagagacc
ttgacacagc tg 223422DNAArtificial Sequenceartificial primer
sequence named IL-4 3' primer; engineered de novo 34gaactcttgc
aggtaatcca gg 223524DNAArtificial Sequenceartificial primer
sequence named IL-10 5' primer; engineered de novo 35ccagtttacc
tggtagaagt gatg 243630DNAArtificial Sequenceartificial primer
sequence named IL-10 3' primer; engineered de novo 36tgtctaggtc
ctggagtcca gcagactcaa 303719DNAArtificial Sequenceartificial primer
sequence named Fe1 d I.1 P1 5' primer; engineered de novo
37aagcttggat gttagacgc 193820DNAArtificial Sequenceartificial
primer sequence named Fe1 d I.1 P2 3' primer; engineered de novo
38ggtaccttaa cacagaggac 203921DNAArtificial Sequenceartificial
primer sequence named Fe1 d I.2 P1 5' primer; engineered de novo
39aagcttggat gaagggggct c 214020DNAArtificial Sequenceartificial
primer sequence named Fe1 d I.2 P2 3' primer; engineered de novo
40ggtaccttaa cacagaggac 204126DNAArtificial Sequenceartificial
primer sequence named Can f 1 P1 5' primer; engineered de novo
41aagcttatga agaccctgct cctcac 264226DNAArtificial
Sequenceartificial primer sequence named Can f 1 P2 3' primer;
engineered de novo 42ggtaccctac tgtcctcctg gagagc
264323DNAArtificial Sequenceartificial primer sequence named Can f
2 P1 5' primer; engineered de novo 43aagcttatgc agctcctact gct
234422DNAArtificial Sequenceartificial primer sequence named Can f
2 P2 3' primer; engineered de novo 44ggtaccctag tctctggaac cc
224525DNAArtificial Sequenceartificial primer sequence named Der p
1 P1 5' primer; engineered de novo 45aagcttaaca tgaaaattgt tttgg
254625DNAArtificial Sequenceartificial primer sequence named Der p
1 P2 3' primer; engineered de novo 46ggtaccgttt agagaatgac aacat
254720DNAArtificial Sequenceartificial primer sequence named Ara h
II P1 5' primer; engineered de novo 47aagcttctca tgcagaagat
204820DNAArtificial Sequenceartificial primer sequence named Ara h
II P2 3' primer; engineered de novo 48ggtaccttag tatctgtctc
204920DNAArtificial Sequenceartificial primer sequence named Ara h
5 P1 5' primer; engineered de novo 49aagcttatgt cgtggcaaac
205020DNAArtificial Sequenceartificial primer sequence named Ara h
5 P2 3' primer; engineered de novo 50ggtacctaaa gacccgtatc
205125DNAArtificial Sequenceartificial primer sequence named Cry j
l. 1 P1 5' primer; engineered de novo 51aagcttatgg attccccttg cttat
255223DNAArtificial Sequenceartificial primer sequence named Cry j
l. 1 P2 3' primer; engineered de novo 52ggtaccatca acaacgttta gag
235325DNAArtificial Sequenceartificial primer sequence named Cry j
l.2 P1 5' primer; engineered de novo 53aagcttatgg attccccttg cttag
255426DNAArtificial Sequenceartificial primer sequence named Cry j
l.2 P2 3' primer; engineered de novo 54ggtacctcaa caacgtttag agagag
265520DNAArtificial Sequenceartificial primer sequence named Blo t
5 P1 5' primer; engineered de novo 55aagcttacaa tgaagttcgc
205620DNAArtificial Sequenceartificial primer sequence named Blo t
5 P2 3' primer; engineered de novo 56ggtaccaatt tttattgggt
205721DNAArtificial Sequenceartificial primer sequence named Gata 3
5' primer; engineered de novo 57ggaggcatcc agacccgaaa c
215819DNAArtificial Sequenceartificial primer sequence named GATA 3
3' primer; engineered de novo 58accatggcgg tgaccatgc
195920DNAArtificial Sequenceartificial primer sequence named T-bet
5' primer; engineered de novo 59tgaagcccac actcctaccc
206020DNAArtificial Sequenceartificial primer sequence named T-bet
3' primer; engineered de novo 60gcggcatttt ctcagttggg
2061489DNAArtificial SequenceK-FSA1 insert which includes FSA
coding sequence linked to a Kozak sequence 61gccgccacca tggaagatat
ttggaaagtt aataaaaaat gtacatcagg tggaaaaaat 60caagatagaa aactcgatca
aataattcaa aaaggccaac aagttaaaat ccaaaatatt 120tgcaaattaa
tacgggataa accacataca aatcaagaga aagaaaaatg tatgaaattt
180agcaaaaaag tttgcaaagg ttatagagga gcttgtgatg gcaatatttg
ctactgcagc 240aggccaagta atttaggtcc tgattggaaa gtaagcaaag
aatgcaaaga tcccaataac 300aaagattctc ggcctacgga aatagttcca
tatcggcagc aattagcaat tccaaatatt 360tgcaaactaa aaaattcagg
gaccaatgaa gattccaaat gcaaaaaaca ttgcaaagaa 420aaatgtcgtg
gtggaaatga tgctggatgt gatggaaact tttgttattg tcggccaaaa 480aataaataa
489
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