U.S. patent application number 13/123510 was filed with the patent office on 2016-08-25 for recombinant protein bodies as immunogen-specific adjuvants.
This patent application is currently assigned to Era Biotech, S.A.. The applicant listed for this patent is Francois Devesa, Inga Isabel Hitzeroth, Pablo Marzabal Luna, Ann Elizabeth Meyers, Peter Ohlschlager, Edward Peter Rybicki. Invention is credited to Francois Devesa, Inga Isabel Hitzeroth, Pablo Marzabal Luna, Ann Elizabeth Meyers, Peter Ohlschlager, Edward Peter Rybicki.
Application Number | 20160243213 13/123510 |
Document ID | / |
Family ID | 41611154 |
Filed Date | 2016-08-25 |
United States Patent
Application |
20160243213 |
Kind Code |
A9 |
Rybicki; Edward Peter ; et
al. |
August 25, 2016 |
Recombinant Protein Bodies as Immunogen-Specific Adjuvants
Abstract
An immunogen-specific is adjuvant for a vaccine or inoculum is
disclosed. The adjuvant is comprised of particulate recombinant
protein body-like assemblies (RPBLAs) that contain a recombinant
fusion protein that contains two portions peptide-linked together.
A first portion is a protein body-inducing sequence (PBIS) and a
second portion is a T-cell stimulating immunogenic polypeptide
whose sequence is that of a pathogenic polypeptide sequence present
in or induced by a vaccine or inoculum. The adjuvant, when used as
an inoculum in a host animal without a prior priming vaccination or
inoculation, does not induce production of antibodies or T cell
activation to the pathogenic sequence.
Inventors: |
Rybicki; Edward Peter;
(Pinelands, ZA) ; Meyers; Ann Elizabeth;
(Plumstead, ZA) ; Devesa; Francois; (Launaguet,
FR) ; Marzabal Luna; Pablo; (Barcelona, ES) ;
Hitzeroth; Inga Isabel; (Cape Town, ZA) ;
Ohlschlager; Peter; (Wurselen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rybicki; Edward Peter
Meyers; Ann Elizabeth
Devesa; Francois
Marzabal Luna; Pablo
Hitzeroth; Inga Isabel
Ohlschlager; Peter |
Pinelands
Plumstead
Launaguet
Barcelona
Cape Town
Wurselen |
|
ZA
ZA
FR
ES
ZA
DE |
|
|
Assignee: |
Era Biotech, S.A.
Barcelona
ES
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20110262478 A1 |
October 27, 2011 |
|
|
Family ID: |
41611154 |
Appl. No.: |
13/123510 |
Filed: |
October 9, 2009 |
PCT Filed: |
October 9, 2009 |
PCT NO: |
PCT/EP2009/063223 PCKC 00 |
371 Date: |
July 5, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11709527 |
Feb 22, 2007 |
8163880 |
|
|
13123510 |
|
|
|
|
60776391 |
Feb 23, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/55516
20130101; A61P 43/00 20180101; A61P 31/18 20180101; A61K 39/21
20130101; A61K 2039/5258 20130101; A61K 2039/572 20130101; A61K
39/12 20130101; C12N 15/625 20130101; C12N 15/8258 20130101; C12N
7/00 20130101; C07K 14/425 20130101; A61K 2039/53 20130101; A61K
2039/6031 20130101; A61K 2039/575 20130101; C12N 2740/16022
20130101; C12N 2740/16034 20130101; A61K 2039/585 20130101; C12N
15/8257 20130101; C12N 2740/16322 20130101; C12N 2710/20071
20130101; A61P 31/20 20180101; C07K 14/415 20130101; A61P 37/04
20180101; C12N 2740/16122 20130101; C12N 2740/16234 20130101; C07K
14/005 20130101; C07K 2319/04 20130101; C12N 2710/20022 20130101;
C12N 15/8218 20130101; A61K 2039/54 20130101; C12N 15/8216
20130101; C12N 2710/20034 20130101; A61K 39/385 20130101; C12N
2740/16222 20130101 |
International
Class: |
A61K 39/12 20060101
A61K039/12; C07H 21/04 20060101 C07H021/04; A61K 39/39 20060101
A61K039/39; A61K 39/21 20060101 A61K039/21; C12N 7/00 20060101
C12N007/00; A61P 31/20 20060101 A61P031/20; A61P 31/18 20060101
A61P031/18; A61P 37/04 20060101 A61P037/04; A61K 39/385 20060101
A61K039/385; A61K 39/00 20060101 A61K039/00; C07K 19/00 20060101
C07K019/00 |
Claims
1. A method for inducing a T-cell mediated immune response in a
subject in need thereof against an immunogenic peptide, the method
comprising administration to a subject in need thereof of a vaccine
comprising (i) a particulate recombinant protein body-like assembly
(RPBLA) that comprises a recombinant fusion protein, said
recombinant fusion protein comprising a protein body-inducing
sequence (PBIS) and an immunogenic polypeptide or (ii) a nucleic
acid molecule that encodes a recombinant fusion protein, said
recombinant fusion protein comprising a protein body-inducing
sequence (PBIS) and an immunogenic polypeptide.
2. The method as defined in claim 1 wherein the PBIS comprises a
prolamin.
3. The method as defined in claim 2 wherein the prolamin is
selected from the group consisting of ganuna-zein, alpha-zein,
delta-zein, beta-zein, rice prolamin and gamma-gliadin.
4. The method as defined in claim 1 wherein the PBIS sequence
further comprises a signal peptide sequence.
5. The method as defined in claim 1 wherein the immunogenic
polypeptide sequence is selected from the group consisting of (i) a
polypeptide encoded by the HPV E7 gene, (ii) a polypeptide encoded
by the HIV-1 gag, gene and (iii) a polypeptide encoded by the HIV-1
p01 gene.
6. (canceled)
7. The method as defined in claim 1 wherein the administration is
preceded by administration of a priming vaccination or inoculation
using a composition comprising the immunogenic polypeptide or a
nucleic acid encoding the immunogenic polypeptide.
8. The method as defined in claim 7 wherein the composition
comprising the immunogenic polypeptide used in the priming
vaccination or inoculation is selected from the group consisting of
(i) a particulate recombinant protein body-like assembly (RPBLA)
comprising a recombinant fusion protein, said recombinant fusion
protein comprising a protein body-inducing sequence (PBIS) and an
immunogenic polypeptide, (ii) a: nucleic acid molecule that encodes
the immunogenic polypeptide and (iii) a nucleic acid molecule that
encodes a recombinant fusion protein, said recombinant fusion
protein containing a protein body-inducing sequence (PBIS) and the
immunogenic polypeptide.
9. The method as defined in claim 1 wherein the vaccine is
administered intramuscularly.
10. An immunogen-specific adjuvant for a vaccine or inoculum
comprising a particulate recombinant protein body-like assembly
(RPBLA) comprising a recombinant fusion protein, said recombinant
fusion protein comprising a protein body-inducing sequence (PBIS)
and a T-cell stimulating immunogenic polypeptide whose sequence is
that of a pathogenic polypeptide sequence present in or induced by
a vaccine or inoculum, said adjuvant at the concentration used in
an inoculum without a prior priming vaccination or inoculation not
inducing production of antibodies or T cell activation to the
pathogenic sequence.
11. The immunogen-specific adjuvant according to claim 10, whose
PBIS comprises a prolamin.
12. The immunogen-specific adjuvant according to claim 10, whose
PBIS further comprises a signal peptide.
13. The immunogen-specific adjuvant according to claim 11, wherein
said prolamin is selected from the group consisting of gamma-zein,
alpha-zein, delta-zein, beta-zein, rice prolamin and
gamma-gliadin.
14. The immunogen-specific adjuvant according to claim 10, wherein
said T-cell stimulating immunogenic polypeptide sequence is encoded
by the HPV E7 gene.
15. The immunogen-specific adjuvant according to claim 10, wherein
said T-cell stimulating immunogenic polypeptide sequence is present
in HIV-1.
16. The immunogen-specific adjuvant according to claim 15, wherein
the polypeptide present in HIV-1 is encoded by the HIV-1 gag
gene.
17. (canceled)
18. An adjuvant composition comprising the adjuvant according to
claim 10 present in an adjuvant-effective amount dissolved or
dispersed in a pharmaceutically acceptable diluent.
19. The adjuvant composition according to claim 18, wherein the
diluent is aqueous-based.
20. An immunogen-specific adjuvant for a vaccine or inoculum that
is comprised of a nucleic acid molecule that encodes a recombinant
fusion protein, said recombinant fusion protein comprising a
protein body-inducing sequence (PBIS) and a T-cell stimulating
immunogenic polypeptide whose sequence is that of a pathogenic
polypeptide sequence present in or induced by a vaccine or
inoculum, said adjuvant when used as an inoculum without a prior
priming vaccination or inoculation not inducing production of
antibodies or T cell activation to the pathogenic sequence.
21. The immunogen-specific adjuvant according to claim 20, wherein
the PBIS comprises a prolamin.
22. The immunogen-specific adjuvant according to claim 20, wherein
the PBIS further comprises a signal peptide.
23. The immunogen-specific adjuvant according to claim 21, wherein
said prolamin selected from the group consisting of gamma-zein,
alpha-zein, delta-zein; beta-zein, rice prolamin and
gamma-gliadin.
24. A vaccine or inoculum comprising a nucleic acid molecule that
encodes a recombinant fusion protein, said recombinant fusion
protein comprising a protein body-inducing sequence (PBIS) and an
immunogenic polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of priority of U.S.
Application Ser. No. 61/104,403 filed on 10 Oct. 2008, whose
disclosures are incorporated by reference.
TECHNICAL FIELD
[0002] The present invention provides an immunogen-specific
adjuvant for a vaccine or inoculum. More specifically, the
invention provides a vaccine or inoculum adjuvant comprising
recombinant protein body-like assemblies (RPBLAs) that contain a
recombinant fusion protein. The recombinant fusion protein contains
two sequences that are peptide-linked together in which one
sequence is a protein body-inducing sequence (PBIS) and the other
is a T-cell stimulatory polypeptide that corresponds to a portion
of a pathogenic polypeptide sequence present in or encoded by a
vaccine or inoculum.
BACKGROUND ART
[0003] Protein bodies (PBs) are subcellular organelles (or large
vesicles, about 1-3 microns in diameter, surrounded by a membrane)
that specialize in protein accumulation. They are naturally formed
in some specific plant tissues, like seeds, and serve as principal
source of amino acids for germination and seedling growth.
[0004] The storage proteins are co-translationally inserted into
the lumen of the endoplasmic reticulum (ER) via a signal peptide to
be packaged either in the ER or into the vacuoles (Galili et al.,
1993 Trends Cell Biol. 3:437-443) and assembled into multimeric
units inside these subcellular compartments, developing specific
organelles called (ER)-derived protein bodies (PBs) or protein
storage vacuoles (PSV) (Okita and Rogers, 1996 Annu. Rev. Plant
Physiol Mol. Biol. 47:327-350; Herman and Larkins, 1999 Plant Cell
11:601-613; Sanderfoot and Raikel, 1999 Plant Cell 11:629-642).
[0005] The storage proteins dicotiledoneous plants are primarily
soluble proteins such as the 7S globulin or vicilin type, 11S
globulins or legumin-type proteins and are sequestered in PSVs
together with other proteins (i.e., protease inhibitors,
proteolytic enzymes, lectins and the like), sugars and salts.
[0006] In contrast to PSVs, PBs (1-3 microns) sequester
predominantly prolamins, which are highly hydrophobic storage
proteins of cereals (such as zeins of maize and gliadins of wheat),
and lack of other auxiliary proteins (Herman et al., 1999 Plant
Cell 11:601-613).
[0007] At present, no PBs have been found in tissues other than
plant seeds, with the exception of the ER bodies. The ER bodies are
small in size (0.2-0.4 micrometers) and are formed in Arabidopsis
leaves only by wounding and chewing by insects but do not develop
under normal conditions (Matsushima et al., 2003 Plant J.
33:493-502).
[0008] Genetic engineering approaches have been used to study plant
PBs formation, storage protein assembly and targeting. It has been
shown that when recombinant proteins, predominantly plant storage
proteins are expressed and packaged in Arabidopsis and tobacco,
plant tissues that did not contain PBs (as vegetative tissues),
develop these organelles "de novo" (Bagga et al., 1997 Plant Cell
9:1683-1696 and Bagga et al., 1995 Plant Physiol. 107:13-23, and
U.S. Pat. No. 5,990,384, No. 5,215,912, and No. 5,589,616; and Geli
et al., 1994 Plant Cell 6:1911-1922).
[0009] Maize beta-zein when expressed in transgenic tobacco plants
was correctly targeted in new formed ER-derived PBs in leaf cells
(Bagga et al., 1995 Plant Physiol. 107:13-23). Maize gamma-zein
and, truncated gamma-zein cDNAs expressed in Arabidopsis plants
also accumulate in a novel ER-derived PBs in leaves (Geli et al.,
1994 Plant Cell 6:1911-1922). Lysine-rich gamma-zeins expressed in
maize endosperms (Torrent et al. 1997 Plant Mol. Biol.
34(1):139-149) accumulate in maize PBs and co-localized with
endogenous zeins. Transgenic tobacco plants expressing alpha-zein
gene demonstrated that alpha-zein was not able to form PBs.
However, when alpha- and gamma-zein were co-expressed, the
stability of alpha-zein increased and both proteins co-localized in
ER-derived protein bodies (Coleman et al., 1996 Plant Cell
8:2335-2345). Formation of novel PBs has been also described in
transgenic soybean transformed with methionine-rich 10 kDa
delta-zein (Bagga et al., 2000 Plant Sci. 150:21-28).
[0010] Recombinant storage proteins are also assembled in PBs-like
organelles about 100 to about 400 nm in diameter in a non-plant
host system such as Xenopus oocytes and in yeast. Rosenberg et al.,
1993 Plant Physiol 102:61-69 reported the expression of wheat
gamma-gliadin in yeast. The gene expressed correctly and the
protein was accumulated in ER-derived PBs. In Xenopus oocytes,
Torrent et al., 1994 Planta 192:512-518 demonstrated that gamma
zein also accumulates in PB-like organelles when transcripts
encoding the protein were microinjected into oocytes. Hurkman et
al., 1981 J. Cell Biol. 87:292-299 with alfa-zeins and Altschuler
et al., 1993 Plant Cell 5:443-450 with gamma-gliadins had similar
results in Xenopus oocytes.
[0011] One of the fundamental achievements of the field of the
biotechnology (genetic engineering) is the ability to genetically
manipulate an organism to produce a protein for therapeutic,
nutraceutical or industrial uses. Methods are provided for
producing and recovering recombinant proteins from fermentation
broth of bacteria, yeast, crop plants and mammalian cell cultures.
Different approaches for protein expression in host cells have been
described. The essential objectives of these approaches are:
protein expression level, protein stability and protein recovery
(Menkhaus et al., 2004 Biotechnol. Prog. 20: 1001-1014; Evangelista
et al., 1998 Biotechnol. Prog. 14:607-614).
[0012] One strategy that can solve a problem with protein recovery
is secretion. However, secretion involves some times poor
expression levels and product instability. Another strategy is the
accumulation of the recombinant protein in the most beneficial
location in the cell. This strategy has been extensively used by
directing recombinant proteins to the ER by engineering C-terminal
extension of a tetrapeptide (HDEL/KDEL) (Conrad and Fiedler, 1998
Plant Mol. Biol. 38:101-109).
[0013] Fusion proteins containing a plant storage protein or
storage protein domains fused to the heterologous protein have been
an alternative approach to direct recombinant proteins to the ER
(WO 2004003207). One interesting fusion strategy is the production
of recombinant proteins fused to oleosins, constitutive protein of
plant oil bodies. The specific characteristics of oil bodies
benefit of the easy recovery of proteins using a two-phase system
(van Rooijen and Moloney, 1995 Bio/Technology 13:72-77).
[0014] Heterologous proteins have been successfully expressed in
plant cells (reviews Horn et al., 2004 Plant Cell Rep. 22:711-720;
Twyman et al., 2003, Trends in Biotechnology 21:570-578; Ma et al.,
1995, Science 268: 716-719; Richter et al., 2000 Nat. Biotechnol.
18:1167-1171), and in some, the expression of the recombinant
protein has been directed to ER-derived PB or PSV (PSV). Yang et
al., 2003 Planta 216:597-603, expressed human lysozyme in rice
seeds using the seed-specific promoters of glutelin and globulin
storage proteins. Immunocytochemistry results indicated that the
recombinant protein was located in ER-PBs and accumulated with
endogenous rice globulins and glutelins. The expression of
glycoprotein B of the human cytomegalovirus (hCMV) in transgenic
tobacco plants has been carried out using a glutelin promoter of
rice. Tackaberry et al., 1999 Vaccine 17:3020-3029. Recently,
Arcalis et al., 2004 Plant Physiology 136:1-10 expressed human
serum albumin (HSA) with a C-terminal extension (KDEL) in rice
seeds. The recombinant HSA accumulated in PSVs with the endogenous
rice storage proteins.
[0015] One obstacle for the application of plants as biofactories
is the need for more research regarding the downstream processing.
Protein purification from plants is a difficult task due to the
complexity of the plant system. Plant solids of the extract are
large, dense and relative elevated (9-20 percent by weight) (see
review Menkhaus et al., 2004 Biotechnol. Prog. 20:1001-1014). At
present, recombinant protein purification techniques include
clarification of the extracts, treatment with solvents to remove
lipids and pigments and protein or peptides purification by several
ion-exchange and gel-filtration chromatography columns. The
existing protocols rely upon the use of specific solvents or
aqueous solutions for each plant-host system and recombinant
protein. There is a need in the art for efficient and general
procedures for recombinant protein recovery from transformed hosts.
This need is especially relevant in cases where recombinant
proteins produced in plant hosts must to be isolated. The diversity
of hosts and proteins and the different physical-chemical traits
between them required an efficient method to concentrate and
recover recombinant products.
[0016] Immunologic adjuvants are agents that enhance specific
immune responses to vaccines and inocula. An immunologic adjuvant
can be defined as any substance or formulation that, when
incorporated into a vaccine or inoculum, acts generally or
specifically to accelerate, prolong, or enhance the quality of
specific immune responses to the immunogenic materials in the
preparation.
[0017] The word adjuvant is derived from the Latin verb adjuvare,
which means to help or aid. Adjuvant mechanisms of action include
the following: (1) increasing the biological or immunologic
half-life of vaccine or inoculum immunogens; (2) improving
immunogen delivery to antigen (immunogen)-presenting cells (APCs),
as well as antigen (immunogen) processing and presentation by the
APCs; and (3) inducing the production of immunomodulatory
cytokines.
[0018] Possession of biological activity that resembles an activity
of a natural pathogen or other agent is particularly relevant for
vaccines or inocula, which must induce a correct immune response in
an immunized human or other animal to be effective. Several new
vaccines and inocula are composed of synthetic, recombinant, or
highly purified subunit immunogens (antigens) that are thought to
be safer than whole-inactivated or live-attenuated vaccines.
However, pathogen-related immunomodulatory adjuvant components that
are typically associated with attenuated or killed pathogen
vaccines are absent from such synthetic, recombinant, or highly
purified subunit immunogens, which often results in weaker
immunogenicity for such preparations.
[0019] Phagocytosis involves the entry of large particles, such us
apoptotic cells or whole microbes, into another cell. The capacity
of the cells to engulf large particles likely appeared as a
nutritional function in unicellular organisms; however complex
organisms have taken advantage of the phagocytic machinery to
fulfill additional functions. For instance, the phagocytosis of
immunogens undertaken by the macrophages, the B-cells or the
dendritic cells represents a key process in innate and adaptive
immunity. Indeed, phagocytosis and the subsequent killing of
microbes in phagosomes form the basis of an organism's innate
defense against intracellular pathogens. Furthermore, the
degradation of pathogens in the phagosome lumen and the production
of antigenic peptides, which are presented by phagocytic cells to
activate specific lymphocytes, also link phagocytosis to adaptive
immunity (Jutras et al., 2005 Annual Review in Cell Development
Biology. 21:511-527).
[0020] The proteins present on and in engulfed particles encounter
an array of degrading proteases in phagosomes. Yet, this
destructive environment generates peptides that are capable of
binding to MHC class II molecules. Newly formed immunogen-MHC class
II complexes are delivered to the cell surface for presentation to
CD4+ T cells (Boes et al., 2002 Nature 418:983-988). The activation
of these cells induces the Th2 subset of cytokines such as IL-4 and
IL-5 that help B cells to proliferate and differentiate, and is
associated with humoral-type immune response.
[0021] A large body of evidence indicates that, in addition to the
clear involvement of the MHC class II pathway in the immune
response against phagocytosed pathogens, immunogens from pathogens,
including mycobacteria, Salmonella, Brucella, and Leishmania, can
elicit an immunogen cross-presentation. That is to say, the
presentation of an engulfed immunogen by phagocytosis by the MHC
class I-dependent response promotes the proliferation of CD8+
cytotoxic T cells (Ackerman et al., 2004 Nature Immunology
5(7):678-684; Kaufmann et al., 2005 Current Opinions in Immunology
17(1):79-87).
[0022] Dendritic cells play a central immunogen presentation role
to induce the immune system (Blander et al., Nature Immunology 2006
10:1029-1035). Although rare, dendritic cells are the most highly
specialized APC, with ability both to instigate and regulate immune
reactivity (Lau et al. 2003 Gut 52:307-314). Although dendritic
cells are important in presenting immunogens, particularly to
initiate primary immune responses, macrophages are the APC type
most prominent in inflammatory sites and specialized for clearing
necrotic and apoptotic material. Macrophages can act not only as
APC, but can also perform either pro- or anti-inflammatory roles,
dependent on the means by which they are activated.
[0023] Considering that APCs play a central role in the induction
and regulation of the adaptive immunity (humoral and cellular), the
recognition and phagocytosis of the immunogen by those cells can be
considered a key step in the immunization process. A wide variety
of techniques based on the uptake of fluorescent particles have
been developed to study phagocytosis by the macrophages (Vergne et
al., 1998 Analytical Biochemistry 255:127-132).
[0024] An important aspect in veterinary vaccines is the genetic
diversity of the species being considered and the requirement for
generic systems that work across different species. To a large
degree, this diversity limits the use of molecular targeting
techniques to cell surface markers and immune modulators such as
cytokines, because for many species including wildlife, only
minimal knowledge of these molecules is available. Thus, adjuvants
that rely on universal activation signals of the innate immune
response (i.e. that are identical in different species) are to be
preferred. Taking these requirements into consideration,
particulate vaccine delivery systems are well suited for veterinary
and wildlife vaccine strategies (Scheerlinck et al., 2004 Methods
40:118-124). In Third World countries, cervical cancer (cc) is one
of the major causes of cancer-related deaths. About 80% of women
dying from this disease originate from low-budget countries where
screening programs for early detection and the medical
infrastructure for treatment are not available. In contrast, in the
more developed world the mortality was reduced (by 70% in the US)
during the last 50 years as a consequence of cytological screening
programs [American Cancer Society, Cancer facts and figures 2004.
Atlanta, Ga.] Treatment of cc patients by surgery, radiotherapy or
chemotherapy results in a significant loss of quality of life. Even
when optimal treatment is available about 40% of all cc patients
die of this disease [Gatta et al., 1998 Eur J Cancer 34(14 Spec.
No.):2218-2225]. Therefore, the development of an effective and
save therapeutic vaccine is needed.
[0025] A necessary event for the development of premalignancies
like cervical intraepithelial neoplasia (CIN) and cc is infection
by hr-HPVs [Walboomers et al., 1999 J Pathol 189(1):12-19]. So far
over 120 HPV types are identified [de Villiers et al., 2004
Virology 324(1):17-27], 18 of which were found to be associated
with cc [Munoz et al., 2003 N Engl J Med 348(6):518-527].
[0026] HPV-16 is responsible for about 50% of the cases [Bosch et
al., 1995 J Natl Cancer Inst 87(11):796-802]. Due to the fact that
the oncoprotein E7 of the hr-HPVs is exclusively and consistently
expressed by HPV-infected tumor cells [von Knebel Doeberitz et al.,
1994 J Virol 68(5):2811-2821], that protein represents a specific
target for an immune therapy directed against cc and its
premalignant dysplasia. The E7 protein, however, is an oncoprotein
with transforming activity that operates by interfering with the
cell cycle control. The E7 alters cell growth regulation by
inactivating the pRB (retinoblastoma) tumor suppressor protein
[Dyson et al., 1989 Science 243(4893):934-937; Munger et al., 1992
Cancer Surv 12:197-217] and contains two metal-binding motifs
(C-XX-C) [Edmonds et al., 1989 J Virol 63(6):2650-2656; Watanabe et
al., 1990 J Virol 64(1):207-214].
[0027] For safety reasons a functional oncogene cannot be applied
to humans. Therefore, efforts were made to inactivate the oncogenic
properties of the HPV-16 E7. Some investigators have introduced
point mutations into the sites of the E7-oncogene that are
associated with transforming potential [Shi et al., 1999 J Virol
73(9):7877-7881; Smahel et al., 2001 Virology 281(2):231-238],
whereas others have used HLA- (human leukocyte antigen) restricted
singular epitopes [Doan et al., 2000 Cancer Res 60(11):2810-2815;
Velders et al., 2001 J Immunol 166(9):5366-5373]. These approaches,
however, can lead to an unwanted loss of a naturally occurring
epitope that is potentially associated with a decrease in vaccine
efficacy.
[0028] An aim of the present inventors was to supply several to all
potential naturally occurring T cell epitopes, covering the broad
range of MHC restriction. In consequence, prior knowledge of the
patient's HLA-haplotype is not required. This is especially
important in the outbred human population.
[0029] In addition, a more potent immune response may be induced,
involving all occurring HLA-restriction elements in the vaccine. A
"proof-of-principle" was generated in a study using an artificial
HPV-16 E7 gene (HPV-16 E7SH) of the first generation [Osen et al.,
2001 Vaccine 19(30):4276-4286]. It was shown in that study that an
oncoprotein with a rearranged primary sequence still induces
E7WT-specific CTLs in mice but is devoid of transforming
properties. That study took advantage of the earlier finding that
fusion with the VP22 gene of Herpes Simplex Virus Type 1 strongly
enhances the CTL response in mice [Michel et al., 2002 Virology
294(1):47-59].
[0030] The HIV-1 virus is comprised of several layers of proteins
and glycoproteins that surround its RNA, and its associated
proteins integrase and reverse transcriptase. The RNA is
encapsidated by a capsid protein (CA), p24. The capsid environment
also contains other viral proteins such as integrase and reverse
transcriptase. The capsid is in turn encapsidated by a layer of
matrix protein (MA), p17. This matrix protein is associated with a
lipid bilayer or envelope.
[0031] The great diversity among human immunodeficiency virus type
1 (HIV-1) subtypes, which are prevalent in various regions of the
world, is a major impediment to the development of broad-based
prophylactic HIV-1 vaccines. Thus, it may be necessary to develop
vaccines that match local epidemics more closely (Morris et al.,
2001). In southern Africa, subtype C infections predominate
(UNAIDS, 2006), and isolates of this subtype have been selected for
the development of a DNA vaccine in South Africa (Williamson et
al., 2003). This candidate vaccine has been constructed and
characterized (van Harmelen et al., 2003) and is scheduled to be
evaluated in clinical trials shortly.
[0032] DNA vaccines encoding HIV or simian immunodeficiency virus
(SIV)/simian-human immunodeficiency virus (SHIV) antigens have been
studied extensively and shown to induce both humoral and cellular
immune responses in animal models as well as in humans [Boyer et
al., 1997 J Infect Dis. 176(6):1501-1509; Calarota et al., 1998
Lancet 351(9112):1320-1325; Estcourt et al., Immunol. Rev. 2004
199:144-155; Letvin et al., 1997 Proc Natl Acad Sci USA.
94(17):9378-9383; Yasutomi et al., 1996 J Virol. 70(1):678-681].
However, although DNA vaccines have been shown to be safe,
immunization generates low and transient levels of immune
responses. Various approaches to augment DNA vaccines have been
tested [Barouch et al., 2000 Intervirology 43(4-6):282-287; Hemmi
et al., 2003 J Immunol 170(6):3059-3064; Raviprakash et al., 2003
Virology. 315(2):345-352], including their use in heterologous
prime-boost immunization regimens [Casimiro et al., 2003 J. Virol.
77(13):7663-7668; Cherpelis et al., 2001 Immunol Lett.
79(1-2):47-55; Leung et al., 2004 AIDS 18(7):991-1001; Pal et al.,
2006 Virology 348(2):341-353; Robinson et al., 1999 Int J Mol. Med.
4(5):549-555; Suh et al., 2006 Vaccine 24(11):1811-1820. Epub 2005
Oct. 25].
[0033] The HIV-1 Gag gene encodes the precursor protein Pr55 Gag,
which is the major protein that makes up the structure of the HIV
viral particle. On maturation of the viral particle, Gag is cleaved
by the viral protease into several smaller proteins that include
the capsid (CA) protein p24, the matrix protein p17, as well as
proteins p7 and p6.
[0034] HIV-1 Pr55.sup.gag precursor protein possesses an ability to
self-assemble into non-replicating and non-infectious virus-like
particles (VLPs) [Deml et al., 1997 Virology 235(1):26-39; Mergener
et al., 1992 Virology 186(1):25-39; Sakuragi et al., 2002 Proc Natl
Acad Sci USA 99(12):7956-7961; Wagner et al., 1994 Behring Inst
Mitt (95):23-34; Wagner et al., 1996 Virology 220(1):128-140], and
elicits strong humoral and cellular immune responses in animals
[Deml et al., 1997 Virology 235(1):26-39; Deml et al., 2004 Methods
Mol Med 94:133-157; Jaffray et al., 2004 J Gen Virol. 85(Pt
2):409-413], including non-human primates (NHPs) (Montefiori et
al., 2001 J. Virol. 75(21):10200-10207; Paliard et al., 2000 AIDS
Res Hum Retroviruses 16(3):273-282]. Recently, Chege et al., J Gen
Virol 2008 89:2214-2227 have shown that subtype C Pr55.sup.gag VLPs
can very efficiently boost baboons primed with a matched DNA
vaccine.
[0035] In addition, HIV-1 Pr55.sup.gag VLPs are safe, easy to
produce and have the potential of including chimeric immunogens
(Doan et al., 2005; Halsey et al., 2008). Their particulate nature
and size, which approximates that of HIV-1, make HIV-1 Pr55.sup.gag
VLPs more likely to stimulate the immune system better than
non-particulate immunogens.
[0036] As above, the p24 protein forms the outer capsid layer of
the viral particle. This protein has a high density of cytotoxic
T-lymphocyte (CTL) epitopes compared to other parts of the HIV
proteome (Novitsky et al., J. Virol. 2002 76(20):10155-10168),
which make it more effective in inducing a broad immune response
when used as a vaccine candidate. It has also been shown that the
risk of AIDS is greatly increased in individuals with falling
titres of p24 antibodies. This suggests that high anti-p24 antibody
titres might be necessary to maintain a disease-free state.
[0037] In addition, HIV-1 Pr55.sup.gag VLPs are safe, easy to
produce and have the potential of including chimeric immunogens
[Doan et al., 2005 Rev Med. Virol. 15(2):75-88; Halsey et al., 2008
Virus Res. 2008 133(2):259-268. Epub 2008 Mar. 10]. Their
particulate nature and size, which approximates that of HIV-1, make
HIV-1 Pr55.sup.gag VLPs more likely to stimulate the immune system
better than non-particulate immunogens.
[0038] As above, the p24 protein forms the outer capsid layer of
the viral particle. This protein has a high density of cytotoxic
T-lymphocyte (CTL) epitopes compared to other parts of the HIV
proteome (Novitsky et al., J. Virol. 2002 76(20):10155-10168),
which make it more effective in inducing a broad immune response
when used as a vaccine candidate. It has also been shown that the
risk of AIDS is greatly increased in individuals with falling
titres of p24 antibodies. This suggests that high anti-p24 antibody
titres might be necessary to maintain a disease-free state.
[0039] The matrix protein, p17, facilitates the intra-membrane
associations that are required for viral assembly and release
(Dorfman et al., 1994 J Virol 68(12):8180-8187]. Protein p17 is
also involved in the transport of the viral pre-integration complex
into the nucleus (Burkinsky et al., 1993). Fused together with p24,
this p17/p24 (p41) complex contains the highest density of CTL
epitopes in the HIV-1 genome (Novitsky et al., J. Virol. 2002
76(20):10155-10168).
[0040] HIV-1 reverse transcriptase (RT) is an RNA-dependent DNA
polymerase that makes DNA templates and synthesises DNA from RNA.
It is essential for viral replication. HIV-1 RT is cleaved from the
Pr160.sup.gag-pol polyprotein by the HIV-1 protease (PR). Several
CTL epitopes against HIV-1 have been identified in RT, although
they appear to be subdominant to Gag-specific epitopes [Dela Cruz
et al., 2000 Int Immunol 12(9):1293-1302].
[0041] Several studies have indicated that enhanced immune
responses can be achieved by heterologous prime-boost inoculation
regimens. It has been shown that a HIV-1 DNA vaccine denominated
pTHGagC used as a prime inoculation of mice is boosted effectively
by Pr55.sup.Gag virus-like particles (VLPs) (Chege et al., J. Gen.
Virol. 2008 89:2214-2227). Because p24 has the highest density of
cytotoxic T-lymphocyte (CTL) epitopes compared to other parts of
the HIV proteome it was thought that the particulate nature of
protein bodies containing p24 may have a similar boosting effect to
expand immune responses after the immune system has been primed. It
has also been thought that the use of combinations of protein
bodies containing different HIV-1 antigens such as p41 and RT, may
broaden the immune response such as has been shown in the use of
the multigene DNA vaccine "grttn" (Burgers et al., AIDS Research
and Human Retroviruses 2008 24(2):195-206) that contains five AIDS
genes, the gag, reverse transcriptase, tat and nef genes that are
expressed as a polyprotein and a truncated env gene (gp150).
BRIEF SUMMARY OF THE INVENTION
[0042] The present invention contemplates an immunogen-specific
adjuvant for a vaccine or inoculum. The adjuvant is comprised of
particulate recombinant protein body-like assemblies (RPBLAs) that
contain a recombinant fusion protein. The recombinant fusion
protein contains two sequences peptide-linked together in which one
sequence is a protein body-inducing sequence (PBIS) such as a
prolamin sequence and the other is a T-cell stimulating immunogenic
polypeptide whose sequence is that of a pathogenic polypeptide
sequence present in or induced by a vaccine or inoculum. The
adjuvant when used in an inoculum without a prior priming
vaccination or inoculation does not induce production of antibodies
or T cell activation to the pathogenic sequence. A contemplated
adjuvant is typically used in an adjuvant-effective amount
dissolved or dispersed in a pharmaceutically acceptable
diluent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] In the drawings forming a part of this disclosure,
[0044] FIG. 1 in three panels (FIG. 1A, FIG. 1B and FIG. 1C) shows
the analysis by western blot of RPBLA fractions isolated from
tobacco plants agroinfiltrated with RX3-p24, RX3-p41 and RX3-RT.
The presence of full length RX3 fusion proteins in the
corresponding RPBLA fraction preparation was checked by using the
following antibodies: (i) .alpha.R8 which recognizes RX3, (ii)
.alpha.p24 which recognizes p41 and p24 antigens and (iii)
.alpha.RT which recognizes RT antigen.
[0045] FIG. 2 contains two graphs that show an IFN-.gamma. (FIG.
2A) and IL-2 (FIG. 2B) ELISPOT analysis of p24 cell responses after
vaccination of BALB/c mice. Inoculations with the indicated
immunogens were given as specified in the methods. Reactions in the
corresponding ELISPOT assay were done in triplicate with the
indicated Gag peptides, an irrelevant peptide (Irrel pept) or
absence of peptide (Med), and bars are the average number of spot
forming units (sfu).+-.SD/106 splenocytes. Data are from a
representative study with splenocytes pooled from 5 mice per
group.
[0046] FIG. 3 shows western blot detection of anti-Gag antibodies
in mouse serum. The content of anti-Gag antibody in mouse serum was
detected using commercial western blot strips as described in the
methods. Pos, positive control serum; Neg, negative control serum;
d40, mouse serum taken at day 40 after inoculation as indicated and
described in methods; d0, pre-inoculation mouse serum. The
inoculation regimen for each set of strips is indicated on the
right of the strips: these were (i) single gag DNA inoculation
(pTHGagx1), (ii) gag DNA prime-gag DNA boost (pTHGagx2), (iii) gag
DNA prime--RX3-p24 boost (pTHGagC+RX3-p24), (iv) single RX3-p24
inoculation (RX3-p24).
[0047] FIG. 4 contains two graphs that show an IFN-.gamma. (FIG.
4A) and IL-2 (FIG. 4B) ELISPOT analysis of p41 cell responses after
vaccination of BALB/c mice. Inoculations with the indicated
immunogens were given as specified in the methods. Reactions in the
corresponding ELISPOT assay were done in triplicate with the
indicated Gag peptides, an irrelevant peptide TYSTVASSL (SEQ ID
NO:1; irrel pept) or absence of peptide (Med) and bars are the
average number of spot forming units (sfu).+-.SD/106 splenocytes.
Data are from a representative study with splenocytes pooled from 5
mice per group.
[0048] FIG. 5 contains two graphs that show an IL-2 (FIG. 5A) and
IFN-.gamma. (FIG. 5B) ELISPOT analysis of RT cell responses after
vaccination of BALB/c mice. Inoculations with the indicated
immunogens were given as specified in the methods. Reactions in the
corresponding ELISPOT assay were done in triplicate with the
indicated Gag peptides, an irrelevant peptide TYSTVASSL (SEQ ID
NO:1; irrel pept) or absence of peptide (Med) and bars are the
average number of spot forming units (sfu).+-.SD/106 splenocytes.
Data are from a representative study with splenocytes pooled from 5
mice per group.
[0049] FIG. 6 is a map of the artificial HPV-16 E7SH gene. The
HPV-16 E7 wild-type gene (E7WT, above) was dissected at the
positions corresponding to the pRB binding site (nt 72/73) and
between the two C-X-X-C motifs (nt 177/178 and nt 276/277). The
resulting four fragments a, b, c and d were rearranged ("shuffled")
forming the core element with the sequence a, d, c, b. To avoid
loss of putative CTL epitopes at the junctions a-b, b-c and c-d,
these sequences (3.times.27 nt=3.times.9 amino acids) were added as
an appendix forming the complete HPV-16 E7SH gene. To minimize the
potential risk of "back-to-wild-type recombination" the codons of
the core element were optimized for expression in humans according
to the Kazusa codon usage database that can be found at
kazusa.or.jp/codon/. A Kozak sequence was added in front of the
gene to enhance translation.
[0050] FIG. 7 shows the analysis by western blot of RPBLA fractions
isolated from tobacco plants agroinfiltrated with RX3-E7SH. The
presence of full length RX3 fusion proteins in the corresponding
RPBLA fraction preparation was checked by using E7SH antibody.
[0051] FIG. 8 contains two graphs (FIG. 8A and FIG. 8B) that
illustrate CTL responses in C57BL/6 mice after DNA and RPBLAs
immunization. Four mice per group were immunized once
intra-muscularly in each musculus tibialis anterior with: (i) 50
.mu.g empty plasmid (pTHamp), (ii) 50 .mu.g plasmid expressing E7SH
(pTHamp-E7SH), (iii) or subcutaneously into the flank with 5 .mu.g
of RPBLAs containing RX3-Gfp fusion protein (RX3-Gfp), (iv) 5 .mu.g
of RPBLAs containing RX3-E7SH fusion protein (RX3-E7SH) or (v) 5
.mu.g of RPBLAs containing RX3-E7SH fusion protein and 100 .mu.l of
IFA (5 .mu.g RX3-E7SH in 100 .mu.l buffer+100 .mu.l IFA). Ex vivo
IFN-.gamma. and Granzyme B Elispot assays were performed and each
bar represents the number of activated T cells from an individual
animal.
[0052] FIG. 9 is a graph of CTL responses in C57BL/6 mice after
RPBLAs immunization. Four mice per group were immunized once
intramuscularly or sc (as above) with: (i) 5 .mu.g of RPBLAs
containing RX3-Gfp fusion protein (RX3-Gfp), (ii) 5 .mu.g of RPBLAs
containing RX3-E7SH fusion protein (RX3-E7SH), (iii) 5 .mu.g of
RPBLAs containing RX3-E7SH fusion protein and 100 .mu.l of IFA
(RX3-E7SH/IFA), (iv) 5 .mu.g of ovalbumin (OVA) or (v) 5 .mu.g of
ovalbumin and 100 .mu.l of IFA (OVA/IFA) in each musculus tibialis
anterior. Ex vivo Granzyme B Elispot assays were performed and each
bar represents the number of activated T cells from an individual
animal.
[0053] FIG. 10 in two parts as FIG. 10A and FIG. 10B illustrate
growth of C3 tumors in C57BL/6 mice after immunization with: (i)
100 .mu.g empty plasmid (pTHamp), (ii) 100 .mu.g plasmid expressing
E7SH (pTHamp-E7SH), (iii) 5 .mu.g of RPBLAs containing RX3-Gfp
fusion protein (RX3-Gfp), (iv) 5 .mu.g of RPBLAs containing
RX3-E7SH fusion protein (RX3-E7SH) or (v) 5 .mu.g of RPBLAs
containing RX3-E7SH fusion protein and 100 .mu.l of IFA
(RX3-E7SH/IFA). Data shown provide the surface area tumor size from
days 0 to 14. FIG. 10A illustrates a comparison of DNA vs RPBLAs
immunization effect on tumor regression, whereas FIG. 10B
illustrates that there is no unspecific tumor regression effect in
DNA and RPBLAs immunizations lacking the E7SH antigen.
[0054] FIG. 11 is a graph showing the results of tumor growth on
rechallenge studies after immunization with: (i) 100 .mu.g plasmid
expressing E7SH (pTHamp-E7SH), (ii) 5 .mu.g of RPBLAs containing
RX3-E7SH fusion protein (RX3-E7SH) or (iii) 5 .mu.g of RPBLAs
containing RX3-E7SH fusion protein and 100 .mu.l of IFA
(RX3-E7SH/IFA). Those mice that showed complete regression after
the tumor regression study of FIG. 10 were injected again with
0.5.times.10.sup.6 C3 cells s.c. in 100 .mu.l PBS into the flank 3
weeks after completion of the tumor regression experiment. As a
control, the same number of non-immunized mice received the same
treatment. Twenty days after this injection, all control mice
showed tumor growing, whereas none of the immunized mice developed
tumors.
Definitions
[0055] The word "antigen" has been used historically to designate
an entity that is bound by an antibody or receptor, and also to
designate the entity that induces the production of the antibody or
cellular response such as that of a CD4+ T cell. More current usage
limits the meaning of antigen to that entity bound by an antibody
or receptor, whereas the word "immunogen" is used for the entity
that induces antibody production or cellular response. Where an
entity discussed herein is both immunogenic and antigenic,
reference to it as either an immunogen or antigen is typically made
according to its intended utility.
[0056] "Antigenic determinant" refers to the actual structural
portion of the antigen that is immunologically bound by an antibody
combining site or T-cell receptor. The term is also used
interchangeably with "epitope".
[0057] As used herein, the term "fusion protein" designates a
polypeptide that contains at least two amino acid residue sequences
not normally found linked together in nature that are operatively
linked together end-to-end (head-to-tail) by a peptide bond between
their respective carboxy- and amino-terminal amino acid residues. A
fusion protein of the present invention is a chimer of a protein
body-inducing sequence (PBIS) linked to a second sequence that is a
T-cell stimulating polypeptide (e.g., peptide or protein) that is
present in the pathogen (target) at which the vaccine or inoculum
is directed.
[0058] The term "immunogen-specific" is used herein to distinguish
the adjuvanticity of a contemplated recombinant adjuvant and a more
general adjuvant. More particularly, a contemplated
immunogen-specific adjuvant enhances the cellular (T-cell) immune
response toward an immunogen that includes an amino acid residue
sequence of the adjuvant and does not generally activate the immune
system. Thus, the vaccine or inoculum shares an amino acid residue
sequence or encodes a shared sequence with the adjuvant.
[0059] An "inoculum" is a composition that comprises an
immunogenically effective amount of immunogenic chimer particles
dissolved or dispersed in a pharmaceutically acceptable diluent
composition that typically also contains water. When administered
to a host animal in need of immunization or in which antibodies or
activated T cells are desired to be induced such as a mammal (e.g.,
a mouse, dog, goat, sheep, horse, bovine, monkey, ape, or human) or
bird (e.g., a chicken, turkey, duck or goose), an inoculum induces
a B cell and/or T cell response (stimulation) in an inoculated host
animal such as production of antibodies that immunoreact with the
immunogen of the chimer and/or induces T cells that respond to the
immunogen. A "vaccine" is a type of inoculum in which the
vaccine-induced antibodies not only immunoreact with the immunogen
or activated T cells respond to that immunogen, but also
immunoreact with the pathogen from which the immunogen is derived
in vivo, and provide protection from that disease state.
[0060] The expression "T-Cell-mediated immunity" refers to an
immune response that does not involve antibodies or complement but
rather involves the activation of macrophages, natural killer cells
(NK), antigen-specific cytotoxic T-lymphocytes, and the release of
various cytokines in response to an antigen. Historically, the
immune system was separated into two branches: humoral immunity,
for which the protective function of immunization could be found in
the humor (cell-free bodily fluid or serum) and cellular immunity,
for which the protective function of immunization was associated
with cells. CD4 cells or helper T cells provide protection against
different pathogens. T-Cell-mediated immunity is an immune response
produced when T cells, especially cytotoxic T cells, that are
sensitized to foreign antigens attack and lyse target cells. In
addition to direct cytotoxicity, T cells can stimulate the
production of lymphokines that activate macrophages. Cell-mediated
immune responses are important in defense against pathogens,
autoimmune diseases, some acquired allergies, viral infection, some
tumors and other immune reactions.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0061] The present invention contemplates an immunogen-specific
adjuvant for a vaccine or inoculum. The adjuvant is comprised of
particulate recombinant protein body-like assemblies (RPBLAs) that
contain a recombinant fusion protein. The recombinant fusion
protein contains two sequences peptide-linked together in which one
sequence is a protein body-inducing sequence (PBIS) such as a
preferred prolamin sequence and the other is a T-cell stimulating
immunogenic polypeptide whose sequence (a) is present in a
pathogenic polypeptide sequence present in a polypeptide-containing
vaccine or inoculum or (b) is encoded by a nucleic acid vaccine or
inoculum. The adjuvant, at the concentration used in an inoculum in
a host animal without a prior priming by vaccination or inoculation
of a host animal or additional immunogen, does not induce
production of antibodies in that host animal that immunoreact with
or induce T cell activation to the pathogenic sequence.
[0062] The invention also contemplates a method for inducing an
T-cell mediated immune response in a subject in need thereof
against an immunogenic peptide which comprises the administration
to a subject in need thereof of a vaccine selected from the group
of [0063] (i) a particulate recombinant protein body-like
assemblies (RPBLAs) that contain a recombinant fusion protein, said
recombinant fusion protein containing two portions peptide-linked
together in which a first portion is a protein body-inducing
sequence (PBIS) and a second portion is a immunogenic polypeptide
and [0064] (ii) a nucleic acid molecule that encodes a fusion
protein, said recombinant fusion protein containing two portions
peptide-linked together in which a first portion is a protein
body-inducing sequence (PBIS) and a second portion is a immunogenic
polypeptide.
[0065] In a preferred embodiment, the method of the invention is
carried out using a RPBLA wherein the PBIS forming part of the
first portion includes a prolamin sequence. In a still more
preferred embodiment, the prolamin sequence is present in a
prolamin selected from the group consisting of gamma-zein,
alpha-zein, delta-zein, beta-zein, rice prolamin and
gamma-gliadin.
[0066] In a preferred embodiment, the PBIS sequence further
includes a signal peptide sequence that directs a protein towards
the endoplasmic reticulum (ER) of the RPBLA-expressing cell.
[0067] In a preferred embodiment, the immunogenic peptide used in
fusion protein forming the RPBLA is a peptide capable of
stimulating the T-cell immune response.
[0068] In another preferred embodiment, the method of the invention
is carried out using a RPBLA comprising a second portion wherein
the immunogenic polypeptide sequence is selected from the group of
[0069] (i) a polypeptide encoded by the HPV E7 gene, [0070] (ii) a
polypeptide encoded by the HIV-1 gag gene and [0071] (iii) a
polypeptide encoded by the HIV-1 pol gene
[0072] In a preferred embodiment, the method of the invention is
carried out using a particulate recombinant protein body-like
assemblies (RPBLAs) are assembled in vitro from the purified
recombinant fusion protein.
[0073] In a more preferred embodiment, the administration step of
the method of the invention is preceded by a priming vaccination or
inoculation step using a composition comprising immunogenic
polypeptide or a nucleic acid encoding said immunogenic
polypeptide.
[0074] In a still more preferred embodiment, the composition
comprising the immunogenic polypeptide used in the priming
vaccination or stimulation step is selected from the group of
[0075] (i) a particulate recombinant protein body-like assemblies
(RPBLAs) that contain a recombinant fusion protein, said
recombinant fusion protein containing two portions peptide-linked
together in which a first portion is a protein body-inducing
sequence (PBIS) and a second portion is the T-cell stimulating
immunogenic polypeptide, [0076] (ii) a nucleic acid molecule that
encodes the immunogenic polypeptide and [0077] (iii) a nucleic acid
molecule that encodes a fusion protein, said recombinant fusion
protein containing two portions peptide-linked together in which a
first portion is a protein body-inducing sequence (PBIS) and a
second portion is the immunogenic polypeptide.
[0078] In a preferred embodiment, the immunogenic peptide used in
fusion protein forming the RPBLA is a peptide capable of
stimulating the T-cell immune response.
[0079] In a more preferred embodiment, the vaccine is administered
intramuscularly.
[0080] In another aspect, the invention relates to vaccine for use
in a method for inducing an T-cell mediated immune response in a
subject in need thereof against an immunogenic peptide wherein the
vaccine is selected from the group of [0081] (i) a particulate
recombinant protein body-like assemblies (RPBLAs) that contain a
recombinant fusion protein, said recombinant fusion protein
containing two portions peptide-linked together in which a first
portion is a protein body-inducing sequence (PBIS) and a second
portion is a immunogenic polypeptide and [0082] (ii) a nucleic acid
molecule that encodes a fusion protein, said recombinant fusion
protein containing two portions peptide-linked together in which a
first portion is a protein body-inducing sequence (PBIS) and a
second portion is a immunogenic polypeptide.
[0083] A contemplated adjuvant can be administered along with or
separately as a boost to an anti-pathogen vaccine or inoculum. Such
a vaccine or inoculum can contain an attenuated live or killed
pathogen such as a bacterium or virus, a subunit vaccine or
inoculum that contains only a protein portion of a pathogen, or a
vaccine or inoculum that contains an immunogen that is comprised of
a polypeptide linked to a carrier, wherein the immunogenic portion
of the vaccine or inoculum contains a polypeptide sequence that is
also present in the adjuvant. Where the vaccine or inoculum is a
nucleic acid preparation such as a DNA or RNA vaccine, the nucleic
acid encodes an immunogenic amino acid sequence that is also
present in the adjuvant. Nucleic acid vaccines and inocula are
themselves well known.
[0084] A contemplated adjuvant can be administered as a preparation
of expressed RPBLAs, or as a nucleic acid preparation, such as a
single or double stranded DNA sequence, that encodes the RPBLAs. In
the latter circumstance, the RPBLAs are expressed in vivo in the
host animal. In either situation, media in which the expressed
RPBLAs or nucleic acids are dissolved or dispersed to form adjuvant
compositions are also well known.
[0085] Illustrative nucleic acid sequences are provided hereinafter
that encode specific portions of the RPBLAs. As is well known in
the art, particular codons are preferred for encoding amino acid
residues in different animals, and as a consequence the skilled
worker can revise specific nucleic acid sequences to provide
desired degrees of expression. In addition, several vectors are
well known for expressing foreign nucleic acids and their encoded
proteins in animal hosts, including humans. On expression, the
polypeptides encoded self-assemble in vivo to form RPBLAs.
[0086] T-cell stimulating immunogenic polypeptide portions of a
number of illustrative adjuvants are discussed hereinafter that
relate to the HIV-1 virus. In those adjuvants, a DNA vaccine that
comprises all of parts of the gag gene is utilized. The HIV-1 gag
gene encodes four proteins: the P24 capsid (CA), P17 matrix (MA),
and two nucleocapsid proteins (NC) P6 and P9. Illustrative
adjuvants' fusion proteins contain the gag-encoded P24 sequence or
the P41 sequence that results from fusion of the P24 and P17
sequences, and the reverse transcriptase (RT) that is encoded by
the HIV-1 pol gene.
[0087] Analogously, a vaccine or inoculum against hepatitis B virus
(HBV) the utilizes one or more of the surface (HBsAg) proteins as
immunogen can utilize an adjuvant whose T-cell stimulating
immunogenic polypeptide portion includes a sequence illustrated of
a surface protein that includes the PreS1 and/or PreS2 portions of
the surface protein in the table of T Cell Epitopes that follows.
One such vaccine is that sold under the name RECOMBIVAX HB.RTM.
hepatitis B vaccine that is a non-infectious subunit viral vaccine
derived from the hepatitis B surface antigen (HBsAg) produced in
yeast cells and developed in the Merck Research Laboratories.
Similarly, the HBV core-based vaccine of U.S. Pat. No. 7,351,413,
can be provided an adjuvant by utilization of one or more core
sequences set out in the table of T Cell Epitopes that follows.
Additional adjuvants can be prepared as discussed herein using the
sequences in that following table or other T cell epitopes obtained
from the literature.
[0088] A contemplated adjuvant is typically used in an
adjuvant-effective amount dissolved or dispersed in a
pharmaceutically acceptable diluent as an adjuvant composition. The
amount utilized can vary widely in different host animals, with the
T-cell stimulating immunogenic polypeptide portion used, and the
construct used. Typical amounts are about 1 microgram (.mu.g) of
RPBLAs per kilogram (kg) of host body weight (.mu.g/kg) to about 1
milligram (mg) of RPBLAs per kilogram of host body weight (mg/kg).
More usual amounts are about 5 .mu.g/kg of host body weight to
about 0.5 mg/kg host body weight.
[0089] The diluent is typically aqueous-based and can include one
or more additional adjuvants, buffers, salts and viscosity
enhancing agents. The ingredients of the diluent are those
materials that are often present in a vaccine or inoculum as are
discussed hereinafter.
Protein Bodies and Protein Body-Inducing Sequences
[0090] Inasmuch as protein bodies (PBs) are appropriately so-named
only in seeds, similar structures produced in other plant organs
and in non-higher plants are referred to generally as synthetic PBs
or "recombinant protein body-like assemblies" (RPBLAs). Such RPBLAs
are membrane-enclosed fusion proteins that are found associated
with the endoplasmic reticulum (ER) of a cell.
[0091] "Purified RPBLAs" are membrane free preparations of RPBLAs
in which the membrane has been removed, usually by chemical
reduction as with a mercaptan-containing reagent, and the fusion
protein purified as by chromatographic means to free the fusion
protein from the membrane and other expression-associated
impurities. The resulting purified protein is then reassembled in
vitro to form purified RPBLA particles. That reformation of
particles typically takes place in an aqueous composition in the
presence of salts and an oxidizing environment. The formation of
such purified RPBLAs is illustrated hereinafter.
[0092] A contemplated RPBLA is a recombinantly prepared fusion
protein (polypeptide) that is expressed in a cell foreign to the
nucleic acids used to transform the cell. The cell(s) in which the
polypeptide is expressed is a host cell(s), and can be a cell
preparation or cells of an intact organism. The intact organism can
itself be a group of single celled organisms such as bacteria or
fungi, or multi-celled plants or animals, including humans. When a
human is the host, the person is the recipient of a nucleic
acid-encoded form of the adjuvant and the adjuvant is administered
as part of a treatment regimen.
[0093] In living organisms, the amino acid residue sequence of a
protein or polypeptide is directly related via the genetic code to
the deoxyribonucleic acid (DNA) sequence of the gene that codes for
the protein. Thus, through the well-known degeneracy of the genetic
code additional DNAs and corresponding RNA sequences (nucleic
acids) can be prepared as desired that encode the same fusion
protein amino acid residue sequences, but are sufficiently
different from a before-discussed gene sequence that the two
sequences do not hybridize at high stringency, but do hybridize at
moderate stringency.
[0094] High stringency conditions can be defined as comprising
hybridization at a temperature of about 50.degree.-55.degree. C. in
6.times.SSC and a final wash at a temperature of 68.degree. C. in
1-3.times.SSC. Moderate stringency conditions comprise
hybridization at a temperature of about 50.degree. C. to about
65.degree. C. in 0.2 to 0.3 M NaCl, followed by washing at about
50.degree. C. to about 55.degree. C. in 0.2.times.SSC, 0.1% SDS
(sodium dodecyl sulfate).
[0095] A nucleic sequence (DNA sequence or an RNA sequence) that
(1) itself encodes, or its complement encodes, a fusion protein
containing a protein body-inducing sequence (PBIS) and a
polypeptide of interest is also contemplated herein. As is
well-known, a nucleic acid sequence such as a contemplated nucleic
acid sequence is expressed when operatively linked to an
appropriate promoter in an appropriate expression system as
discussed elsewhere herein.
[0096] Different hosts often have preferences for a particular
codon to be used for encoding a particular amino acid residue. Such
codon preferences are well known and a DNA sequence encoding a
desired fusion protein sequence can be altered, using in vitro
mutagenesis for example, so that host-preferred codons are utilized
for a particular host in which the fusion protein is to be
expressed.
[0097] The RPBLAs are usually present in a generally spherical form
having a diameter of about 0.5 to about 3 microns (.mu.) and
usually about 1.mu.. In some instances, RPBLAs are amorphous in
shape and can vary widely in dimensions, but are still found
associated with the ER.
[0098] The density of RPBLAs is typically greater than that of
substantially all of the endogenous host cell proteins, and is
typically about 1.1 to about 1.35 g/ml. The high density of
contemplated RPBLAs is due to the general ability of the
recombinant fusion proteins to assemble as multimers and
accumulate.
[0099] A contemplated RPBLA used as an adjuvant need not be
expressed in a plant. Rather, as disclosed in published US
application 20060121573, RPBLAs can be expressed in other
transformed eukaryotes, particularly in transformed mammalian
cells.
[0100] A fusion protein of the adjuvant RPBLAs contains two
proteinaceous sequences linked together by a peptide bond as is
found in a naturally occurring protein or in a polypeptide
expressed by a genetically engineered nucleic acid. In a
contemplated fusion protein, one sequence is a protein
body-inducing sequence (PBIS) such as that of a prolamin, and the
other is a biologically active immunogenic polypeptide. Either of
the two portions can be at the N-terminus of the fusion protein.
However, it is preferred to have the PBIS at the N-terminus.
[0101] A contemplated protein body-inducing sequence (PBIS) is
preferably in whole or part from a higher plant. Illustrative,
non-limiting examples of PBIS include storage proteins or modified
storage proteins, as for instance, prolamins or modified prolamins,
prolamin domains or modified prolamin domains. Prolamins are
reviewed in Shewry et al., 2002 J. Exp. Bot. 53(370):947-958. A
preferred PBIS sequence is present in a prolamin compound sequence
such as gamma-zein, alpha-zein, delta-zein, beta-zein, rice
prolamin and gamma-gliadin that are discussed hereinafter.
[0102] A PBIS includes a sequence that directs a protein towards
the endoplasmic reticulum (ER) of the RPBLA-expressing cell. That
sequence often referred to as a leader sequence or signal peptide
can be from the same plant as the remainder of the PBIS or from a
different plant or an animal or fungus. Illustrative signal
peptides are the 19 residue gamma-zein signal peptide sequence
shown in WO 2004003207 (US 20040005660), the 19 residue signal
peptide sequence of alpha-gliadin or 21 residue gamma-gliadin
signal peptide sequence (see, Altschuler et al., 1993 Plant Cell
5:443-450; Sugiyama et al., 1986 Plant Sci. 44:205-209; and
Rafalski et al., 1984 EMBO J. 3(6):1409-11415 and the citations
therein). The pathogenesis-related protein of PR10 class includes a
25 residue signal peptide sequence that is also useful herein.
Similarly functioning signal peptides from other plants and animals
are also reported in the literature.
[0103] The characteristics of the signal peptides responsible for
directing the protein to the ER have been extensively studied (von
Heijne et al., 2001 Biochim. Biophys. Acta December 12
1541(1-2):114-119). The signal peptides do not share homology at a
primary structure, but have a common tripartite structure: a
central hydrophobic h-region and hydrophilic N- and C-terminal
flanking regions. These similarities, and the fact that proteins
are translocated through the ER membrane using apparently common
pathways, permits interchange of the signal peptides between
different proteins or even from different organisms belonging to
different phyla (See, Martoglio et al., 1998 Trends Cell Biol.
October; 8(10):410-415). Thus, a PBIS can include a signal peptide
of a protein from a phylum different from higher plants.
[0104] It is to be understood that an entire prolamin sequence is
not required to be used. Rather, as is discussed hereinafter, only
portions are needed although an entire prolamin sequence can be
used.
[0105] Gamma-Zein, a maize storage protein whose DNA and amino acid
residue sequences are shown hereinafter, is one of the four maize
prolamins and represents 10-15 percent of the total protein in the
maize endosperm. As other cereal prolamins, alpha- and gamma-zeins
are biosynthesized in membrane-bound polysomes at the cytoplasmic
side of the rough ER, assembled within the lumen and then
sequestered into ER-derived protein bodies (Herman et al., 1999
Plant Cell 11:601-613; Ludevid et al., 1984 Plant Mol. Biol.
3:277-234; Torrent et al., 1986 Plant Mol. Biol. 7:93-403).
[0106] Gamma-Zein is composed of four characteristic domains: i) a
peptide signal of 19 amino acids, ii) the repeat domain containing
eight units of the hexapeptide PPPVHL (SEQ ID NO:2) [(53 amino acid
residues (aa)], iii) the ProX domain where proline residues
alternate with other amino acids (29 aa) and iv) the hydrophobic
cysteine rich C-terminal domain (lll aa).
[0107] The ability of gamma-zein to assemble in ER-derived RPBLAs
is not restricted to seeds. In fact, when gamma-zein-gene was
constitutively expressed in transgenic Arabidopsis plants, the
storage protein accumulated within ER-derived PBLS in leaf mesophyl
cells (Geli et al., 1994 Plant Cell 6:1911-1922). Looking for a
signal responsible for the gamma-zein deposition into the
ER-derived protein bodies (prolamins do not have KDEL signal for
ER-retention), it has been demonstrated that the proline-rich
N-terminal domain including the tandem repeat domain was necessary
for ER retention. In this work, it was also suggested that the
C-terminal domain could be involved in protein body formation,
however, recent data (WO2004003207A1) demonstrate that the
proline-rich N-terminal domain is necessary and sufficient to
retain in the ER and to induce the protein body formation. However,
the mechanisms by which these domains promote the protein body
assembly are still unknown, but evidence from in vitro studies
suggests that the N-terminal portion of gamma-zein is able to
self-assemble into ordered structures.
[0108] It is preferred that a gamma-zein-based PBIS include at
least one repeat and the amino-terminal nine residues of the ProX
domain, and more preferably the entire Pro-X domain. The C-terminal
portion of gamma-zein is not needed, but can be present. Those
sequences are shown in US 20040005660 and designated as RX3 and P4,
respectively, and are noted hereinafter.
[0109] Zeins are of four distinct types: alpha, beta, delta, and
gamma. They accumulate in a sequential manner in the ER-derived
protein bodies during endosperm development. Beta-zein and
delta-zein do not accumulate in large amount in maize PBs, but they
were stable in the vegetative tissues and were deposited in
ER-derived protein body-like structures when expressed in tobacco
plants (Bagga et al., 1997 Plant Cell September 9(9):1683-1696).
This result indicates that beta-zein, as well as delta-zein, can
induce ER retention and PB formation.
[0110] The wheat prolamin storage proteins, gliadins, are a group
of K/HDEL-less proteins whose transport via the ER appears to be
complex. These proteins sequester in to the ER where they are
either retained and packaged into dense protein bodies, or are
transported from the ER via the Golgi into vacuoles. (Altschuler et
al., 1993 Plant Cell 5:443-450.)
[0111] The gliadins appear to be natural chimeras, containing two
separately folded autonomous regions. The N-terminus is composed of
about 7 to about 16 tandem repeats rich in glutamine and proline.
The sequence of the tandem repeats varies among the different
gliadins, but are based on one or the other consensus sequences
PQQPFPQ (SEQ ID NO:3), PQQQPPFS (SEQ ID NO:4) and PQQPQ (SEQ ID
NO:5). The C-terminal region of the protein contains six to eight
cysteines that form intramolecular disulfide bonds. The work of the
Altschuler et al. indicates that the N-terminal region and
consensus sequences are responsible for PB formation in the ER from
gamma-gliadin. (Altschuler et al., 1993 Plant Cell 5:443-450.)
[0112] Illustrative useful prolamin-type sequences are shown in the
Table below along with their GenBank identifiers.
TABLE-US-00001 PROTEIN NAME GENBANK ID .alpha.-Zein (22 kD) M86591
Albumin (32 kD) X70153 .gamma.-Zein (27 kD) X53514 .gamma.-Zein (50
kD) AF371263 .delta.-Zein (18 kD) AF371265 7S Globulin or Vicilin
type NM_113163 11S Globulin or Legumin type DQ256294 Prolamin 13 kD
AB016504 Prolamin 16 kD AY427574 Prolamin 10 kD AF294580
.gamma.-Gliadin M36999 .gamma.-Gliadin precursor AAA34272
[0113] Further useful sequences are obtained by carrying out a
BLAST search in the all non-redundant GenBank CDS
translations+PDB+SwissProt+PIR+PRF (excluding environmental
samples) data base as described in Altschul et al., 1997 Nucleic
Acids Res. 25:3389-3402 using a query such as those shown
below:
RX3 Query
TABLE-US-00002 [0114] SEQ ID NO: 6
PPPPVHLPPPVHLPPPVHLPPPVHLPPPVHLPPPVHLPPPVHVPPPVHL PPPP
Alpha-Zein
TABLE-US-00003 [0115] SEQ ID NO: 7
QQQQQFLPALSQLDVVNPVAYLQQQLLASNPLALANVAAYQQQQQLQQF
LPALSQLAMVNPAAYL
Rice Prolamin Query
TABLE-US-00004 [0116] SEQ ID NO: 8
QQVLSPYNEFVRQQYGIAASPFLQSATFQLRNNQVWQQLALVAQQSHCQ
DINIVQAIAQQLQLQQFGDLY
[0117] An illustrative modified prolamin includes (a) a signal
peptide sequence, (b) a sequence of one or more copies of the
repeat domain hexapeptide PPPVHL (SEQ ID NO: 2) of the protein
gamma-zein, the entire domain containing eight hexapeptide units;
and (c) a sequence of all or part of the ProX domain of gamma-zein.
Illustrative specific modified prolamins include the polypeptides
identified below as R3, RX3 and P4 whose DNA and amino acid residue
sequences are also shown below.
[0118] Particularly preferred prolamins include gamma-zein and its
component portions as disclosed in published application
WO2004003207, the rice rP13 protein and the 22 kDa maize alpha-zein
and its N-terminal fragment. The DNA and amino acid residue
sequences of the gamma-zein, rice and alpha-zein proteins are shown
below.
Gamma-Zein of 27 kD
DNA Sequence:
TABLE-US-00005 [0119] SEQ ID NO: 9 atgagggtgt tgctcgttgc cctcgctctc
ctggctctcg 40 ctgcgagcgc cacctccacg catacaagcg gcggctgcgg 80
ctgccagcca ccgccgccgg ttcatctacc gccgccggtg 120 catctgccac
ctccggttca cctgccacct ccggtgcatc 160 tcccaccgcc ggtccacctg
ccgccgccgg tccacctgcc 200 accgccggtc catgtgccgc cgccggttca
tctgccgccg 240 ccaccatgcc actaccctac tcaaccgccc cggcctcagc 280
ctcatcccca gccacaccca tgcccgtgcc aacagccgca 320 tccaagcccg
tgccagctgc agggaacctg cggcgttggc 360 agcaccccga tcctgggcca
gtgcgtcgag tttctgaggc 400 atcagtgcag cccgacggcg acgccctact
gctcgcctca 440 gtgccagtcg ttgcggcagc agtgttgcca gcagctcagg 480
caggtggagc cgcagcaccg gtaccaggcg atcttcggct 520 tggtcctcca
gtccatcctg cagcagcagc cgcaaagcgg 560 ccaggtcgcg gggctgttgg
cggcgcagat agcgcagcaa 600 ctgacggcga tgtgcggcct gcagcagccg
actccatgcc 640 cctacgctgc tgccggcggt gtcccccacg cc 672
Protein Sequence:
TABLE-US-00006 [0120] SEQ ID NO: 10 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Thr His Thr
Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30 Pro Val His Leu
Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu 35 40 45 Pro Pro
Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val 50 55 60
His Leu Pro Pro Pro Val His Val Pro Pro Pro Val His Leu Pro Pro 65
70 75 80 Pro Pro Cys His Tyr Pro Thr Gln Pro Pro Arg Pro Gln Pro
His Pro 85 90 95 Gln Pro His Pro Cys Pro Cys Gln Gln Pro His Pro
Ser Pro Cys Gln 100 105 110 Leu Gln Gly Thr Cys Gly Val Gly Ser Thr
Pro Ile Leu Gly Gln Cys 115 120 125 Val Glu Phe Leu Arg His Gln Cys
Ser Pro Thr Ala Thr Pro Tyr Cys 130 135 140 Ser Pro Gln Cys Gln Ser
Leu Arg Gln Gln Cys Cys Gln Gln Leu Arg 145 150 155 160 Gln Val Glu
Pro Gln His Arg Tyr Gln Ala Ile Phe Gly Leu Val Leu 165 170 175 Gln
Ser Ile Leu Gln Gln Gln Pro Gln Ser Gly Gln Val Ala Gly Leu 180 185
190 Leu Ala Ala Gln Ile Ala Gln Gln Leu Thr Ala Met Cys Gly Leu Gln
195 200 205 Gln Pro Thr Pro Cys Pro Tyr Ala Ala Ala Gly Gly Val Pro
His Ala 210 215 220
RX3
DNA Sequence:
TABLE-US-00007 [0121] SEQ ID NO: 11 atgagggtgt tgctcgttgc
cctcgctctc ctggctctcg 40 ctgcgagcgc cacctccacg catacaagcg
gcggctgcgg 80 ctgccagcca ccgccgccgg ttcatctacc gccgccggtg 120
catctgccac ctccggttca cctgccacct ccggtgcatc 160 tcccaccgcc
ggtccacctg ccgccgccgg tccacctgcc 200 accgccggtc catgtgccgc
cgccggttca tctgccgccg 240 ccaccatgcc actaccctac tcaaccgccc
cggcctcagc 280 ctcatcccca gccacaccca tgcccgtgcc aacagccgca 320
tccaagcccg tgccagacc 339
Protein Sequence:
TABLE-US-00008 [0122] SEQ ID NO: 12 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Thr His Thr
Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30 Pro Val His Leu
Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu 35 40 45 Pro Pro
Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val 50 55 60
His Leu Pro ProPro Val His Val Pro Pro Pro Val His Leu Pro Pro 65
70 75 80 Pro Pro Cys His Tyr Pro Thr Gln Pro Pro Arg Pro Gln Pro
His Pro 85 90 95 Gln Pro His Pro Cys Pro Cys Gln Gln Pro His Pro
Ser Pro Cys Gln 100 105 110 Tyr
R3
DNA Sequence:
TABLE-US-00009 [0123] SEQ ID NO: 13 atgagggtgt tgctcgttgc
cctcgctctc ctggctctcg 40 ctgcgagcgc cacctccacg catacaagcg
gcggctgcgg 80 ctgccagcca ccgccgccgg ttcatctacc gccgccggtg 120
catctgccac ctccggttca cctgccacct ccggtgcatc 160 tcccaccgcc
ggtccacctg ccgccgccgg tccacctgcc 200 accgccggtc catgtgccgc
cgccggttca tctgccgccg 240
Protein Sequence:
TABLE-US-00010 [0124] SEQ ID NO: 14 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Thr His Thr
Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30 Pro Val His Leu
Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu 35 40 45 Pro Pro
Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val 50 55 60
His Leu Pro Pro Pro Val His Val Pro Pro Pro Val His Leu Pro Pro 65
70 75 80 Pro Pro Cys His Tyr Pro Thr Gln Pro Pro Arg Tyr 85 90
P4
DNA Sequence:
TABLE-US-00011 [0125] SEQ ID NO: 15 atgagggtgt tgctcgttgc
cctcgctctc ctggctctcg 40 ctgcgagcgc cacctccacg catacaagcg
gcggctgcgg 80 ctgccagcca ccgccgccgg ttcatctgcc gccgccacca 120
tgccactacc ctacacaacc gccccggcct cagcctcatc 160 cccagccaca
cccatgcccg tgccaacagc cgcatccaag 200 cccgtgccag acc 213
Protein Sequence:
TABLE-US-00012 [0126] SEQ ID NO: 16 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Thr His Thr
Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30 Pro Val His Leu
Pro Pro Pro Pro Cys His Tyr Pro Thr Gln Pro Pro 35 40 45 Arg Pro
Gln Pro His Pro Gln Pro His Pro Cys Pro Cys Gln Gln Pro 50 55 60
His Pro Ser Pro Cys Gln Tyr 65 70
X10
DNA Sequence:
TABLE-US-00013 [0127] SEQ ID NO: 17 atgagggtgt tgctcgttgc
cctcgctctc ctggctctcg 40 ctgcgagcgc cacctccacg catacaagcg
gcggctgcgg 80 ctgccaatgc cactacccta ctcaaccgcc ccggcctcag 120
cctcatcccc agccacaccc atgcccgtgc caacagccgc 160 atccaagccc
gtgccagacc 180
Protein Sequence:
TABLE-US-00014 [0128] SEQ ID NO: 18 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala Leu Ala Ala Ser 1 5 10 15 Ala Thr Ser Thr His Thr
Ser Gly Gly Cys Gly Cys Gln Cys His Tyr 20 25 30 Pro Thr Gln Pro
Pro Arg Pro Gln Pro His Pro Gln Pro His Pro Cys 35 40 45 Pro Cys
Gln Gln Pro His Pro Ser Pro Cys Gln Tyr 50 55 60
rP13--rice prolamin of 13 kD homologous to the clone--AB016504 Sha
et al., 1996 Biosci. Biotechnol. Biochem. 60(2):335-337; Wen et
al., 1993 Plant Physiol. 101(3):1115-1116; Kawagoe et al., 2005
Plant Cell 17(4):1141-1153; Mullins et al., 2004 J. Agric. Food
Chem. 52(8):2242-2246; Mitsukawa et al., 1999 Biosci. Biotechnol.
Biochem. 63(11):1851-1858
Protein Sequence:
TABLE-US-00015 [0129] SEQ ID NO: 19
MKIIFVFALLAIAACSASAQFDVLGQSYRQYQLQSPVLLQQQVLSPYNEF
VRQQYGIAASPFLQSATFQLRNNQVWQQLALVAQQSHCQDINIVQAIAQQ
LQLQQFGDLYFDRNLAQAQALLAFNVPSRYGIYPRYYGAPSTITTLGGVL
DNA Sequence:
TABLE-US-00016 [0130] SEQ ID NO: 20
atgaagatcattttcgtctttgctctccttgctattgctgcatgcagcg
cctctgcgcagtttgatgttttaggtcaaagttataggcaatatcagct
gcagtcgcctgtcctgctacagcaacaggtgcttagcccatataatgag
ttcgtaaggcagcagtatggcatagcggcaagccccttcttgcaatcag
ctacgtttcaactgagaaacaaccaagtctggcaacagctcgcgctggt
ggcgcaacaatctcactgtcaggacattaacattgttcaggccatagcg
cagcagctacaactccagcagtttggtgatctctactttgatcggaatc
tggctcaagctcaagctctgttggcttttaacgtgccatctagatatgg
tatctaccctaggtactatggtgcacccagtaccattaccacccttggc ggtgtcttg
22aZt N-terminal fragment of the maize alpha-zein of 22 kD--V01475
Kim et al., 2002 Plant Cell 14(3):655-672; Woo et al., 2001 Plant
Cell 13(10):2297-2317; Matsushima et al., 1997 Biochim. Biophys.
Acta 1339(1):14-22; Thompson et al., 1992 Plant Mol. Biol.
18(4):827-833.
Protein Sequence (Full Length):
TABLE-US-00017 [0131] SEQ ID NO: 21
MATKILALLALLALFVSATNAFIIPQCSLAPSAIIPQFLPPVTSMGFEHL
AVQAYRLQQALAASVLQQPINQLQQQSLAHLTIQTIATQQQQQFLPALSQ
LDVVNPVAYLQQQLLASNPLALANVAAYQQQQQLQQFLPALSQL
DNA Sequence (Full Length):
TABLE-US-00018 [0132] SEQ ID NO: 22
atggctaccaagatattagccctccttgcgcttcttgccctttttgtgag
cgcaacaaatgcgttcattattccacaatgctcacttgctcctagtgcca
ttataccacagttcctcccaccagttacttcaatgggcttcgaacaccta
gctgtgcaagcctacaggctacaacaagcgcttgcggcaagcgtcttaca
acaaccaattaaccaattgcaacaacaatccttggcacatctaaccatac
aaaccatcgcaacgcaacagcaacaacagttcctaccagcactgagccaa
ctagatgtggtgaaccctgtcgcctacttgcaacagcagctgcttgcatc
caacccacttgctctggcaaacgtagctgcataccaacaacaacaacaat
tgcagcagtttctgccagcgctcagtcaacta
Gamma-Gliadin precursor--AAA34272--Scheets et al., 1988 Plant Sci.
57:141-150.
Protein Sequence:
TABLE-US-00019 [0133] SEQ ID NO: 23 NMQVDPSGQV QWPQQQPFPQ
PQQPFCQQPQ RTIPQPHQTF HHQPQQTFPQ PQQTYPHQPQ QQFPQPQQPQ QPFPQPQQTF
PQQPQLPFPQ QPQQPFPQPQ QPQQPFPQSQ QPQQPFPQPQ QQFPQPQQPQ QSFPQQQQPA
IQSFLQQQMN PCKNFLLQQC NHVSLVSSLV SIILPRSDCQ VMQQQCCQQL AQIPQQLQCA
AIHSVAHSII MQQEQQQGVP ILRPLFQLAQ GLGIIQPQQP AQLEGIRSLV LKTLPTMCNV
YVPPDCSTIN VPYANIDAGI GGQ
DNA Sequence (M36999)
TABLE-US-00020 [0134] SEQ ID NO: 24 gcatgcattg tcaaagtttg
tgaagtagaa ttaataacct tttggttatt gatcactgta tgtatcttag atgtcccgta
gcaacggtaa gggcattcac ctagtactag tccaatatta attaataact tgcacagaat
tacaaccatt gacataaaaa ggaaatatga tgagtcatgt attgattcat gttcaacatt
actacccttg acataaaaga agaatttgac gagtcgtatt agcttgttca tcttaccatc
atactatact gcaagctagt ttaaaaaaga atyaaagtcc agaatgaaca gtagaatagc
ctgatctatc tttaacaaca tgcacaagaa tacaaattta gtcccttgca agctatgaag
atttggttta tgcctaacaa catgataaac ttagatccaa aaggaatgca atctagataa
ttgtttgact tgtaaagtcg ataagatgag tcagtgccaa ttataaagtt ttcgccactc
ttagatcata tgtacaataa aaaggcaact ttgctgacca ctccaaaagt acgtttgtat
gtagtgccac caaacacaac acaccaaata atcagtttga taagcatcga atcactttaa
aaagtgaaag aaataatgaa aagaaaccta accatggtag ctataaaaag cctgtaatat
gtacactcca taccatcatc catccttcac acaactagag cacaagcatc aaatccaagt
aagtattagt t aacgcaaat ccaccatgaa gaccttactc atcctaacaa tccttgcgat
ggcaacaacc atcgccaccg ccaatatgca agtcgacccc agcggccaag tacaatggcc
acaacaacaa ccattccccc agccccaaca accattctgc cagcaaccac aacgaactat
tccccaaccc catcaaacat tccaccatca accacaacaa acatttcccc aaccccaaca
aacatacccc catcaaccac aacaacaatt tccccagacc caacaaccac aacaaccatt
tccccagccc caacaaacat tcccccaaca accccaacta ccatttcccc aacaacccca
acaaccattc ccccagcctc agcaacccca acaaccattt ccccagtcac aacaaccaca
acaacctttt ccccagcccc aacaacaatt tccgcagccc caacaaccac aacaatcatt
cccccaacaa caacaaccgg cgattcagtc atttctacaa caacagatga acccctgcaa
gaatttcctc ttgcagcaat gcaaccatgt gtcattggtg tcatctctcg tgtcaataat
tttgccacga agtgattgcc aggtgatgca gcaacaatgt tgccaacaac tagcacaaat
tcctcaacag ctccagtgcg cagccatcca cagcgtcgcg cattccatca tcatgcaaca
agaacaacaa caaggcgtgc cgatcctgcg gccactattt cagctcgccc agggtctggg
tatcatccaa cctcaacaac cagctcaatt ggaggggatc aggtcattgg tattgaaaac
tcttccaacc atgtgcaacg tgtatgtgcc acctgactgc tccaccatca acgtaccata
tgccaacata gacgctggca ttggtggcca atgaaaaatg caagatcatc attgcttagc
tgatgcacca atcgttgtag cgatgacaaa taaagtggtg tgcaccatca tgtgtgaccc
cgaccagtgc tagttcaagc ttgggaataa aagacaaaca aagttcttgt ttgctagcat
tgcttgtcac tgttacattc actttttatt tcgatgttca tccctaaccg caatcctagc
cttacacgtc aatagctagc tgcttgtgct ggcaggttac tatataatct atcaattaat
ggtcgaccta ttaatccaag taataggcta ttgatagact gctcccaagc cgaccgagca
cctatcagtt acggatttct tgaacattgc acactataat aattcaacgt atttcaacct
ctagaagtaa agggcatttt agtagc
Beta zein--AF371264--Woo et al., (2001) Plant Cell 13 (10),
2297-2317.
DNA
TABLE-US-00021 [0135] SEQ ID NO: 25
atgaagatggtcatcgttctcgtcgtgtgcctggctctgtcagctgccag
cgcctctgcaatgcagatgccctgcccctgcgcggggctgcagggcttgt
acggcgctggcgccggcctgacgacgatgatgggcgccggcgggctgtac
ccctacgcggagtacctgaggcagccgcagtgcagcccgctggcggcggc
gccctactacgccgggtgtgggcagccgagcgccatgttccagccgctcc
ggcaacagtgctgccagcagcagatgaggatgatggacgtgcagtccgtc
gcgcagcagctgcagatgatgatgcagcttgagcgtgccgctgccgccag
cagcagcctgtacgagccagctctgatgcagcagcagcagcagctgctgg
cagcccagggtctcaaccccatggccatgatgatggcgcagaacatgccg
gccatgggtggactctaccagtaccagctgcccagctaccgcaccaaccc
ctgtggcgtctccgctgccattccgccctactactga
Protein
TABLE-US-00022 [0136] SEQ ID NO: 26
MKMVIVLVVCLALSAASASAMQMPCPCAGLQGLYGAGAGLTTMMGAGGLY
PYAEYLRQPQCSPLAAAPYYAGCGQPSAMFQPLRQQCCQQQMRMMDVQSV
AQQLQMMMQLERAAAASSSLYEPALMQQQQQLLAAQGLNPMAMMMAQNMP
AMGGLYQYQLPSYRTNPCGVSAAIPPYY
Delta zein 10 kD--AF371266--Woo et al., (2001) Plant Cell 13 (10),
2297-2317. and Kirihara et al., (1988) Gene. November 30;
71(2):359-70.
DNA
TABLE-US-00023 [0137] SEQ ID NO: 27
atggcagccaagatgcttgcattgttcgctctcctagctctttgtgcaag
cgccactagtgcgacgcatattccagggcacttgccaccagtcatgccat
tgggtaccatgaacccatgcatgcagtactgcatgatgcaacaggggctt
gccagcttgatggcgtgtccgtccctgatgctgcagcaactgttggcctt
accgcttcagacgatgccagtgatgatgccacagatgatgacgcctaaca
tgatgtcaccattgatgatgccgagcatgatgtcaccaatggtcttgccg
agcatgatgtcgcaaatgatgatgccacaatgtcactgcgacgccgtctc
gcagattatgctgcaacagcagttaccattcatgttcaacccaatggcca
tgacgattccacccatgttcttacagcaaccctttgttggtgctgcattc tag
Protein
TABLE-US-00024 [0138] SEQ ID NO: 28
MAAKMLALFALLALCASATSATHIPGHLPPVMPLGTMNPCMQYCMMQQGL
ASLMACPSLMLQQLLALPLQTMPVMMPQMMTPNMMSPLMMPSMMSPMVLP
SMMSQMMMPQCHCDAVSQIMLQQQLPFMFNPMAMTIPPMFLQQPFVGAAF
Signal Peptides
Gamma-Zein
TABLE-US-00025 [0139] SEQ ID NO: 29 Met Arg Val Leu Leu Val Ala Leu
Ala Leu Leu Ala Leu Ala Ala Ser Ala Thr Ser
Alpha-Gliadin
TABLE-US-00026 [0140] SEQ ID NO: 30 Met Lys Thr Phe Leu Ile Leu Val
Leu Leu Ala Ile Val Ala Thr Thr Ala Thr Thr Ala
Gamma-Gliadin
TABLE-US-00027 [0141] SEQ ID NO: 31 Met Lys Thr Leu Leu Ile Leu Thr
Ile Leu Ala Met Ala Ile Thr Ile Gly Thr Ala Asn Met
PR10
TABLE-US-00028 [0142] SEQ ID NO: 32 Met Asn Phe Leu Lys Ser Phe Pro
Phe Tyr Ala Phe Leu Cys Phe Gly Gln Tyr Phe Val Ala Val Thr His
Ala
T-Cell Stimulating Immunogenic Polypeptides
[0143] A large number of T-cell-stimulating immunogenic polypeptide
sequences have been identified in the literature. A partial list is
provided below in the table below using the single letter code.
TABLE-US-00029 T Cell Epitopes SEQ Organism Protein Sequence*
Citation ID NO HIV P24 GPKEPFRDY- 1 33 VDRFYKC Corynebacterium
toxin FQVVHNSYN- 2 34 diptheriae RPAYSPGC Borrelia ospA
VEIKEGTVTLKRE- 3 35 burgdorferi IDKNGKVTVSLC TLSKNISKSG- 4 36
EVSVELNDC Influenza Virus HA SSVSSFERFEC 5 37 A8/PR8 LIDALLGDPC 6
38 TLIDALLGC 6 39 NP FWRGENGRKTRS- 7 40 AYERMCNILKGK LRVLSFIRGTKV-
7 41 SPRGKLSTRG SLVGIDPFKLLQ- 7 42 NSQVYSLIRP AVKGVGTMVMEL- 7 43
IRMIKRGINDRN Trypanosoma SHNFTLVASVII- 8 44 cruzi EEAPSGNTC
Plasmodium MSP1 SVQIPKVPYPNGIVYC 9 45 falciparum DFNHYYTLKTGLEADC
46 PSDKHIEQYKKI- 10 47 KNSISC EYLNKIQNSLST- 11 48 EWSPCSVT P. vivax
YLDKVRATVGTE- 22 49 WTPCSVT P. yoelii EFVKQISSQLTE- 22 50 EWSQCSVT
Streptococcus AgI/II KPRPIYEAKL- 12 51 sobrinus AQNQKC AKADYEAKLA-
52 QYEKDLC LCMV (lymphocytic NP RPQASGVYM- 13 53 choriomeningitis
virus) GNLTAQC Clostridium tox QYIKANSKFIG- 14 54 tetani ITELC
Neisseria PorB AIWQVEQKASIAGTDSGWC 21 55 meningitidis
NYKNGGFFVQYGGAYKRHC 21 56 HNSQTEVAATLAYRFGNVC 21 57 PorB
TPRVSYAHGFKGLVDDADC 21 58 RFGNAVPRISYAHGFDFIC 21 59
AFKYARHANVGRNAFELFC 21 60 SGAWLKRNTGIGNYTQINAC 21 61
AGEFGTLRAGRVANQC 21 62 IGNYTQINAASVGLRC 21 63 GRNYQLQLTEQPSRTC 21
64 SGSVQFVPAQNSKSAC 21 65 HANVGRDAFNLFLLGC 21 66 LGRIGDDDEAKGTDPC
21 67 SVQFVPAQNSKSAYKC 21 68 NYAFKYAKHANVGRDC 21 69
AHGFDFIERGKKGENC 21 70 GVDYDFSKRTSAIVSC 21 71 HDDMPVSVRYDSPDFC 21
72 RFGNAVPRISYAHGFD3 FIERGKKGENC 21 73 NYAFKYAKHANVGRDA- 21 74
FNLFLLGC SGAWLKRNTGIGNYTQ- 21 75 INAASVGLRC SGSVQFVPAQNSKSAYTPAC 21
76 OpaB TGANNTSTVSDYFRNRITC 21 77 IYDFKLNDKFDKFKPYIGC 21 78 Opa-5d
LSAIYDFKLNDKFKPYIGC 21 79 Opac NGWYINPWSEVKFDLNSRC 21 80 Hepatitis
B Surface MGTNLSVPN- 15, 16 81 PreS1 PLGFFPDHQLDP PLGFFPDH 82
PLGFFPDHQL 83 PreS2 MQWNSTAFHQ- 15 84 TLQDPRVRG- LYLPAGG MQWSTAFHQ-
85 TLQDP MQWNSTALHQ- 86 ALQDP QDPRVR 17 87 Core MDIDPYKEFGAT- 18 88
VELLSFLP RDLLDTASALYR- 18 89 EALESPEHCSPHH TWVGVNLEDPAS- 18 90
RDLVVSYVNTNMG VVSYVNTNMGL- 18 91 KFRQL LLWFHISCLTF- 18 92
GRETVIEYLV LLWFHISCLTF- 18 93 VSFGVWIRTPP- 18 94 AYRPPNAPIL
VSFGVWIRTPPA 18 95 PPAYRPPNAPIL 18 96 WIRTPPAYRPPN 18 97
PHHTALRQAIL- 19 98 CWGELMTLA M. tuberculosis 65 KD Protein
AVLEDPYILLVSSKV 20 99 LLVSSKVSTVKDLLP 20 100 LLPLLEKVIGAGKPL 20 101
AILTGGQVISEEVGL 20 102 IAFNSGLEPGVVAEK 20 103 ARRGLERGLNAL- 20 104
ADAVKV EKIGAELVKEVAKK 20 105 GLKRGIEKAVEKVETL 20 106
IEDAVRNAKAAVEEG 20 107 HPV-16 E6 Protein TIHDIILEC 23 121
FAFRDLCIVY 23 122 E7 Protein YMLDLQPETT 23 123 LEDLLMGTL 23 124
DLYCYEQLN 24 125 *Underlined C (C) is not from the native
sequence.
CITATIONS
[0144] 1. U.S. Pat. No. 5,639,854. [0145] 2. EPO 399001 B1. [0146]
3. Bockenstedt et al. (1996) J. Immunol., 157(12):5496-5502. [0147]
4. Zhong et al. (1996) Eur. J. Immunol., 26(11):2749. [0148] 5.
Brumeanu et al. (1996) Immunotechnology, 2(2):85-95. [0149] 6.
Brown et al. (1993) J. Virol., 67(5):2887-2893. [0150] 7. Brett et
al., (1991) J. Immunol., 147(3):984-991. [0151] 8. Kahn et al.
(1997) J. Immunol., 159(9):4444. [0152] 9. Ohta et al. (1997) Int.
Arch. Allergy Immunol., 114(1):15. [0153] 10. U.S. Pat. No.
4,886,782. [0154] 11. Calvo-Calle et al. (1997) J. Immunol.
159(3):1362-1373. [0155] 12. Staffileno et al. (1990) Arch. Oral
Biol., 35: Suppl. 47S. [0156] 13. Saron et al. (1997) Proc. Natl.
Acad. Sci. USA 94(7):3314-3319. [0157] 14. Yang et al. (1997)
Vaccine, 15(4):377-386. [0158] 15. Neurath et al., (1986) F. Brown
et al. eds., Vaccines 85, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., pp. 185-189. [0159] 16. Milich et al., (1987)
F. Brown et al. eds., Vaccines 87, Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York, pp. 50-55. [0160] 17. Kent et al.,
(1987) F. Brown et al. eds., Vaccines 86, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., pp. 365-369. [0161] 18. U.S.
Pat. No. 4,882,145. [0162] 19. Alexander et al., (1994) Immunity
1:751-761. [0163] 20. U.S. Pat. No. 5,478,726 [0164] 21. WO
03/072731 [0165] 22. U.S. Pat. No. 6,942,866 [0166] 23. U.S. Patent
Publication 20060182763 [0167] 24. U.S. Pat. No. 7,329,498.
[0168] A group of preferred T-cell stimulating immunogenic
sequences are present in HPV-16, in the E7 gene. In order to
translate the previously discussed therapeutic DNA vaccine
candidate [Osen et al., 2001 Vaccine 19(30):4276-4286] into a
vaccine for use in a clinical trial, the safety features were
further enhanced. For this reason, no heterologous genes were
fused. Rather, immunogenicity was enhanced by placing a
Kozak-sequence [Kozak et al., 1987 Nucleic Acids Res
15(20):8125-8148] in front of the gene [Steinberg et al., 2005
Vaccine 23(9):1149-1157]. A plasmid-vector pTHamp [Hanke et al.,
(1998) Vaccine 16(4):426-435] applicable to humans [Hanke et al.,
2000 Nat Med 6(9):951-955] was selected. Many expression vectors
are known and available for use for DNA vaccines. See for example,
U.S. Pat. No. 7,351,813 B2 and EP 1 026 253 B1 and the citations
therein.
[0169] More importantly E7 itself was redesigned. The sequence was
taken apart exactly at the positions that are critical for
transforming properties of the protein (pRB-binding site, C-X-X-C
motifs) and reassembled in a "shuffled" order as "core" gene. This
sequence was codon optimized to humans (almost identical to mice).
The original junctions destroyed by the dissection were added as an
"appendix" with a non-codon optimized sequence to minimize
recombination events reconstituting the wild-type sequences (see
also FIG. 6).
[0170] Tumor protection and regression studies provide a first
impression on immunogenicity and effectivity of tumor vaccines.
Those studies do not fully reflect, however, the responses induced
in humans. "In vitro immunization" of human lymphocytes by
antigen-loaded dendritic cells (DCs) can be used as a model of
human responses [Norm et al., 2003 J Cancer Res Clin Oncol
129(9):511-520]. Loading of DCs by DNA transfection is a suitable
technique [Lohmann et al., 2000 Cancer Gene Ther 7(4):605-614] and
specific T cell priming verifies the potential immunogenicity of
the DNA vaccine candidate.
[0171] The results shown hereinafter illustrate that the HPV-16
E7SH DNA vaccine candidate of the second generation induces
specific immunity in vivo in mice and after in vitro immunization
of human lymphocytes and, therefore, can provide for a safe
therapeutic HPV-vaccine.
[0172] The sequence of the gene that expresses the HPV-16 E7SH
protein is as follows from 5' to 3':
TABLE-US-00030 SEQ ID NO: 126 CCC GCC GCC ACC ATG CAC GGC GAC ACC
CCC ACC CTG CAC GAG TAC ATG CTG GAC CTG CAG CCC GAG ACC ACC GAC CTG
TAC TGC ATC TGC AGC CAG AAA CCC AAG TGC GAC AGC ACC CTG CGG CTG TGC
GTG CAG AGC ACC CAC GTG GAC ATC CGG ACC CTG GAG GAC CTG CTG ATG GGC
ACC CTG GGC ATC GTG TGC CCC TAC GAG CAG CTG AAC GAC AGC AGC GAG GAG
GAG GAT GAG ATC GAC GGC CCC GCC GGC CAG GCT GAG CCC GAC CGG GCC CAC
TAC AAC ATC GTG ACC TTC TGC TGC CAA CCA GAG ACA ACT GAT CTC TAC TGT
TAT GAG CAA TTA AAT GAC AGC TCA GAG CAT TAC AAT ATT GTA ACC TTT TGT
TGC AAG TGT GAC TCT ACG CTT CGG TTG TGC ATG GGC ACA CTA GGA ATT GTG
TGC CCC ATC TGT TCT CAG AAA CCA TAA
[0173] Another group of preferred T-cell stimulating immunogenic
sequences are present in HIV-1. In particularly preferred practice,
a polypeptide sequence present in HIV-1 is encoded by the HIV-1 gag
gene. These sequences are thus present in the P24, P17, P6 or P9
proteins encoded by the gag gene, or a polypeptide such as the P41
polypeptide.
[0174] A particular T-cell stimulating immunogenic sequence need
not itself be present as a distinct polypeptide in HIV-1 or any
other pathogen. Rather, such a sequence is present as a portion of
a distinct polypeptide or proteinaceous material encoded by an open
reading frame of a pathogenic genome.
[0175] Specific T-cell stimulating immunogenic sequences useful
herein are provided below.
p41 DNA Sequence 5' to 3'
TABLE-US-00031 [0176] SEQ ID NO: 108 1 ATGGGTGCTA GAGCTTCTAT
TCTTAGAGGT GAAAAGCTTG ATAAGTGGGA AAAGATTAGA 61 CTTAGACCAG
GTGGTAAGAA GCATTATATG CTTAAGCATA TTGTTTGGGC TTCTAGAGAA 121
CTTGAAAGAT TTGCTCTTAA TCCAGGTTTG CTTGAAACTT CTGAAGGTTG TAAGCAAATT
181 ATGAAGCAAC TTCAACCAGC TCTTCAAACT GGTACTGAAG AACTTAAGTC
TCTTTATAAT 241 ACTGTTGCTA CTCTTTATTG TGTTCATGAA AAGATTGAAG
TTAGAGATAC TAAGGAAGCT 301 CTTGATAAGA TTGAAGAAGA ACAAAATAAG
TGTCAACAAA AGACTCAACA AGCTAAGGCT 361 GCTGATGGTA AGGTTTCTCA
AAATTATCCA ATTGTTCAAA ATCTTCAAGG TCAAATGGTT 421 CATCAAGCTA
TTTCTCCAAG AACTCTTAAT GCTTGGGTTA AGGTTATTGA AGAAAAGGCT 481
TTTTCTCCAG AAGTTATTCC AATGTTTACT GCTCTTTCTG AAGGTGCTAC TCCACAAGAT
541 CTTAATACTA TGCTTAATAC TGTTGGTGGT CATCAAGCTG CTATGCAAAT
GCTTAAGGAT 601 ACTATTAATG AAGAAGCTGC TGAATGGGAT AGACTTCATC
CAGTTCATGC TGGTCCAATT 661 GCTCCAGGTC AAATGAGAGA ACCAAGAGGT
TCTGATATTG CTGGTACTAC TTCTACTCTT 721 CAAGAACAAA TTGCTTGGAT
GACTTCTAAT CCACCAATTC CAGTTGGTGA TATTTATAAG 781 AGATGGATTA
TTCTTGGTCT TAATAAGATT GTTAGAATGT ATTCTCCAGT TTCTATTCTT 841
GATATTAGAC AAGGTCCAAA GGAACCATTT AGAGATTATG TTGATAGATT TTTTAAGACT
901 CTTAGAGCTG AACAAGCTAC TCAAGAAGTT AAGAATTGGA TGACTGATAC
TCTTCTTGTT 961 CAAAATGCTA ATCCAGATTG TAAGACTATT CTTAGGGCTC
TTGGTCCAGG TGCTACTCTT 1021 GAAGAAATGA TGACTGCTTG TCAAGGTGTT
GGTGGTCCAG GTCATAAGGC TAGAGTTCTT 1081 TAA
p41 Amino Acid Sequence
Translation of P41 (1-1083)
[0177] Universal code Total amino acid number: 360, MW=40309 Max
ORF starts at AA pos 1 (may be DNA pos 1) for 360 AA (1080
bases),
MW=40309
Origin
TABLE-US-00032 [0178] SEQ ID NO: 109 1 MGARASILRG EKLDKWEKIR
LRPGGKKHYM LKHIVWASRE LERFALNPGL LETSEGCKQI 61 MKQLQPALQT
GTEELKSLYN TVATLYCVHE KIEVRDTKEA LDKIEEEQNK CQQKTQQAKA 121
ADGKVSQNYP IVQNLQGQMV HQAISPRTLN AWVKVIEEKA FSPEVIPMFT ALSEGATPQD
181 LNTMLNTVGG HQAAMQMLKD TINEEAAEWD RLHPVHAGPI APGQMREPRG
SDIAGTTSTL 241 QEQIAWMTSN PPIPVGDIYK RWIILGLNKI VRMYSPVSIL
DIRQGPKEPF RDYVDRFFKT 301 LRAEQATQEV KNWMTDTLLV QNANPDCKTI
LRALGPGATL EEMMTACQGV GGPGHKARVL 361 *
p24 DNA Sequence 5' to 3'
TABLE-US-00033 [0179] SEQ ID NO: 110 1 ATGCCAATTG TTCAAAATCT
TCAAGGTCAA ATGGTTCATC AAGCTATTTC TCCAAGAACT 61 CTTAATGCTT
GGGTTAAGGT TATTGAAGAA AAGGCTTTTT CTCCAGAAGT TATTCCAATG 121
TTTACTGCTC TTTCTGAAGG TGCTACTCCA CAAGATCTTA ATACTATGCT TAATACTGTT
181 GGTGGTCATC AAGCTGCTAT GCAAATGCTT AAGGATACTA TTAATGAAGA
AGCTGCTGAA 241 TGGGATAGAC TTCATCCAGT TCATGCTGGT CCAATTGCTC
CAGGTCAAAT GAGAGAACCA 301 AGAGGTTCTG ATATTGCTGG TACTACTTCT
ACTCTTCAAG AACAAATTGC TTGGATGACT 361 TCTAATCCAC CAATTCCAGT
TGGTGATATT TATAAGAGAT GGATTATTCT TGGTCTTAAT 421 AAGATTGTTA
GAATGTATTC TCCAGTTTCT ATTCTTGATA TTAGACAAGG TCCAAAGGAA 481
CCATTTAGAG ATTATGTTGA TAGATTTTTT AAGACTCTTA GAGCTGAACA AGCTACTCAA
541 GAAGTTAAGA ATTGGATGAC TGATACTCTT CTTGTTCAAA ATGCTAATCC
AGATTGTAAG 601 ACTATTCTTA GGGCTCTTGG TCCAGGTGCT ACTCTTGAAG
AAATGATGAC TGCTTGTCAA 661 GGTGTTGGTG GTCCAGGTCA TAAGGCTAGA
GTTCTTTAA
p24 Amino Acid Sequence
Translation of p24 (1-699)
[0180] Universal code Total amino acid number: 232, MW=25660 Max
ORF starts at AA pos 1 (may be DNA pos 1) for 232 AA (696
bases),
MW=25660
Origin
TABLE-US-00034 [0181] SEQ ID NO: 111 1 MPIVQNLQGQ MVHQAISPRT
LNAWVKVIEE KAFSPEVIPM FTALSEGATP QDLNTMLNTV 61 GGHQAAMQML
KDTINEEAAE WDRLHPVHAG PIAPGQMREP RGSDIAGTTS TLQEQIAWMT 121
SNPPIPVGDI YKRWIILGLN KIVRMYSPVS ILDIRQGPKE PFRDYVDRFF KTLRAEQATQ
181 EVKNWMTDTL LVQNANPDCK TILRALGPGA TLEEMMTACQ GVGGPGHKAR VL
RT DNA Sequence 5' to 3'
TABLE-US-00035 [0182] SEQ ID NO: 112 1 ATGAGGGTGT TGCTCGTTGC
CCTCGCTCTC CTGGCTCTCG CTGCGAGCGC CACCTCCACG 61 CATACAAGCG
GCGGCTGCGG CTGCCAGCCA CCGCCGCCGG TTCATCTACC GCCGCCGGTG 121
CATCTGCCAC CTCCGGTTCA CCTGCCACCT CCGGTGCATC TCCCACCGCC GGTCCACCTG
181 CCGCCGCCGG TCCACCTGCC ACCGCCGGTC CATGTGCCGC CGCCGGTTCA
TCTGCCGCCG 241 CCACCATGCC ACTACCCTAC TCAACCGCCC CGGCCTCAGC
CTCATCCCCA GCCACACCCA 301 TGCCCGTGCC AACAGCCGCA TCCAAGCCCG
TGCCAGACCA TGGACGACGA TGATAAGTGC 361 GGCAAGAAGG CCATCGGCAC
CGTGCTGGTG GGCCCCACCC CCGTGAACAT CATCGGCCGG 421 AACATGCTGA
CCCAGCTGGG CTGCACCCTG AACTTCCCCA TCAGCCCCAT CGAGACCGTG 481
CCCGTGAAGC TGAAGCCCGG CATGGACGGC CCCAAGGTGA AGCAGTGGCC CCTGACCGAG
541 GTGAAGATCA AGGCCCTGAC CGCCATCTGC GAGGAGATGG AGAAGGAGGG
CAAGATCACC 601 AAGATCGGCC CCGAGAACCC CTACAACACC CCCATCTTCG
CCATCAAGAA GGAGGACAGC 661 ACCAAGTGGC GGAAGCTGGT GGACTTCCGG
GAGCTGAACA AGCGGACCCA GGACTTCTGG 721 GAGGTGCAGC TGGGCATCCC
CCACCCCGCC GGCCTGAAGA AGAAGAAGAG CGTGACCGTG 781 CTGGACGTGG
GCGACGCCTA CTTCAGCGTG CCCCTGGACG AGGGCTTCCG GAAGTACACC 841
GCCTTCACCA TCCCCAGCAT CAACAACGAG ACCCCCGGCA TCCGGTACCA GTACAACGTG
901 CTGCCCCAGG GCTGGAAGGG CAGCCCCGCC ATCTTCCAGG CCAGCATGAC
CAAGATCCTG 961 GAGCCCTTCC GGGCCAAGAA CCCCGAGATC GTGATCTACC
AGTACATGGC CGCCCTGTAC 1021 GTGGGCAGCG ACCTGGAGAT CGGCCAGCAC
CGGGCCAAGA TCGAGGAGCT GCGGGAGCAC 1081 CTGCTGAAGT GGGGCTTCAC
CACCCCCGAC AAGAAGCACC AGAAGGAGCC CCCCTTCCTG 1141 TGGATGGGCT
ACGAGCTGCA CCCCGACAAG TGGACCGTGC AGCCCATCCA GCTGCCCGAG 1201
AAGGACAGCT GGACCGTGAA CGACATCCAG AAGCTGGTGG GCAAGCTGAA CTGGACCAGC
1261 CAGATCTACC CCGGCATCAA GGTGCGGCAG CTGTGCAAGC TGCTGCGGGG
CACCAAGGCC 1321 CTGACCGACA TCGTGCCCCT GACCGAGGAG GCCGAGCTGG
AGCTGGCCGA GAACCGGGAG 1381 ATCCTGAAGG AGCCCGTGCA CGGCGTGTAC
TACGACCCCA GCAAGGACCT GATCGCCGAG 1441 ATCCAGAAGC AGGGCGACGA
CCAGTGGACC TACCAGATCT ACCAGGAGCC CTTCAAGAAC 1501 CTGAAAACCG
GCAAGTACGC CAAGCGGCGG ACCACCCACA CCAACGACGT GAAGCAGCTG 1561
ACCGAGGCCG TGCAGAAGAT CAGCCTGGAG AGCATCGTGA CCTGGGGCAA GACCCCCAAG
1621 TTCCGGCTGC CCATCCAGAA GGAGACCTGG GAGATCTGGT GGACCGACTA
CTGGCAGGCC 1681 ACCTGGATCC CCGAGTGGGA GTTCGTGAAC AGCGGCCGCT
TTCGAATCTA G
RT Amino Acid Sequence
Translation of RT (1-1731)
[0183] Universal code Total amino acid number: 576, MW=65360 Max
ORF starts at AA pos 1 (may be DNA pos 1) for 576 AA (1728
bases),
MW=65360
Origin
TABLE-US-00036 [0184] SEQ ID NO: 113 1 MRVLLVALAL LALAASATST
HTSGGCGCQP PPPVHLPPPV HLPPPVHLPP PVHLPPPVHL 61 PPPVHLPPPV
HVPPPVHLPP PPCHYPTQPP RPQPHPQPHP CPCQQPHPSP CQTMDDDDKC 121
GKKAIGTVLV GPTPVNIIGR NMLTQLGCTL NFPISPIETV PVKLKPGMDG PKVKQWPLTE
181 VKIKALTAIC EEMEKEGKIT KIGPENPYNT PIFAIKKEDS TKWRKLVDFR
ELNKRTQDFW 241 EVQLGIPHPA GLKKKKSVTV LDVGDAYFSV PLDEGFRKYT
AFTIPSINNE TPGIRYQYNV 301 LPQGWKGSPA IFQASMTKIL EPFRAKNPEI
VIYQYMAALY VGSDLEIGQH RAKIEELREH 361 LLKWGFTTPD KKHQKEPPFL
WMGYELHPDK WTVQPIQLPE KDSWTVNDIQ KLVGKLNWTS 421 QIYPGIKVRQ
LCKLLRGTKA LTDIVPLTEE AELELAENRE ILKEPVHGVY YDPSKDLIAE 481
IQKQGDDQWT YQIYQEPFKN LKTGKYAKRR TTHTNDVKQL TEAVQKISLE SIVIWGKTPK
541 FRLPIQKETW EIWWTDYWQA TWIPEWEFVN SGRFRI*
NSs DNA Sequence 5' to 3'
[0185] NSs is a silencing suppressor used in the agroinfiltration
of tobacco plants.
TABLE-US-00037 SEQ ID NO: 114 1 ATGTCTTCAA GTGTTTATGA GTCGATCATT
CAGACAAAAG CTTCAGTCTG GGGATCAACT 61 GCATCTGGTA AAGCTGTTGT
AGATTCTTAC TGGATTCATG AACTTGGTAC TGGTTCTCCA 121 CTAGTTCAAA
CCCAGCTGTA TTCTGATTCA AGAAGCAAAA GTAGCTTTGG CTATACTGCA 181
AAGGTAGGGA ATCTTCCCTG TGAGGAAGAA GAAATTCTTT CTCAGCATGT GTATATCCCT
241 ATTTTTGATG ATGTTGATTT TAGCATCAAT ATTGATGACT CTGTTCTGGC
ACTGTCTGTT 301 TGCTCCAACA CAGTCAATAC TAACGGAGTG AAACATCAAG
GTCATTTGAA AGTTTTGTCT 361 CCTGCTCAGC TCCACTCTAT TGGATCTACC
ATGAACGGAT CTGATATTAC AGACCGATTC 421 CAGCTCCAAG AAAAAGATAT
AATTCCCAAT GACAGGTACA TTGAAGCTGT AAACAAAGGC 481 TCTTTGTCTT
GTGTTAAAGA GCATACCTAT AAGGTCGAGA TGTGCTACAA TCAAGCTTTA 541
GGCAAAGTGA ATGTTCTATC CCCTAACAGA AATGTCCATG AATGGCTGTA CAGTTTCAAG
601 CCAAATTTCA ATCAAGTTGA AAGCAACAAC AGAACTGTAA ATTCTCTTGC
AGTGAAATCT 661 CTGCTCATGT CAGCAGGAAA TAACATCATG CCTAACTCTC
AGGCTTTTGT CAAAGCTTCC 721 ACTGATTCTC ATTTCAAGCT GAGCCTCTGG
CTAAGAGTTC CAAAGGTTTT GAAGCAGATT 781 TCCATTCAGA AATTGTTCAA
AGTTGCAGGA GATGAAACTA ACAAAACATT TTATTTATCT 841 ATTGCTTGCA
TTCCAAACCA TAACAGTGTT GAGACAGCTT TAAACATTTC TGTTATTTGC 901
AAGCATCAGC TCCCAATCCG TAAATTTAAA GCTCCTTTTG AATTATCAAT GATGTTTTCT
961 GATTTAAAGG AGCCTTACAA CATTGTTCAT GATCCTTCAT ATCCTCAGAG
GATTGTTCAT 1021 GCTCTGCTTG AAACTCACAC GTCTTTTGCA CAAGTTCTTT
GCAACAACTT GCAAGAAGAC 1081 GTGATCATCT ACACTTTGAA CAACTATGAG
CTAACTCCTG GAAAGTTAGA TCTAGGTGAA 1141 AGAACCTTAA ATTACAGTGA
AGATGTCTGC AAAAGGAAAT ATTTCCTCTC AAAAACACTT 1201 GAATGTCTTC
CATCTAACAC ACAAACTATG TCTTACTTAG ACAGCATCCA AATCCCTTCC 1261
TGGAAGATAG ACTTTGCTAG GGGAGAAATT AAAATTTCTC CACAATCTGT TTCAGTTGCA
1321 AAATCTTTGT TAAAGCTTGA TTTAAGTGGG ATCAAAAAGA AAGAATCTAA
GATTTCGGAA 1381 GCATGTGCTT CAGGATCAAA ATAA
Translation of NSs (1-1404)
[0186] Universal code Total amino acid number: 467, MW=52121 Max
ORF starts at AA pos 1 (may be DNA pos 1) for 467 AA (1401
bases),
MW=52121
Origin
TABLE-US-00038 [0187] SEQ ID NO: 115 1 MSSSVYESII QTKASVWGST
ASGKAVVDSY WIHELGTGSP LVQTQLYSDS RSKSSFGYTA 61 KVGNLPCEEE
EILSQHVYIP IFDDVDFSIN IDDSVLALSV CSNTVNTNGV KHQGHLKVLS 121
PAQLHSIGST MNGSDITDRF QLQEKDIIPN DRYIEAVNKG SLSCVKEHTY KVEMCYNQAL
181 GKVNVLSPNR NVHEWLYSFK PNFNQVESNN RTVNSLAVKS LLMSAGNNIM
PNSQAFVKAS 241 TDSHFKLSLW LRVPKVLKQI SIQKLFKVAG DETNKTFYLS
IACIPNHNSV ETALNISVIC 301 KHQLPIRKFK APFELSMMFS DLKEPYNIVH
DPSYPQRIVH ALLETHTSFA QVLCNNLQED 361 VIIYTLNNYE LTPGKLDLGE
RTLNYSEDVC KRKYFLSKTL ECLPSNTQTM SYLDSIQIPS 421 WKIDFARGEI
KISPQSVSVA KSLLKLDLSG IKKKESKISE ACASGSK*
Gag CD8 Peptide Amino Acid Sequence
TABLE-US-00039 [0188] AMQMLKDTI SEQ ID NO: 116
Gag CD4 (13) Peptide Amino Acid Sequence
TABLE-US-00040 [0189] NPPIPVGDIYKRWIIGLNK SEQ ID NO: 117
Gag CD4 (17) Peptide Amino Acid Sequence
TABLE-US-00041 [0190] FRDYVDRFFKTLRAEQATQE SEQ ID NO: 118
RT CD4 Peptide Amino Acid Sequence
TABLE-US-00042 [0191] PKVKQWPLTEVKIKALTAI SEQ ID NO: 119
RT CD8 Peptide Amino Acid Sequence
TABLE-US-00043 [0192] VYYDPSKDLIA SEQ ID NO: 120
[0193] T-cell stimulating immunogenicity of a contemplated adjuvant
can be measured by a variety of well known techniques. In usual
practice, a host animal is inoculated with a contemplated RPBLA
vaccine or inoculum, and peripheral mononuclear blood cells (PMBC)
are thereafter collected. Those PMBC are then cultured in vitro in
the presence of the biologically active polypeptide (T cell
immunogen) for a period of about three to five days. The cultured
PMBC are then assayed for proliferation or secretion of a cytokine
such as IL-2, GM-CSF of IFN-.gamma.. Assays for T cell activation
are well known in the art. See, for example, U.S. Pat. No.
5,478,726 and the art cited therein.
[0194] A contemplated adjuvant is typically prepared from a
recovered RPBLA particles by dispersing the RPBLAs in a
physiologically tolerable (acceptable) diluent vehicle such as
water, saline, phosphate-buffered saline (PBS), acetate-buffered
saline (ABS), Ringer's solution, or the like to form an aqueous
composition. The diluent vehicle can also include oleaginous
materials such as peanut oil, squalane, squalene and the like as
are well known.
[0195] The preparation of adjuvants that contain proteinaceous
materials as active ingredients is also well understood in the art.
Typically, such adjuvants are prepared as parenterals, either as
liquid solutions or suspensions; solid forms suitable for solution
in, or suspension in, liquid prior to injection can also be
prepared. The preparation can also be emulsified, which is
particularly preferred.
[0196] The immunogenically active RPBLAs are often mixed with
excipients that are pharmaceutically acceptable and compatible with
the active ingredient. Suitable excipients are, for example,
dextrose, glycerol, ethanol, or the like and combinations thereof.
In addition, if desired, an adjuvant can contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents that enhance the immunogenic effectiveness of the
composition.
[0197] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description and the detailed
examples below, utilize the present invention to its fullest
extent. The following preferred specific embodiments are,
therefore, to be construed as merely illustrative, and not limiting
of the remainder of the disclosure in any way whatsoever.
Example 1
Plasmid Construction and Plant Transformation
[0198] DNA encoding HIV-1 p24, p41 and RT from a cloned South
African HIV isolate Du422 (GenBank accession no. AF544010) was
fused to Zera.RTM. using PCR and subsequently cloned into an A.
tumefaciens binary expression vector pTRAc (Meyers, BMC
Biotechnology 2008 8:53) in E. coli to yield the recombinant clone
pTRAcRX3p24, pTRAcRX3p41 and pTRAcRX3RT.
[0199] The HPV-16 E7SH gene was engineered by three consecutive PCR
reactions as described in [Steinberg et al., 2995 Vaccine
23(9):1149-1157]. The resulting HPV16 E7SH gene was amplified by
PCR and an enterokinase site was added to the 5' end of the
protein. This construct was then fused to RX3 by replacing GFP in
an A. tumefaciens binary vector pTRA C [Mclean et al., 2007 J Gen
Virol 88:1460-1469] that contained a RX3 GFP fusion gene.
[0200] The recombinant clone was purified from E. coli, and
electroporated into competent host A. tumefaciens GV3101::pMP90RK
cells. Recombinant A. tumefaciens cultures containing pTRAcRX3p24,
pTRAcRX3p41, pTRAcRX3RT and pRX3E7SH were injection-infiltrated
into the leaves of 6-week old N. benthamiana plants using a needle
and syringe. The leaves were co-infiltrated with A. tumefaciens
LBA4404 containing a silencing suppressor pBIN-NS to enhance
transient protein expression. The infiltrated plants were grown at
22.degree. C. under a 16 hour: 8 hour light:dark cycle.
[0201] Harvesting and Purification of Transiently-Expressed RX3
Fusion Proteins
[0202] Approximately 10 g of infiltrated leaf tissue was ground up
in liquid nitrogen and resuspended in 20 ml of buffer PBP3 (100 mM
Tris pH8, 50 mM KCl, 6 mM MgCl.sub.2, 10 mM EDTA and 0.4M NaCl).
This suspension was homogenized for 3 minutes and then filtered
through miracloth (a quick filtration material for gelatinous
homogenates and for protoplast isolation that is composed of rayon
polyester with a pore size of 22-25 mm and an acrylic binder that
is available from Calbiochem.RTM., San Diego, Calif.). The filtrate
was loaded on top of a density step gradient. The gradient
comprised of 7 ml volumes of 15, 25, 35 and 45% concentrations of
Optiprep.RTM. density gradient medium made up in buffer PBP3. The
gradient was centrifuged for 2 hours at 80 000Xg in a Beckman SW28
rotor at 4.degree. C. The pellet was resuspended in 500 .mu.l of
buffer PBP3 and an aliquot stored for analysis. The remainder was
stored at -70.degree. C.
Example 2
Immunization of Mice
[0203] HIV Group Study
[0204] Female BALB/c mice (8 to 10 weeks old) were divided into the
appropriate number of groups (5 mice per group):
[0205] Group 1--vDNA prime
[0206] Group 2--vDNA prime+vDNA boost
[0207] Group 3--vDNA prime+RPBLAs boost
[0208] Group 4--RPBLAs prime
[0209] The DNA vaccines (vDNA) used in the prime and boost
inoculations correspond to: (i) pTHGagC that expresses the Du422
HIV-1 subtype C Gag (van Harmelen, 2003), was manufactured by
Aldevron, Fargo, N. Dak., USA and resuspended at 1 mg DNA/ml saline
and (ii) pVRCgrttn, that express five HIV-1 subtype C genes gag,
reverse transcriptase (RT), tat, and nef (Burgers et al., AIDS
Research and Human Retroviruses 2008 24(2):195-206). The vDNA (100
.mu.g DNA/100 .mu.l saline) was administered by injecting 50 .mu.l
into each tibialis anterior muscle.
[0210] For intramuscular inoculation with the RPBLAs containing the
corresponding RX3 fusion proteins (RX3-p24, RX3-p41 and RX3-RT),
100 .mu.l of the corresponding RPBLAs fraction isolated as
described above and resuspended in saline buffer The following
amounts of RPBLAs were injected into the tibialis anterior muscle:
3.6 .mu.g for RX3-p24; 3.1 .mu.g for RX3-p41; and 4 .mu.g for
RX3-RT.
[0211] HPV Group Study
[0212] DNA Vaccination
[0213] Six- to eight-week-old female C57BL/6 mice were injected
with 50 .mu.l of 10 .mu.M cardiotoxin into each tibialis anterior
muscle 5-6 days prior to DNA injection. For vaccination, 50 .mu.l
of plasmid DNA (1 .mu.g/.mu.l in PBS) was injected into each
pretreated muscle. Ten days later, mice were sacrificed and
splenocytes were isolated from the spleen.
[0214] Tumor Protection and Regression Studies
Tumor protection and regression studies were performed essentially
as described in (Ohlschlager et al., Vaccine 2006 24(2):2880-2893).
The specific modifications of the protocol are indicated in the
corresponding experiments.
Example 3
IFN-.gamma. and IL-2 ELISPOT Assay
[0215] HIV Antigens
[0216] A single cell suspension of splenocytes was prepared from
spleens harvested on day 12 or 40 and pooled from 5 mice per group.
IFN-.gamma. ELISPOT responses were measured using a mouse IL-2 or
IFN-.gamma. ELISPOT set (BD Pharmingen). Splenocytes were plated in
triplicate at 5.times.10.sup.5/well in a final volume of 200 .mu.l
R10 culture medium (RPMI with 10% heat inactivated FCS, Gibco,
containing 15 mM .beta.-mercaptoethanol, 100 U penicillin per ml,
and 100 .mu.g streptomycin).
The peptides (>95% pure, Bachem, Switzerland) GagCD8 AMQMLKDTI
(SEQ ID NO:116) gag CD4(13) NPPIPVGDIYKRWIIGLNK (SEQ ID NO:117) gag
CD4(17) FRDYVDRFFKTLRAEQATQE (SEQ ID NO:118), RT(CD8) VYYDPSKDLIA
(SEQ ID NO:120), or RT(CD4) PKVKQWPLTEVKIKALTAI (SEQ ID NO:119),
were used as stimuli in the assay at a final concentration of 4
.mu.g/ml. Reactions containing an irrelevant H-2K.sup.d binding
peptide TYSTVASSL (SEQ ID NO:1), (obtained from Elizabeth Reap,
AlphaVax) or without peptide served as background controls. Spots
were detected with the detection antibody at 22 hours, developed
with Nova Red then counted using a CTL Analyzer (Cellular
Technology, OH, USA) with Immunospot Version 3.0 software. The
average number of spots in triplicate wells was calculated and
results are expressed as the average number of spot-forming units
(SFU) per 10.sup.6 splenocytes.+-.the standard deviation (SD). For
each group of mice, the average background spots obtained in the
absence of peptide and in the presence of the irrelevant peptide
plus one standard deviation of this average was considered as the
cut-off for a positive response.
[0217] HPV Antigens
[0218] In all studies, ELISPOTs were performed ex vivo essentially
as described in (Steinberg et al., 2005 Vaccine 23:1149-1157).
Murine IFN-gamma Elispot assays were performed ex vivo and 5 or 6
days after each in vitro restimulation as described earlier
(Ohlschlager et al., 2006). The granzyme B Elispot assay was
performed similarly to the IFN-gamma Elispot Assay. For this assay,
the anti-mouse granzyme capture antibody (100 ng/well, clone
R4-6A2; PharMingen, San Diego, USA) and the biotinylated anti-mouse
granzyme detection antibody (50 ng/well, clone XMG1.2; PharMingen,
San Diego, USA) were used.
Example 4
Western Blot of Antiserum
[0219] Western blots were carried out using a LAV Blot I commercial
kit (Biorad). Mouse serum from inoculated mice was used to detect
antibodies with goat anti-mouse IgG conjugated to alkaline
phosphatase.
Example 5
Isolation (Purification) of RPBLAs Containing RX3-p24, RX3-p41 or
RX3-RT by Density Gradient from Agroinfiltrated Tobacco Leaves
[0220] Approximately 10 g of leaf tissue agroinfiltrated with the
corresponding construct (pRX3-p24, pRX3-p41 or pRX3-RT) was ground
up in liquid nitrogen and resuspended in 20 ml of buffer PBP3 (100
mM Tris pH8, 50 mM KCl, 6 mM MgCl.sub.2, 10 mM EDTA and 0.4M NaCl).
This was homogenized for 3 minutes on ice using a Polytron
homogenizer and then filtered through miracloth. The corresponding
filtrate was loaded on top of a density step gradient, comprising
of 7 ml volumes of 15, 25, 35 and 45% concentrations of
Optiprep.RTM. density gradient medium made up in buffer PBP3. The
gradient was centrifuged for 2 hours at 80,000.times.g in a Beckman
SW28 rotor at 4.degree. C. The pellet was resuspended in 500 .mu.l
buffer PBP3 to check for the presence of RPBLAs by optic
microscopy, and an aliquot stored for analysis. The remainder was
stored at -70.degree. C.
[0221] To verify that the RPBLAs fraction contained the
corresponding RX3 fusion protein, an aliquot of it was analyzed by
western blot using anti-RX3 and anti-p24 antibodies to verify the
integrity of the fusion protein (FIG. 1). The amount of immunogen
was quantified by densitometric analysis of a western blot
dilutions of HIV-1 p17/p24 (also referred as p41) and HIV-1 RT as
standards. The concentration of the corresponding immunogen was
estimated to 36 ng/.mu.l for RX3-p24, 31 ng/.mu.l for RX3-p41 and
approximately 40 ng/.mu.l for RX-RT.
Example 6
Determination of the Cellular Response Triggered by the
Intramuscular Inoculation of RX3-p24
[0222] To determine the cellular immune response induced by the
administration of RX3-p24 containing RPBLAs, four groups of mice
were inoculated as follows: (i) mice inoculated with the DNA
vaccine (pTHGagx1), (ii) mice inoculated with the DNA vaccine and
boosted with another dose of the same DNA vaccine (pTHGagx2), (iii)
mice inoculated with the DNA vaccine and boosted with RPBLAs
containing RX3-p24 and no further DNA (pTHGag+RX3-p24), and (iv)
mice inoculated exclusively with RPBLAs containing RX3-p24
(RX3-p24).
[0223] IFN-.gamma. and IL-2 ELISPOT assays indicated that mice
inoculated with the DNA vaccine alone (pTHGagx1) induced a cellular
response. As shown in FIG. 2, CD4 as well as CD8 T-cells secreted a
larger amount of IFN-.gamma. and IL-2 when they were incubated with
the stimulating peptides gag CD8, gag CD4(13) or gag CD4(17),
compared to T-cells incubated with unrelated peptide. As expected
and has been shown previously, the mouse group boosted with a
second inoculation of the DNA vaccine (pTHGagx2) showed an even
larger cellular response (4-fold compared to the pTHGag group).
[0224] When the same assays were performed with the mouse group
inoculated exclusively with the RPBLAs containing RX3-p24
(RX3-p24), no significant response was observed. This result
suggested that the immunogen aggregated inside RPBLAs is not able
to trigger the cellular response. Nevertheless, when IFN-.gamma.
and IL-2 ELISPOT assays were performed on T-cells from mice
inoculated with the DNA vaccine and boosted with a second
inoculation consisting of RPBLAs containing RX3-p24 and no further
DNA (pTHGag+RX3-p24), a surprising 3-fold higher cellular response
was observed compared to the pTHGagx1 group. The lack of cellular
response observed in the p24 mouse group probably indicates that a
higher amount of RPBLAs should be inoculated.
[0225] These data indicate that RPBLAs are a suitable immunogen
presentation vehicle able to induce a cellular response.
Example 7
Determination of the Humoral Response Triggered by the
Intramuscular Inoculation of RX3-p24
[0226] It has been shown that the risk of AIDS is greatly increased
in individuals with falling titres of p24 antibodies, suggesting
that high anti-p24 antibody titres might be necessary to maintain a
disease-free state.
[0227] To determine the presence of antibodies against the p24
antigen, strips containing a representation of the HIV virus
proteins [LAV Blot I commercial kit (Biorad)] were incubated with
mouse serum from the four inoculation groups (pTHGagx1, pTHGagx2,
pTHGag+RX3-p24 and RX3-p24). Antibodies against the p24 protein
were detected only in mice inoculated with the DNA vaccine and
boosted with a second round of the DNA inoculation or with RPBLAs
containing the RX3-p24 and no further DNA (FIG. 3; pTHGagx2 and
pTHGag+RX3-p24 mouse groups). Interestingly, the antibodies
generated from this second group recognized the full length Gag
protein (p55) in addition to the p24 protein indicating that a
higher titer of antibodies is produced in pTHGag+RX3-p24 mouse
group compared to the pTHGagx1 one.
Example 8
Determination of the Cellular Response Triggered by the
Intramuscular Inoculation of RX3-p41
[0228] As indicated previously, p41 which results from the fusion
of p17 and p24 fragments (p17/24) of the HIV Gag protein, contains
the highest density of CTL epitopes in the HIV-1 genome (Novitsky
et al., J. Virol. 2002 76(20):10155-10168). In this context the
efficiency of RPBLAs containing the RX3-p41 fusion protein to
trigger the cellular response of the immune system was
examined.
[0229] As occurred previously in Example 6, IFN-.gamma. and IL-2
ELISPOT assays indicated that mice inoculated with a single dose of
the DNA vaccine induced a small cellular response, which was
significantly increased when those mice were boosted with a second
inoculation with the DNA vaccine (compare pTHGagx1 versus pTHGagx2
in FIG. 4). It is interesting to point out that splenocytes from
the mouse group boosted with the RX3-p41 (pTHGag+RX3-p41) secreted
an even larger amount of IFN.gamma. and IL-2 than the pTHGagx2
group when they were incubated with the gagCD4(13) and gagCD4(17)
stimulating peptides (FIG. 4). Although the secretion of IFN.gamma.
and IL-2 was not increased by the incubation of splenocytes from
the pTHGag+RX3-p41 mouse group with gagCD8-stimulating peptides, it
can be concluded that the cellular response of the immune system is
efficiently boosted by the inoculation of RPBLAs containing RX3-p41
fusion protein.
Example 9
Determination of the Cellular Response Triggered by Intramuscular
Inoculation of RX3-RT
[0230] As an effective multivalent vaccine against HIV includes
several antigens, similar studies were performed with HIV viral
protein RT.
[0231] IFN.gamma. ELISPOT assays indicated that mice inoculated
with a single dose of the DNA vaccine (pVRCgrttnx1) induced a very
poor cellular response. Exclusively CD8 T-cells incubated with the
stimulating peptides RT(CD8) secreted a larger amount of
IFN-.gamma. than the same cells incubated with un irrelevant
peptide TYSTVASSL (SEQ ID NO:1; FIG. 5). A boost with a second
inoculation of the DNA vaccine (pVRCgrttnx2) or the RPBLAs
containing RX3--RT (pVRCgrttn+RX3-RT) was needed to observe a
general induction of the cellular response. FIG. 5 shows that CD4
and CD8 T-cells incubated with the corresponding stimulating
peptides secreted a larger amount of IFN-.gamma. and IL-2 compared
to the control treatments (absence or presence of an irrelevant
peptide).
Example 10
Determination of the Immune Response Triggered by the Intramuscular
Inoculation of a DNA Vaccine Expressing RX3-p24, RX3-41 or
RX3-RT
[0232] DNA vaccines encoding HIV antigens have been studied
extensively and shown to induce both humoral and cellular immune
responses in animal models as well as in humans (Estcourt et al.,
Immunol. Rev. 2004 199:144-155). However, although DNA vaccines
have been shown to be safe, immunizations have generated low and
transient levels of immune responses.
[0233] pTHGag was shown in the mouse model to induce a potent
cytotoxic lymphocyte response. Pr55Gag expressed in a variety of
cell systems can assemble and bud through the plasma membrane to
form highly immunogenic virus-like particles (VLPs). RPBLAs can not
been considered as classical VLPs, because their assembly is
induced by the aggregation capacity of RX3, which is not a viral
protein involved in the formation of the virus particles. However,
the suitability of a DNA vaccine expressing RPBLAs containing the
RX3-24, RX3-41, RX3-RT and RX3E7SH was examined. Interestingly,
once the corresponding pTH-derived vectors (pTHRX3-p24, pTHRX3-p41,
pTHRX3-RT and pTHRX3-E7SH) were administrated as the pTHGag in
previous studies, significant humoral and cellular immune responses
were observed. This unexpected result indicates that RPBLAs can be
administered by DNA vaccination; in spite of this organelles are
stored in the ER and are not supposed to bud through the plasma
membrane to form highly immunogenic virus-like particles
(VLPs).
Example 11
Determination of the Immune Response Triggered by the Inoculation
of RPBLAs Assembled In Vitro
[0234] The isolation of RPBLAs by density gradient permits the
recovery of a highly enriched fraction of RPBLAs, but a certain
degree of contaminants are co-purified. To remove as much
contaminants as possible, the RX3 fusion proteins (RX3-p24,
RX3-p41, RX3-RT and RX3-E7SH) were solubilized from the
corresponding RPBLA fraction in 20 mM Tris pH8, 2% DOC, 10 mM DTT
incubated 1 hour at room temperature in soft agitation, and
purified in RP-FPLC. The elution fractions containing the RX3
fusion proteins with more than 95% purity were pooled and
lyophilized. The corresponding pellet was recovered in distilled
water in the presence of 200 mM of NaCl and 50 mM of CaCl. In these
conditions, the fusion proteins containing the RX3 peptide
reassemble spontaneously to reform RPBLAs in vitro, outside of the
plant ER.
[0235] In vitro-assembled RPBLAs containing the corresponding RX3
fusion protein were inoculated into mice and the IFN.gamma., IL-2
and Granzyme B ELISPOT assays showed that RPBLAs boost
significantly the cellular response in equivalent studies as the
those performed using in vivo-formed RPBLAs. This surprising result
indicates that in vitro-assembled RPBLAs maintain the capacity of
inducing the cellular response.
[0236] RX3 fusion proteins can be induced to assemble in vitro and
form RPBLAs in the following conditions: (i) reducing the pH value
of the solution, (ii) increasing salt content, (iii) reducing or
removing the concentration of reducing agents, (iv) adding
oxidizing agents, (v) decreasing the temperature, or a combination
of this factors. Obviously, in vitro RPBLAs are not surrounded by a
membrane. Preferred salts to induce the assembly in vitro are NaCl,
CaCl and KCl and preferred pH values are below 7.
[0237] As indicated before, a double immune response (cellular plus
humoral) produces a more protective effect against AIDS. The
presence of antibodies against the p24, p41 and RT antigens was
shown by using the HIV strips (LAV Blot I commercial kit (Biorad))
in mice primed with the DNA vaccine and boosted with the
corresponding in vitro assembled RPBLAs.
Example 12
Isolation (Purification) of RPBLAs Containing RX3-E7SH by Density
Gradient from Agroinfiltrated Tobacco Leaves
[0238] Approximately 10 g of leaf tissue agroinfiltrated with the
HPV-16 antigen E7SH fused to RX3 (pRX3-E7SH) was ground up in
liquid nitrogen and resuspended in 20 ml of buffer PBP3 (100 mM
Tris pH8, 50 mM KCl, 6 mM MgCl.sub.2, 10 mM EDTA and 0.4M NaCl).
This was homogenized for 3 minutes on ice using a Polytron
homogenizer and then filtered through miracloth. The corresponding
filtrate was loaded on top of a density step gradient, comprising
of 7 ml volumes of 15, 25, 35 and 45% concentrations of
Optiprep.RTM. density gradient medium made up in buffer PBP3. The
gradient was centrifuged for 2 hours at 80,000.times.g in a Beckman
SW28 rotor at 4.degree. C. The pellet was resuspended in 500 .mu.l
buffer PBP3 to check for the presence of RPBLAs by optic
microscopy, and an aliquot stored for analysis. The remainder was
stored at -70.degree. C.
[0239] The gene sequence for the RX2-E7SH fusion protein is shown
below from 5' to 3':
TABLE-US-00044 SEQ ID NO: 129
ATGAGGGTGTTGCTCGTTGCCCTCGCTCTCCTGGCTCTCGCTGCGAGC
GCCACCTCCACGCATACAAGCGGCGGCTGCGGCTGCCAGCCACCGCCG
CCGGTTCATCTACCGCCGCCGGTGCATCTGCCACCTCCGGTTCACCTG
CCACCTCCGGTGCATCTCCCACCGCCGGTCCACCTGCCGCCGCCGGTC
CACCTGCCACCGCCGGTCCATGTGCCGCCGCCGGTTCATCTGCCGCCG
CCACCATGCCACTACCCTACTCAACCGCCCCGGCCTCAGCCTCATCCC
CAGCCACACCCATGCCCGTGCCAACAGCCGCATCCAAGCCCGTGCCAG
ACCATGGACGACGATGATAAGATGCACGGCGACACCCCCACCCTGCAC
GAGTACATGCTGGACCTGCAGCCCGAGACCACCGACCTGTACTGCATC
TGCAGCCAGAAACCCAAGTGCGACAGCACCCTGCGGCTGTGCGTGCAG
AGCACCCACGTGGACATCCGGACCCTGGAGGACCTGCTGATGGGCACC
CTGGGCATCGTGTGCCCCTACGAGCAGCTGAACGACAGCAGCGAGGAG
GAGGATGAGATCGACGGCCCCGCCGGCCAGGCTGAGCCCGACCGGGCC
CACTACAACATCGTGACCTTCTGCTGCCAACCAGAGACAACTGATCTC
TACTGTTATGAGCAATTAAATGACAGCTCAGAGCATTACAATATTGTA
ACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCATGGGCACA
CTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA
[0240] To verify that the RPBLAs fraction contained the
corresponding RX3 fusion protein, an aliquot of it was analyzed by
western blot using anti-RX3 to verify the integrity of the fusion
protein (FIG. 7).
Example 13
Determination of the Cellular Response Triggered by the Inoculation
of RX3-E7SH
[0241] To determine the cellular immune response induced by the
administration of RX3-E7SH containing RPBLAs, five groups of mice
were inoculated as follows: (i) mice inoculated with the DNA
vaccine expressing E7SH antigen (pTHamp-E7SH), (ii) mice inoculated
with the corresponding DNA vaccine negative control (pTHamp) with
the sequence:
TABLE-US-00045 SEQ ID NO: 127
5'GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTAC
AATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTG
TGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAAC
AAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAG
GCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTTTGA
GATTTCTGTCGCCGACTAAATTCATGTCGCGCGATAGTGGTGTTTATC
GCCGATAGAGATGGCGATATTGGAAAAATCGATATTTGAAAATATGGC
ATATTGAAAATGTCGCCGATGTGAGTTTCTGTGTAACTGATATCGCCA
TTTTTCCAAAAGTGATTTTTGGGCATACGCGATATCTGGCGATAGCGC
TTATATCGTTTACGGGGGATGGCGATAGACGACTTTGGTGACTTGGGC
GATTCTGTGTGTCGCAAATATCGCAGTTTCGATATAGGTGACAGACGA
TATGAGGCTATATCGCCGATAGAGGCGACATCAAGCTGGCACATGGCC
AATGCATATCGATCTATACATTGAATCAATATTGGCCATTAGCCATAT
TATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGC
ATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTC
CAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGT
AATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG
TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC
CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAA
TAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTG
CCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA
TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACA
TGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA
TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTG
GATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACG
TCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAAT
GTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTAC
GGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCG
CCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGG
ACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTC
CCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCA
CCCCCTTGGCTTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTA
TACACCCCCGCTTCCTCATGTTATAGGTGATGGTATAGCTTAGCCTAT
AGGTGTGGGTTATTGACCATTATTGACCACTCCCCTATTGGTGACGAT
ACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCTTT
ATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCT
GTATTTTTACAGGATGGGGTCTCATTTATTATTTACAAATTCACATAT
ACAACACCACCGTCCCCAGTGCCCGCAGTTTTTATTAAACATAACGTG
GGATCTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGGCTCTTCT
CCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTCCA
GCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCA
GACTTAGGCACAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGG
CCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGGGAGCGGGCTT
GCACCGCTGACGCATTTGGAAGACTTAAGGCAGCGGCAGAAGAAGATG
CAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCCCG
TTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCG
TTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGAC
TGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCTTGACACGAAG
CTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGA
ATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAG
AGGGCCCTATTCTATAGTGTCACCTAAATGCTAGAGCTCGCTGATCAG
CCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCC
CCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCT
AATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTA
TTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAG
ACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGG
CGGAAAGAACCAGCTGGGGCTCGAGGGGGGATCGATCCCGTCGACCTC
GAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGT
TATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGT
AAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG
CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCAT
TAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC
TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTG
CGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACA
GAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAA
AAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGG
CTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG
TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA
AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC
CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCA
CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGC
TGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGT
AACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTG
GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGT
GCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGG
ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAA
AGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT
GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT
CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAAC
GAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC
TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAA
AGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGT
GAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCC
TGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCT
GGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA
GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGT
GGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG
GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT
GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCT
TCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCC
ATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTC
AGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG
CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACT
GGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCG
AGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC
AGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAA
CTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACT
CGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCT
GGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG
GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT
TGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAA
TGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGA
AAAGTGCCACCTGACGTC3'
(iii) mice inoculated with RPBLAs containing RX3-E7SH(RX3-E7SH),
(iv) mice co-inoculated with RPBLAs containing RX3-E7SH and
incomplete Freund's adjuvant (RX3-E7SH/IFA), and finally (v) mice
inoculated with RPBLAs containing RX3 fused to Gfp (RX3-Gfp) as a
negative control of RPBLAs.
[0242] As expected, IFN-.gamma. and Granzyme B ELISPOT assays
indicated clearly that mice inoculated with the DNA vaccine
(pTHamp-E7SH) induced a cellular response. As shown in FIG. 8,
splenocyte cells coming from mice inoculated with the pTHamp-E7SH
DNA vaccine secreted a significant larger amount of IFN-.gamma. and
Granzyme B, than the ones coming from mice inoculated with the DNA
vaccine in the absence of E7SH antigen (pTHamp).
[0243] Surprisingly, it was also observed that splenocytes isolated
from mice inoculated with RPBLAs containing RX3-E7SH(RX3-E7SH) also
released a large amount of IFN-.gamma. and Granzyme B (equivalent
to pTHamp-E7SH mice group) in the presence or absence of IFA
co-administration FIG. 8. As a control, the negative results
observed in RX3-Gfp group indicate that no unspecific cellular
response against E7SH is triggered by the administration of
RPBLAs.
[0244] These results demonstrate clearly that E7SH antigen
administered in fusion with RX3 in a RPBLAs particle is able to
trigger efficiently a cellular response. The fact that no adjuvant
was needed to achieve the maximum effect indicates that RX3-E7SH in
RPBLAs is an efficient antigen presentation vehicle able to induce
a cellular response. This conclusion was supported by the
observation that it is necessary to co-administer IFA to ovalbumin
(OVA) in order to induce an efficient cellular response FIG. 9.
[0245] The amino acid sequence for ovalbumin in single letter code
is shown below:
TABLE-US-00046 MGSIGAASME FCFDVFKELK VHHANENIFY CPIAIMSALA
MVYLGAKDST RTQINKVVRF DKLPGFGDSI EAQCGTSVNV HSSLRDILNQ ITKPNDVYSF
SLASRLYAEE RYPILPEYLQ CVKELYRGGL EPINFQTAAD QARELINSWV ESQTNGIIRN
VLQPSSVDSQ TAMVLVNAIV FKGLWEKTFK DEDTQAMPFR VTEQESKPVQ MMYQIGLFRV
ASMASEKMKI LELPFASGTM SMLVLLPDEV SGLEQLESII NFEKLTEWTS SNVMEERKIK
VYLPRMKMEE KYNLTSVLMA MGITDVFSSS ANLSGISSAE SLKISQAVHA AHAEINEAGR
EVVGSAEAGV DAASVSEEFR ADHPFLFCIK HIATNAVLFF GRCVSP
Example 14
Determination of the Cytolytic Activity of the Splenocytes Induced
by the Inoculation of with RX3-E7SH
[0246] To determine, if the specifically activated splenocytes had
cytolytic activity, .sup.51Cr-release assays were performed.
.sup.51Cr release assays were performed 5-6 days after an in vitro
restimulation of murine spleen cells as described elsewhere
[Steinberg et al., (2005) Vaccine 23(9):1149-1157.] An animal was
scored positive when the specific lysis of the specific target
(RX3-E7 or pTHamp-E7SH cells) was at least 10% above the lysis of
the control target (RX3-Gfp or pTHamp cells) for the protein and
DNA based vaccines. After a first round of in vitro restimulation
strong specific cytolytic activity against the E7WT-expressing
RMA-E7 transfectants was shown (see table below.)
TABLE-US-00047 TABLE .sup.51C-release assay (after 1.sup.st in
vitro Specific restimulation) Lysis (%) pTHamp 8 .+-. 3 pTHamp-E7SH
12 .+-. 4 RX3-Gfp 26 .+-. 6 RX3-E7SH 33 .+-. 5 RX3-E7SH/IFA 29 .+-.
6
[0247] Surprisingly, the mean of specific lysis of the RMA-E7 cells
was comparable in the RX3-E7SH-group (33%) and the pTHamp-E7SH
immunized animals (26%), and significantly higher than the
corresponding control groups RX3-Gfp (12%) and the pTHamp (8%).
This result indicates that RX3-E7SH RPBLAs was able to induce a
specific cytolytic activity against E7 expressing cells as
efficiently as has already been shown by using the DNA vaccine
pTHamp-E7SH [Ohlschlager et al., (2006) Vaccine 24:2880-2893].
Moreover, the fact that the cytolytic activity was not increased
when the RPBLAs containing RX3-E7SH fusion protein was
co-administered with IFA (see RX3-E7SH/IFA-group in table) suggests
that even a lower dose would be effective to trigger the cytolytic
effect, supporting the idea that RPBLAs provide an efficient
administration vehicle to trigger a specific cytolytic effect.
[0248] It is important to add that it has been widely demonstrated
that a cytolytic response is a crucial element for controlling
tumor growth [Akazawa, 2004 Cancer Res 64:757-764] and viral
infection (Vine et al., 2004 J Immunol 173:5121-5129].
Example 15
Determination of Tumor Growth in Mice Inoculated with RX3-E7SH
[0249] The aim of a therapeutic tumor vaccine is the induction of
an effective immune response eradicating established tumors.
Therefore, vaccination with the E7SH gene was examined to determine
whether a cellular immune response could be induced that was able
to control established E7-expressing tumor cells in vivo. In four
tumor regression studies, a total of 80 animals were transplanted
with a tumorigenic dose of syngeneic C3-tumor cells (day 0). When
the tumors had reached a mean size of 4-9 mm.sup.2 at days 5-18,
the animals were inoculated with: (i) 100 .mu.g of the
E7SH-encoding plasmid (pTHamp-E7SH), (ii) 100 .mu.g of empty pTHamp
vector (pTHamp), (iii) RPBLAs containing 5 .mu.g of
RX3-E7SH(RX3-E7SH), and (iv) the same amount of RX3-E7SH RPBLAs
co-administered with IFA (RX3-E7SH/IFA) (day zero). Tumor size was
determined every two days by measuring with a ruler until the end
of the study (day 14).
[0250] As shown in FIG. 10A, tumor size increased progressively in
mice inoculated with the control DNA vector (pTHamp), reaching a
maximum average size of 110 mm.sup.2 14 days after inoculation.
Tumor growth was significantly reduced in those mice inoculated
with RPBLAs containing RX3-E7SH. Through out the study, the
RX3-E7SH mice group showed tumors with lower size compared to the
control group, reaching a mean value of 40 mm.sup.2 at day 14. It
is interesting to point out that the protective effect of RX3-E7SH
inoculation is comparable to the DNA vaccine pTHamp-E7SH, which has
been reported to be a good therapeutic vaccine against
E7-expressing tumors [Ohlschlager et al., (2006) Vaccine
24:2880-2893]. Moreover, as indicated before, the fact that the
co-administration of RPBLAs containing RX3-E7SH with IFA
(RX3-E7SH/IFA) did not increase the protective effect of the same
amount of RPBLAs containing RX3-E7SH in the absence of an adjuvant
(RX3-E7SH) suggests that a lower amount of RX3-E7SH will be
protective against E7-expressing tumor growth.
[0251] To exclude an unspecific tumor growth reduction due to some
contaminants present in the RPBLAs preparation, or by the RX3
polypeptide by itself, equivalent amounts of RPBLAs containing
RX3-Gfp were inoculated in an independent study, and no effect on
tumor growth was observed when compared to pTHamp DNA control group
(see FIG. 10B).
[0252] It must be pointed out that mice were inoculated only once
with RPBLAs with RX3-E7SH. In a prime boost study it is expected to
have an enhanced therapeutic effect.
Example 16
Protective Effect Against Tumor Growth in Mice Inoculated with
RX3-E7SH
[0253] Taking into consideration a goal of the application of a
protective (prophylactic) vaccine based on RPBLAs in addition to a
therapeutic vaccine, rechallenge studies were undertaken to
determine whether the RPBLAs containing RX3-E7SH were able to
protect animals from an outgrowth of E7-expressing syngeneic
tumors.
[0254] Those mice that showed complete regression after the tumor
regression experiment were injected again with 0.5.times.10.sup.6
C3 cells s.c. in 100 .mu.l PBS into the left flank 3 weeks after
completion of the tumor regression study. The first C3 inoculation
was given into the right flank. As a control, the same number of
non-immunised mice received the same treatment. Twenty days after
this injection, all control mice showed tumors with a size range of
100-400 mm.sup.2, whereas the immunized mice developed no tumors,
and therefore showed clear protection from tumor growth (see FIG.
11).
[0255] Each of the patents, patent applications and articles cited
herein is incorporated by reference. The use of the article "a" or
"an" is intended to include one or more.
[0256] The foregoing description and the examples are intended as
illustrative and are not to be taken as limiting. Still other
variations within the spirit and scope of this invention are
possible and will readily present themselves to those skilled in
the art.
Sequence CWU 1
1
12919PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 1Thr Tyr Ser Thr Val
Ala Ser Ser Leu1 526PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 2Pro
Pro Pro Val His Leu1 537PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE
3Pro Gln Gln Pro Phe Pro Gln1 548PRTARTIFICIAL SEQUENCESYNTHETIC
SEQUENCE 4Pro Gln Gln Gln Pro Pro Phe Ser1 555PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 5Pro Gln Gln Pro Gln1 5653PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 6Pro Pro Pro Pro Val His Leu Pro Pro Pro
Val His Leu Pro Pro Pro1 5 10 15Val His Leu Pro Pro Pro Val His Leu
Pro Pro Pro Val His Leu Pro 20 25 30Pro Pro Val His Leu Pro Pro Pro
Val His Val Pro Pro Pro Val His 35 40 45Leu Pro Pro Pro Pro
50765PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 7Gln Gln Gln Gln Gln
Phe Leu Pro Ala Leu Ser Gln Leu Asp Val Val1 5 10 15Asn Pro Val Ala
Tyr Leu Gln Gln Gln Leu Leu Ala Ser Asn Pro Leu 20 25 30Ala Leu Ala
Asn Val Ala Ala Tyr Gln Gln Gln Gln Gln Leu Gln Gln 35 40 45Phe Leu
Pro Ala Leu Ser Gln Leu Ala Met Val Asn Pro Ala Ala Tyr 50 55
60Leu65870PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 8Gln Gln Val Leu
Ser Pro Tyr Asn Glu Phe Val Arg Gln Gln Tyr Gly1 5 10 15Ile Ala Ala
Ser Pro Phe Leu Gln Ser Ala Thr Phe Gln Leu Arg Asn 20 25 30Asn Gln
Val Trp Gln Gln Leu Ala Leu Val Ala Gln Gln Ser His Cys 35 40 45Gln
Asp Ile Asn Ile Val Gln Ala Ile Ala Gln Gln Leu Gln Leu Gln 50 55
60Gln Phe Gly Asp Leu Tyr65 709672DNAARTIFICIAL SEQUENCESYNTHETIC
SEQUENCE 9atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc
cacctccacg 60catacaagcg gcggctgcgg ctgccagcca ccgccgccgg ttcatctacc
gccgccggtg 120catctgccac ctccggttca cctgccacct ccggtgcatc
tcccaccgcc ggtccacctg 180ccgccgccgg tccacctgcc accgccggtc
catgtgccgc cgccggttca tctgccgccg 240ccaccatgcc actaccctac
tcaaccgccc cggcctcagc ctcatcccca gccacaccca 300tgcccgtgcc
aacagccgca tccaagcccg tgccagctgc agggaacctg cggcgttggc
360agcaccccga tcctgggcca gtgcgtcgag tttctgaggc atcagtgcag
cccgacggcg 420acgccctact gctcgcctca gtgccagtcg ttgcggcagc
agtgttgcca gcagctcagg 480caggtggagc cgcagcaccg gtaccaggcg
atcttcggct tggtcctcca gtccatcctg 540cagcagcagc cgcaaagcgg
ccaggtcgcg gggctgttgg cggcgcagat agcgcagcaa 600ctgacggcga
tgtgcggcct gcagcagccg actccatgcc cctacgctgc tgccggcggt
660gtcccccacg cc 67210224PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE
10Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser1
5 10 15Ala Thr Ser Thr His Thr Ser Gly Gly Cys Gly Cys Gln Pro Pro
Pro 20 25 30Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val
His Leu 35 40 45Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu Pro
Pro Pro Val 50 55 60His Leu Pro Pro Pro Val His Val Pro Pro Pro Val
His Leu Pro Pro65 70 75 80Pro Pro Cys His Tyr Pro Thr Gln Pro Pro
Arg Pro Gln Pro His Pro 85 90 95Gln Pro His Pro Cys Pro Cys Gln Gln
Pro His Pro Ser Pro Cys Gln 100 105 110Leu Gln Gly Thr Cys Gly Val
Gly Ser Thr Pro Ile Leu Gly Gln Cys 115 120 125Val Glu Phe Leu Arg
His Gln Cys Ser Pro Thr Ala Thr Pro Tyr Cys 130 135 140Ser Pro Gln
Cys Gln Ser Leu Arg Gln Gln Cys Cys Gln Gln Leu Arg145 150 155
160Gln Val Glu Pro Gln His Arg Tyr Gln Ala Ile Phe Gly Leu Val Leu
165 170 175Gln Ser Ile Leu Gln Gln Gln Pro Gln Ser Gly Gln Val Ala
Gly Leu 180 185 190Leu Ala Ala Gln Ile Ala Gln Gln Leu Thr Ala Met
Cys Gly Leu Gln 195 200 205Gln Pro Thr Pro Cys Pro Tyr Ala Ala Ala
Gly Gly Val Pro His Ala 210 215 22011339DNAARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 11atgagggtgt tgctcgttgc cctcgctctc
ctggctctcg ctgcgagcgc cacctccacg 60catacaagcg gcggctgcgg ctgccagcca
ccgccgccgg ttcatctacc gccgccggtg 120catctgccac ctccggttca
cctgccacct ccggtgcatc tcccaccgcc ggtccacctg 180ccgccgccgg
tccacctgcc accgccggtc catgtgccgc cgccggttca tctgccgccg
240ccaccatgcc actaccctac tcaaccgccc cggcctcagc ctcatcccca
gccacaccca 300tgcccgtgcc aacagccgca tccaagcccg tgccagacc
33912113PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 12Met Arg Val Leu
Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser1 5 10 15Ala Thr Ser
Thr His Thr Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30Pro Val
His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val His Leu 35 40 45Pro
Pro Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro Pro Val 50 55
60His Leu Pro Pro Pro Val His Val Pro Pro Pro Val His Leu Pro Pro65
70 75 80Pro Pro Cys His Tyr Pro Thr Gln Pro Pro Arg Pro Gln Pro His
Pro 85 90 95Gln Pro His Pro Cys Pro Cys Gln Gln Pro His Pro Ser Pro
Cys Gln 100 105 110Tyr13240DNAARTIFICIAL SEQUENCESYNTHETIC SEQUENCE
13atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccacg
60catacaagcg gcggctgcgg ctgccagcca ccgccgccgg ttcatctacc gccgccggtg
120catctgccac ctccggttca cctgccacct ccggtgcatc tcccaccgcc
ggtccacctg 180ccgccgccgg tccacctgcc accgccggtc catgtgccgc
cgccggttca tctgccgccg 2401492PRTARTIFICIAL SEQUENCESYNTHETIC
SEQUENCE 14Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala
Ala Ser1 5 10 15Ala Thr Ser Thr His Thr Ser Gly Gly Cys Gly Cys Gln
Pro Pro Pro 20 25 30Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro
Pro Val His Leu 35 40 45Pro Pro Pro Val His Leu Pro Pro Pro Val His
Leu Pro Pro Pro Val 50 55 60His Leu Pro Pro Pro Val His Val Pro Pro
Pro Val His Leu Pro Pro65 70 75 80Pro Pro Cys His Tyr Pro Thr Gln
Pro Pro Arg Tyr 85 9015213DNAARTIFICIAL SEQUENCESYNTHETIC SEQUENCE
15atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc cacctccacg
60catacaagcg gcggctgcgg ctgccagcca ccgccgccgg ttcatctgcc gccgccacca
120tgccactacc ctacacaacc gccccggcct cagcctcatc cccagccaca
cccatgcccg 180tgccaacagc cgcatccaag cccgtgccag acc
2131671PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 16Met Arg Val Leu
Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser1 5 10 15Ala Thr Ser
Thr His Thr Ser Gly Gly Cys Gly Cys Gln Pro Pro Pro 20 25 30Pro Val
His Leu Pro Pro Pro Pro Cys His Tyr Pro Thr Gln Pro Pro 35 40 45Arg
Pro Gln Pro His Pro Gln Pro His Pro Cys Pro Cys Gln Gln Pro 50 55
60His Pro Ser Pro Cys Gln Tyr65 7017180DNAARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 17atgagggtgt tgctcgttgc cctcgctctc
ctggctctcg ctgcgagcgc cacctccacg 60catacaagcg gcggctgcgg ctgccaatgc
cactacccta ctcaaccgcc ccggcctcag 120cctcatcccc agccacaccc
atgcccgtgc caacagccgc atccaagccc gtgccagacc 1801860PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 18Met Arg Val Leu Leu Val Ala Leu Ala
Leu Leu Ala Leu Ala Ala Ser1 5 10 15Ala Thr Ser Thr His Thr Ser Gly
Gly Cys Gly Cys Gln Cys His Tyr 20 25 30Pro Thr Gln Pro Pro Arg Pro
Gln Pro His Pro Gln Pro His Pro Cys 35 40 45Pro Cys Gln Gln Pro His
Pro Ser Pro Cys Gln Tyr 50 55 6019150PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 19Met Lys Ile Ile Phe Val Phe Ala Leu
Leu Ala Ile Ala Ala Cys Ser1 5 10 15Ala Ser Ala Gln Phe Asp Val Leu
Gly Gln Ser Tyr Arg Gln Tyr Gln 20 25 30Leu Gln Ser Pro Val Leu Leu
Gln Gln Gln Val Leu Ser Pro Tyr Asn 35 40 45Glu Phe Val Arg Gln Gln
Tyr Gly Ile Ala Ala Ser Pro Phe Leu Gln 50 55 60Ser Ala Thr Phe Gln
Leu Arg Asn Asn Gln Val Trp Gln Gln Leu Ala65 70 75 80Leu Val Ala
Gln Gln Ser His Cys Gln Asp Ile Asn Ile Val Gln Ala 85 90 95Ile Ala
Gln Gln Leu Gln Leu Gln Gln Phe Gly Asp Leu Tyr Phe Asp 100 105
110Arg Asn Leu Ala Gln Ala Gln Ala Leu Leu Ala Phe Asn Val Pro Ser
115 120 125Arg Tyr Gly Ile Tyr Pro Arg Tyr Tyr Gly Ala Pro Ser Thr
Ile Thr 130 135 140Thr Leu Gly Gly Val Leu145 15020450DNAARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 20atgaagatca ttttcgtctt tgctctcctt
gctattgctg catgcagcgc ctctgcgcag 60tttgatgttt taggtcaaag ttataggcaa
tatcagctgc agtcgcctgt cctgctacag 120caacaggtgc ttagcccata
taatgagttc gtaaggcagc agtatggcat agcggcaagc 180cccttcttgc
aatcagctac gtttcaactg agaaacaacc aagtctggca acagctcgcg
240ctggtggcgc aacaatctca ctgtcaggac attaacattg ttcaggccat
agcgcagcag 300ctacaactcc agcagtttgg tgatctctac tttgatcgga
atctggctca agctcaagct 360ctgttggctt ttaacgtgcc atctagatat
ggtatctacc ctaggtacta tggtgcaccc 420agtaccatta ccacccttgg
cggtgtcttg 45021144PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 21Met
Ala Thr Lys Ile Leu Ala Leu Leu Ala Leu Leu Ala Leu Phe Val1 5 10
15Ser Ala Thr Asn Ala Phe Ile Ile Pro Gln Cys Ser Leu Ala Pro Ser
20 25 30Ala Ile Ile Pro Gln Phe Leu Pro Pro Val Thr Ser Met Gly Phe
Glu 35 40 45His Leu Ala Val Gln Ala Tyr Arg Leu Gln Gln Ala Leu Ala
Ala Ser 50 55 60Val Leu Gln Gln Pro Ile Asn Gln Leu Gln Gln Gln Ser
Leu Ala His65 70 75 80Leu Thr Ile Gln Thr Ile Ala Thr Gln Gln Gln
Gln Gln Phe Leu Pro 85 90 95Ala Leu Ser Gln Leu Asp Val Val Asn Pro
Val Ala Tyr Leu Gln Gln 100 105 110Gln Leu Leu Ala Ser Asn Pro Leu
Ala Leu Ala Asn Val Ala Ala Tyr 115 120 125Gln Gln Gln Gln Gln Leu
Gln Gln Phe Leu Pro Ala Leu Ser Gln Leu 130 135
14022432DNAARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 22atggctacca
agatattagc cctccttgcg cttcttgccc tttttgtgag cgcaacaaat 60gcgttcatta
ttccacaatg ctcacttgct cctagtgcca ttataccaca gttcctccca
120ccagttactt caatgggctt cgaacaccta gctgtgcaag cctacaggct
acaacaagcg 180cttgcggcaa gcgtcttaca acaaccaatt aaccaattgc
aacaacaatc cttggcacat 240ctaaccatac aaaccatcgc aacgcaacag
caacaacagt tcctaccagc actgagccaa 300ctagatgtgg tgaaccctgt
cgcctacttg caacagcagc tgcttgcatc caacccactt 360gctctggcaa
acgtagctgc ataccaacaa caacaacaat tgcagcagtt tctgccagcg
420ctcagtcaac ta 43223283PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE
23Asn Met Gln Val Asp Pro Ser Gly Gln Val Gln Trp Pro Gln Gln Gln1
5 10 15Pro Phe Pro Gln Pro Gln Gln Pro Phe Cys Gln Gln Pro Gln Arg
Thr 20 25 30Ile Pro Gln Pro His Gln Thr Phe His His Gln Pro Gln Gln
Thr Phe 35 40 45Pro Gln Pro Gln Gln Thr Tyr Pro His Gln Pro Gln Gln
Gln Phe Pro 50 55 60Gln Thr Gln Gln Pro Gln Gln Pro Phe Pro Gln Pro
Gln Gln Thr Phe65 70 75 80Pro Gln Gln Pro Gln Leu Pro Phe Pro Gln
Gln Pro Gln Gln Pro Phe 85 90 95Pro Gln Pro Gln Gln Pro Gln Gln Pro
Phe Pro Gln Ser Gln Gln Pro 100 105 110Gln Gln Pro Phe Pro Gln Pro
Gln Gln Gln Phe Pro Gln Pro Gln Gln 115 120 125Pro Gln Gln Ser Phe
Pro Gln Gln Gln Gln Pro Ala Ile Gln Ser Phe 130 135 140Leu Gln Gln
Gln Met Asn Pro Cys Lys Asn Phe Leu Leu Gln Gln Cys145 150 155
160Asn His Val Ser Leu Val Ser Ser Leu Val Ser Ile Ile Leu Pro Arg
165 170 175Ser Asp Cys Gln Val Met Gln Gln Gln Cys Cys Gln Gln Leu
Ala Gln 180 185 190Ile Pro Gln Gln Leu Gln Cys Ala Ala Ile His Ser
Val Ala His Ser 195 200 205Ile Ile Met Gln Gln Glu Gln Gln Gln Gly
Val Pro Ile Leu Arg Pro 210 215 220Leu Phe Gln Leu Ala Gln Gly Leu
Gly Ile Ile Gln Pro Gln Gln Pro225 230 235 240Ala Gln Leu Glu Gly
Ile Arg Ser Leu Val Leu Lys Thr Leu Pro Thr 245 250 255Met Cys Asn
Val Tyr Val Pro Pro Asp Cys Ser Thr Ile Asn Val Pro 260 265 270Tyr
Ala Asn Ile Asp Ala Gly Ile Gly Gly Gln 275 280242086DNAARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 24gcatgcattg tcaaagtttg tgaagtagaa
ttaataacct tttggttatt gatcactgta 60tgtatcttag atgtcccgta gcaacggtaa
gggcattcac ctagtactag tccaatatta 120attaataact tgcacagaat
tacaaccatt gacataaaaa ggaaatatga tgagtcatgt 180attgattcat
gttcaacatt actacccttg acataaaaga agaatttgac gagtcgtatt
240agcttgttca tcttaccatc atactatact gcaagctagt ttaaaaaaga
atyaaagtcc 300agaatgaaca gtagaatagc ctgatctatc tttaacaaca
tgcacaagaa tacaaattta 360gtcccttgca agctatgaag atttggttta
tgcctaacaa catgataaac ttagatccaa 420aaggaatgca atctagataa
ttgtttgact tgtaaagtcg ataagatgag tcagtgccaa 480ttataaagtt
ttcgccactc ttagatcata tgtacaataa aaaggcaact ttgctgacca
540ctccaaaagt acgtttgtat gtagtgccac caaacacaac acaccaaata
atcagtttga 600taagcatcga atcactttaa aaagtgaaag aaataatgaa
aagaaaccta accatggtag 660ctataaaaag cctgtaatat gtacactcca
taccatcatc catccttcac acaactagag 720cacaagcatc aaatccaagt
aagtattagt taacgcaaat ccaccatgaa gaccttactc 780atcctaacaa
tccttgcgat ggcaacaacc atcgccaccg ccaatatgca agtcgacccc
840agcggccaag tacaatggcc acaacaacaa ccattccccc agccccaaca
accattctgc 900cagcaaccac aacgaactat tccccaaccc catcaaacat
tccaccatca accacaacaa 960acatttcccc aaccccaaca aacatacccc
catcaaccac aacaacaatt tccccagacc 1020caacaaccac aacaaccatt
tccccagccc caacaaacat tcccccaaca accccaacta 1080ccatttcccc
aacaacccca acaaccattc ccccagcctc agcaacccca acaaccattt
1140ccccagtcac aacaaccaca acaacctttt ccccagcccc aacaacaatt
tccgcagccc 1200caacaaccac aacaatcatt cccccaacaa caacaaccgg
cgattcagtc atttctacaa 1260caacagatga acccctgcaa gaatttcctc
ttgcagcaat gcaaccatgt gtcattggtg 1320tcatctctcg tgtcaataat
tttgccacga agtgattgcc aggtgatgca gcaacaatgt 1380tgccaacaac
tagcacaaat tcctcaacag ctccagtgcg cagccatcca cagcgtcgcg
1440cattccatca tcatgcaaca agaacaacaa caaggcgtgc cgatcctgcg
gccactattt 1500cagctcgccc agggtctggg tatcatccaa cctcaacaac
cagctcaatt ggaggggatc 1560aggtcattgg tattgaaaac tcttccaacc
atgtgcaacg tgtatgtgcc acctgactgc 1620tccaccatca acgtaccata
tgccaacata gacgctggca ttggtggcca atgaaaaatg 1680caagatcatc
attgcttagc tgatgcacca atcgttgtag cgatgacaaa taaagtggtg
1740tgcaccatca tgtgtgaccc cgaccagtgc tagttcaagc ttgggaataa
aagacaaaca 1800aagttcttgt ttgctagcat tgcttgtcac tgttacattc
actttttatt tcgatgttca 1860tccctaaccg caatcctagc cttacacgtc
aatagctagc tgcttgtgct ggcaggttac 1920tatataatct atcaattaat
ggtcgaccta ttaatccaag taataggcta ttgatagact 1980gctcccaagc
cgaccgagca cctatcagtt acggatttct tgaacattgc acactataat
2040aattcaacgt atttcaacct ctagaagtaa agggcatttt agtagc
208625537DNAARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 25atgaagatgg
tcatcgttct cgtcgtgtgc ctggctctgt cagctgccag cgcctctgca 60atgcagatgc
cctgcccctg cgcggggctg cagggcttgt acggcgctgg cgccggcctg
120acgacgatga tgggcgccgg cgggctgtac ccctacgcgg agtacctgag
gcagccgcag 180tgcagcccgc tggcggcggc gccctactac gccgggtgtg
ggcagccgag cgccatgttc 240cagccgctcc ggcaacagtg ctgccagcag
cagatgagga tgatggacgt gcagtccgtc 300gcgcagcagc tgcagatgat
gatgcagctt gagcgtgccg ctgccgccag cagcagcctg 360tacgagccag
ctctgatgca gcagcagcag cagctgctgg cagcccaggg tctcaacccc
420atggccatga tgatggcgca gaacatgccg gccatgggtg gactctacca
gtaccagctg 480cccagctacc gcaccaaccc ctgtggcgtc tccgctgcca
ttccgcccta ctactga 53726178PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE
26Met Lys Met Val Ile Val Leu Val Val Cys Leu Ala Leu Ser Ala Ala1
5 10 15Ser Ala Ser Ala Met Gln Met Pro Cys Pro Cys Ala Gly Leu Gln
Gly 20 25 30Leu Tyr Gly Ala Gly Ala Gly Leu Thr Thr Met Met Gly Ala
Gly Gly 35 40 45Leu Tyr Pro Tyr Ala Glu Tyr Leu Arg Gln Pro Gln Cys
Ser Pro Leu 50 55 60Ala Ala Ala Pro Tyr Tyr Ala Gly Cys Gly Gln Pro
Ser Ala Met Phe65 70 75 80Gln Pro Leu Arg Gln Gln Cys Cys Gln Gln
Gln
Met Arg Met Met Asp 85 90 95Val Gln Ser Val Ala Gln Gln Leu Gln Met
Met Met Gln Leu Glu Arg 100 105 110Ala Ala Ala Ala Ser Ser Ser Leu
Tyr Glu Pro Ala Leu Met Gln Gln 115 120 125Gln Gln Gln Leu Leu Ala
Ala Gln Gly Leu Asn Pro Met Ala Met Met 130 135 140Met Ala Gln Asn
Met Pro Ala Met Gly Gly Leu Tyr Gln Tyr Gln Leu145 150 155 160Pro
Ser Tyr Arg Thr Asn Pro Cys Gly Val Ser Ala Ala Ile Pro Pro 165 170
175Tyr Tyr27453DNAARTIFICIAL SEQUENCESYNTHETIC SEQUENCE
27atggcagcca agatgcttgc attgttcgct ctcctagctc tttgtgcaag cgccactagt
60gcgacgcata ttccagggca cttgccacca gtcatgccat tgggtaccat gaacccatgc
120atgcagtact gcatgatgca acaggggctt gccagcttga tggcgtgtcc
gtccctgatg 180ctgcagcaac tgttggcctt accgcttcag acgatgccag
tgatgatgcc acagatgatg 240acgcctaaca tgatgtcacc attgatgatg
ccgagcatga tgtcaccaat ggtcttgccg 300agcatgatgt cgcaaatgat
gatgccacaa tgtcactgcg acgccgtctc gcagattatg 360ctgcaacagc
agttaccatt catgttcaac ccaatggcca tgacgattcc acccatgttc
420ttacagcaac cctttgttgg tgctgcattc tag 45328150PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 28Met Ala Ala Lys Met Leu Ala Leu Phe
Ala Leu Leu Ala Leu Cys Ala1 5 10 15Ser Ala Thr Ser Ala Thr His Ile
Pro Gly His Leu Pro Pro Val Met 20 25 30Pro Leu Gly Thr Met Asn Pro
Cys Met Gln Tyr Cys Met Met Gln Gln 35 40 45Gly Leu Ala Ser Leu Met
Ala Cys Pro Ser Leu Met Leu Gln Gln Leu 50 55 60Leu Ala Leu Pro Leu
Gln Thr Met Pro Val Met Met Pro Gln Met Met65 70 75 80Thr Pro Asn
Met Met Ser Pro Leu Met Met Pro Ser Met Met Ser Pro 85 90 95Met Val
Leu Pro Ser Met Met Ser Gln Met Met Met Pro Gln Cys His 100 105
110Cys Asp Ala Val Ser Gln Ile Met Leu Gln Gln Gln Leu Pro Phe Met
115 120 125Phe Asn Pro Met Ala Met Thr Ile Pro Pro Met Phe Leu Gln
Gln Pro 130 135 140Phe Val Gly Ala Ala Phe145 1502919PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 29Met Arg Val Leu Leu Val Ala Leu Ala
Leu Leu Ala Leu Ala Ala Ser1 5 10 15Ala Thr Ser3020PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 30Met Lys Thr Phe Leu Ile Leu Val Leu
Leu Ala Ile Val Ala Thr Thr1 5 10 15Ala Thr Thr Ala
203121PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 31Met Lys Thr Leu
Leu Ile Leu Thr Ile Leu Ala Met Ala Ile Thr Ile1 5 10 15Gly Thr Ala
Asn Met 203225PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 32Met Asn
Phe Leu Lys Ser Phe Pro Phe Tyr Ala Phe Leu Cys Phe Gly1 5 10 15Gln
Tyr Phe Val Ala Val Thr His Ala 20 253316PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 33Gly Pro Lys Glu Pro Phe Arg Asp Tyr
Val Asp Arg Phe Tyr Lys Cys1 5 10 153417PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 34Phe Gln Val Val His Asn Ser Tyr Asn
Arg Pro Ala Tyr Ser Pro Gly1 5 10 15Cys3525PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 35Val Glu Ile Lys Glu Gly Thr Val Thr
Leu Lys Arg Glu Ile Asp Lys1 5 10 15Asn Gly Lys Val Thr Val Ser Leu
Cys 20 253619PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 36Thr Leu Ser
Lys Asn Ile Ser Lys Ser Gly Glu Val Ser Val Glu Leu1 5 10 15Asn Asp
Cys3711PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 37Ser Ser Val Ser
Ser Phe Glu Arg Phe Glu Cys1 5 103810PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 38Leu Ile Asp Ala Leu Leu Gly Asp Pro
Cys1 5 10399PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 39Thr Leu Ile
Asp Ala Leu Leu Gly Cys1 54024PRTARTIFICIAL SEQUENCESYNTHETIC
SEQUENCE 40Phe Trp Arg Gly Glu Asn Gly Arg Lys Thr Arg Ser Ala Tyr
Glu Arg1 5 10 15Met Cys Asn Ile Leu Lys Gly Lys 204122PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 41Leu Arg Val Leu Ser Phe Ile Arg Gly
Thr Lys Val Ser Pro Arg Gly1 5 10 15Lys Leu Ser Thr Arg Gly
204222PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 42Ser Leu Val Gly
Ile Asp Pro Phe Lys Leu Leu Gln Asn Ser Gln Val1 5 10 15Tyr Ser Leu
Ile Arg Pro 204324PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 43Ala
Val Lys Gly Val Gly Thr Met Val Met Glu Leu Ile Arg Met Ile1 5 10
15Lys Arg Gly Ile Asn Asp Arg Asn 204421PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 44Ser His Asn Phe Thr Leu Val Ala Ser
Val Ile Ile Glu Glu Ala Pro1 5 10 15Ser Gly Asn Thr Cys
204516PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 45Ser Val Gln Ile
Pro Lys Val Pro Tyr Pro Asn Gly Ile Val Tyr Cys1 5 10
154616PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 46Asp Phe Asn His
Tyr Tyr Thr Leu Lys Thr Gly Leu Glu Ala Asp Cys1 5 10
154718PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 47Pro Ser Asp Lys
His Ile Glu Gln Tyr Lys Lys Ile Lys Asn Ser Ile1 5 10 15Ser
Cys4820PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 48Glu Tyr Leu Asn
Lys Ile Gln Asn Ser Leu Ser Thr Glu Trp Ser Pro1 5 10 15Cys Ser Val
Thr 204919PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 49Tyr Leu Asp
Lys Val Arg Ala Thr Val Gly Thr Glu Trp Thr Pro Cys1 5 10 15Ser Val
Thr5020PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 50Glu Phe Val Lys
Gln Ile Ser Ser Gln Leu Thr Glu Glu Trp Ser Gln1 5 10 15Cys Ser Val
Thr 205116PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 51Lys Pro Arg
Pro Ile Tyr Glu Ala Lys Leu Ala Gln Asn Gln Lys Cys1 5 10
155217PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 52Ala Lys Ala Asp
Tyr Glu Ala Lys Leu Ala Gln Tyr Glu Lys Asp Leu1 5 10
15Cys5316PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 53Arg Pro Gln Ala
Ser Gly Val Tyr Met Gly Asn Leu Thr Ala Gln Cys1 5 10
155416PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 54Gln Tyr Ile Lys
Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Cys1 5 10
155519PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 55Ala Ile Trp Gln
Val Glu Gln Lys Ala Ser Ile Ala Gly Thr Asp Ser1 5 10 15Gly Trp
Cys5619PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 56Asn Tyr Lys Asn
Gly Gly Phe Phe Val Gln Tyr Gly Gly Ala Tyr Lys1 5 10 15Arg His
Cys5719PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 57His Asn Ser Gln
Thr Glu Val Ala Ala Thr Leu Ala Tyr Arg Phe Gly1 5 10 15Asn Val
Cys5819PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 58Thr Pro Arg Val
Ser Tyr Ala His Gly Phe Lys Gly Leu Val Asp Asp1 5 10 15Ala Asp
Cys5919PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 59Arg Phe Gly Asn
Ala Val Pro Arg Ile Ser Tyr Ala His Gly Phe Asp1 5 10 15Phe Ile
Cys6019PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 60Ala Phe Lys Tyr
Ala Arg His Ala Asn Val Gly Arg Asn Ala Phe Glu1 5 10 15Leu Phe
Cys6120PRTARTIFICIAL SEQUENCESYNTHEIC SEQUENCE 61Ser Gly Ala Trp
Leu Lys Arg Asn Thr Gly Ile Gly Asn Tyr Thr Gln1 5 10 15Ile Asn Ala
Cys 206216PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 62Ala Gly Glu
Phe Gly Thr Leu Arg Ala Gly Arg Val Ala Asn Gln Cys1 5 10
156316PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 63Ile Gly Asn Tyr
Thr Gln Ile Asn Ala Ala Ser Val Gly Leu Arg Cys1 5 10
156416PRTARTIFICIAL SEQUENCESYNTHEIC SEQUENCE 64Gly Arg Asn Tyr Gln
Leu Gln Leu Thr Glu Gln Pro Ser Arg Thr Cys1 5 10
156516PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 65Ser Gly Ser Val
Gln Phe Val Pro Ala Gln Asn Ser Lys Ser Ala Cys1 5 10
156616PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 66His Ala Asn Val
Gly Arg Asp Ala Phe Asn Leu Phe Leu Leu Gly Cys1 5 10
156716PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 67Leu Gly Arg Ile
Gly Asp Asp Asp Glu Ala Lys Gly Thr Asp Pro Cys1 5 10
156816PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 68Ser Val Gln Phe
Val Pro Ala Gln Asn Ser Lys Ser Ala Tyr Lys Cys1 5 10
156916PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 69Asn Tyr Ala Phe
Lys Tyr Ala Lys His Ala Asn Val Gly Arg Asp Cys1 5 10
157016PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 70Ala His Gly Phe
Asp Phe Ile Glu Arg Gly Lys Lys Gly Glu Asn Cys1 5 10
157116PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 71Gly Val Asp Tyr
Asp Phe Ser Lys Arg Thr Ser Ala Ile Val Ser Cys1 5 10
157216PRTARTIFICIAL SEQUENCEARTIFICIAL SEQUENCE 72His Asp Asp Met
Pro Val Ser Val Arg Tyr Asp Ser Pro Asp Phe Cys1 5 10
157327PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 73Arg Phe Gly Asn
Ala Val Pro Arg Ile Ser Tyr Ala His Gly Phe Asp1 5 10 15Phe Ile Glu
Arg Gly Lys Lys Gly Glu Asn Cys 20 257424PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 74Asn Tyr Ala Phe Lys Tyr Ala Lys His
Ala Asn Val Gly Arg Asp Ala1 5 10 15Phe Asn Leu Phe Leu Leu Gly Cys
207526PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 75Ser Gly Ala Trp
Leu Lys Arg Asn Thr Gly Ile Gly Asn Tyr Thr Gln1 5 10 15Ile Asn Ala
Ala Ser Val Gly Leu Arg Cys 20 257620PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 76Ser Gly Ser Val Gln Phe Val Pro Ala
Gln Asn Ser Lys Ser Ala Tyr1 5 10 15Thr Pro Ala Cys
207719PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 77Thr Gly Ala Asn
Asn Thr Ser Thr Val Ser Asp Tyr Phe Arg Asn Arg1 5 10 15Ile Thr
Cys7819PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 78Ile Tyr Asp Phe
Lys Leu Asn Asp Lys Phe Asp Lys Phe Lys Pro Tyr1 5 10 15Ile Gly
Cys7919PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 79Leu Ser Ala Ile
Tyr Asp Phe Lys Leu Asn Asp Lys Phe Lys Pro Tyr1 5 10 15Ile Gly
Cys8019PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 80Asn Gly Trp Tyr
Ile Asn Pro Trp Ser Glu Val Lys Phe Asp Leu Asn1 5 10 15Ser Arg
Cys8121PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 81Met Gly Thr Asn
Leu Ser Val Pro Asn Pro Leu Gly Phe Phe Pro Asp1 5 10 15His Gln Leu
Asp Pro 20828PRTARTIFICIAL PROTEINSYNTHETIC SEQUENCE 82Pro Leu Gly
Phe Phe Pro Asp His1 58310PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE
83Pro Leu Gly Phe Phe Pro Asp His Gln Leu1 5 108426PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 84Met Gln Trp Asn Ser Thr Ala Phe His
Gln Thr Leu Gln Asp Pro Arg1 5 10 15Val Arg Gly Leu Tyr Leu Pro Ala
Gly Gly 20 258514PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 85Met Gln
Trp Ser Thr Ala Phe His Gln Thr Leu Gln Asp Pro1 5
108615PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 86Met Gln Trp Asn
Ser Thr Ala Leu His Gln Ala Leu Gln Asp Pro1 5 10
15876PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 87Gln Asp Pro Arg Val
Arg1 58820PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 88Met Asp Ile
Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu1 5 10 15Ser Phe
Leu Pro 208925PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 89Arg Asp
Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu1 5 10 15Ser
Pro Glu His Cys Ser Pro His His 20 259025PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 90Thr Trp Val Gly Val Asn Leu Glu Asp
Pro Ala Ser Arg Asp Leu Val1 5 10 15Val Ser Tyr Val Asn Thr Asn Met
Gly 20 259116PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 91Val Val Ser
Tyr Val Asn Thr Asn Met Gly Leu Lys Phe Arg Gln Leu1 5 10
159221PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 92Leu Leu Trp Phe
His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val1 5 10 15Ile Glu Tyr
Leu Val 209311PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 93Leu Leu
Trp Phe His Ile Ser Cys Leu Thr Phe1 5 109421PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 94Val Ser Phe Gly Val Trp Ile Arg Thr
Pro Pro Ala Tyr Arg Pro Pro1 5 10 15Asn Ala Pro Ile Leu
209512PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 95Val Ser Phe Gly
Val Trp Ile Arg Thr Pro Pro Ala1 5 109612PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 96Pro Pro Ala Tyr Arg Pro Pro Asn Ala
Pro Ile Leu1 5 109712PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 97Trp
Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn1 5 109820PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 98Pro His His Thr Ala Leu Arg Gln Ala
Ile Leu Cys Trp Gly Glu Leu1 5 10 15Met Thr Leu Ala
209915PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 99Ala Val Leu Glu
Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys Val1 5 10
1510015PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 100Leu Leu Val Ser
Ser Lys Val Ser Thr Val Lys Asp Leu Leu Pro1 5 10
1510115PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 101Leu Leu Pro Leu
Leu Glu Lys Val Ile Gly Ala Gly Lys Pro Leu1 5 10
1510215PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 102Ala Ile Leu Thr
Gly Gly Gln Val Ile Ser Glu Glu Val Gly Leu1 5 10
1510315PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 103Ile Ala Phe Asn
Ser Gly Leu Glu Pro Gly Val Val Ala Glu Lys1 5 10
1510418PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 104Ala Arg Arg Gly
Leu Glu Arg Gly Leu Asn Ala Leu Ala Asp Ala Val1 5 10 15Lys
Val10514PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 105Glu Lys Ile Gly
Ala Glu Leu Val Lys Glu Val Ala Lys Lys1 5 1010616PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 106Gly Leu Lys Arg Gly Ile Glu Lys Ala
Val Glu Lys Val Glu Thr Leu1 5 10 1510715PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 107Ile Glu Asp Ala Val Arg Asn Ala Lys
Ala Ala Val Glu Glu Gly1 5 10 151081083DNAARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 108atgggtgcta gagcttctat tcttagaggt
gaaaagcttg ataagtggga aaagattaga 60cttagaccag gtggtaagaa gcattatatg
cttaagcata ttgtttgggc ttctagagaa 120cttgaaagat ttgctcttaa
tccaggtttg cttgaaactt ctgaaggttg taagcaaatt 180atgaagcaac
ttcaaccagc tcttcaaact ggtactgaag aacttaagtc tctttataat
240actgttgcta ctctttattg tgttcatgaa aagattgaag ttagagatac
taaggaagct 300cttgataaga ttgaagaaga acaaaataag tgtcaacaaa
agactcaaca agctaaggct 360gctgatggta aggtttctca aaattatcca
attgttcaaa atcttcaagg tcaaatggtt 420catcaagcta tttctccaag
aactcttaat gcttgggtta aggttattga agaaaaggct 480ttttctccag
aagttattcc aatgtttact gctctttctg aaggtgctac tccacaagat
540cttaatacta tgcttaatac tgttggtggt catcaagctg ctatgcaaat
gcttaaggat 600actattaatg aagaagctgc tgaatgggat agacttcatc
cagttcatgc tggtccaatt 660gctccaggtc aaatgagaga accaagaggt
tctgatattg ctggtactac ttctactctt 720caagaacaaa ttgcttggat
gacttctaat ccaccaattc cagttggtga tatttataag 780agatggatta
ttcttggtct taataagatt gttagaatgt attctccagt ttctattctt
840gatattagac aaggtccaaa ggaaccattt agagattatg ttgatagatt
ttttaagact 900cttagagctg aacaagctac tcaagaagtt aagaattgga
tgactgatac tcttcttgtt 960caaaatgcta atccagattg taagactatt
cttagggctc ttggtccagg tgctactctt 1020gaagaaatga tgactgcttg
tcaaggtgtt ggtggtccag gtcataaggc tagagttctt 1080taa
1083109360PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 109Met Gly Ala
Arg Ala Ser Ile Leu Arg Gly Glu Lys Leu Asp Lys Trp1 5 10 15Glu Lys
Ile Arg Leu Arg Pro Gly Gly Lys Lys
His Tyr Met Leu Lys 20 25 30His Ile Val Trp Ala Ser Arg Glu Leu Glu
Arg Phe Ala Leu Asn Pro 35 40 45Gly Leu Leu Glu Thr Ser Glu Gly Cys
Lys Gln Ile Met Lys Gln Leu 50 55 60Gln Pro Ala Leu Gln Thr Gly Thr
Glu Glu Leu Lys Ser Leu Tyr Asn65 70 75 80Thr Val Ala Thr Leu Tyr
Cys Val His Glu Lys Ile Glu Val Arg Asp 85 90 95Thr Lys Glu Ala Leu
Asp Lys Ile Glu Glu Glu Gln Asn Lys Cys Gln 100 105 110Gln Lys Thr
Gln Gln Ala Lys Ala Ala Asp Gly Lys Val Ser Gln Asn 115 120 125Tyr
Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile 130 135
140Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys
Ala145 150 155 160Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu
Ser Glu Gly Ala 165 170 175Thr Pro Gln Asp Leu Asn Thr Met Leu Asn
Thr Val Gly Gly His Gln 180 185 190Ala Ala Met Gln Met Leu Lys Asp
Thr Ile Asn Glu Glu Ala Ala Glu 195 200 205Trp Asp Arg Leu His Pro
Val His Ala Gly Pro Ile Ala Pro Gly Gln 210 215 220Met Arg Glu Pro
Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu225 230 235 240Gln
Glu Gln Ile Ala Trp Met Thr Ser Asn Pro Pro Ile Pro Val Gly 245 250
255Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg
260 265 270Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro
Lys Glu 275 280 285Pro Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr
Leu Arg Ala Glu 290 295 300Gln Ala Thr Gln Glu Val Lys Asn Trp Met
Thr Asp Thr Leu Leu Val305 310 315 320Gln Asn Ala Asn Pro Asp Cys
Lys Thr Ile Leu Arg Ala Leu Gly Pro 325 330 335Gly Ala Thr Leu Glu
Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly 340 345 350Pro Gly His
Lys Ala Arg Val Leu 355 360110699PRTARTIFICIAL SEQUENCESYNTHETIC
SEQUENCE 110Ala Thr Gly Cys Cys Ala Ala Thr Thr Gly Thr Thr Cys Ala
Ala Ala1 5 10 15Ala Thr Cys Thr Thr Cys Ala Ala Gly Gly Thr Cys Ala
Ala Ala Thr 20 25 30Gly Gly Thr Thr Cys Ala Thr Cys Ala Ala Gly Cys
Thr Ala Thr Thr 35 40 45Thr Cys Thr Cys Cys Ala Ala Gly Ala Ala Cys
Thr Cys Thr Thr Ala 50 55 60Ala Thr Gly Cys Thr Thr Gly Gly Gly Thr
Thr Ala Ala Gly Gly Thr65 70 75 80Thr Ala Thr Thr Gly Ala Ala Gly
Ala Ala Ala Ala Gly Gly Cys Thr 85 90 95Thr Thr Thr Thr Cys Thr Cys
Cys Ala Gly Ala Ala Gly Thr Thr Ala 100 105 110Thr Thr Cys Cys Ala
Ala Thr Gly Thr Thr Thr Ala Cys Thr Gly Cys 115 120 125Thr Cys Thr
Thr Thr Cys Thr Gly Ala Ala Gly Gly Thr Gly Cys Thr 130 135 140Ala
Cys Thr Cys Cys Ala Cys Ala Ala Gly Ala Thr Cys Thr Thr Ala145 150
155 160Ala Thr Ala Cys Thr Ala Thr Gly Cys Thr Thr Ala Ala Thr Ala
Cys 165 170 175Thr Gly Thr Thr Gly Gly Thr Gly Gly Thr Cys Ala Thr
Cys Ala Ala 180 185 190Gly Cys Thr Gly Cys Thr Ala Thr Gly Cys Ala
Ala Ala Thr Gly Cys 195 200 205Thr Thr Ala Ala Gly Gly Ala Thr Ala
Cys Thr Ala Thr Thr Ala Ala 210 215 220Thr Gly Ala Ala Gly Ala Ala
Gly Cys Thr Gly Cys Thr Gly Ala Ala225 230 235 240Thr Gly Gly Gly
Ala Thr Ala Gly Ala Cys Thr Thr Cys Ala Thr Cys 245 250 255Cys Ala
Gly Thr Thr Cys Ala Thr Gly Cys Thr Gly Gly Thr Cys Cys 260 265
270Ala Ala Thr Thr Gly Cys Thr Cys Cys Ala Gly Gly Thr Cys Ala Ala
275 280 285Ala Thr Gly Ala Gly Ala Gly Ala Ala Cys Cys Ala Ala Gly
Ala Gly 290 295 300Gly Thr Thr Cys Thr Gly Ala Thr Ala Thr Thr Gly
Cys Thr Gly Gly305 310 315 320Thr Ala Cys Thr Ala Cys Thr Thr Cys
Thr Ala Cys Thr Cys Thr Thr 325 330 335Cys Ala Ala Gly Ala Ala Cys
Ala Ala Ala Thr Thr Gly Cys Thr Thr 340 345 350Gly Gly Ala Thr Gly
Ala Cys Thr Thr Cys Thr Ala Ala Thr Cys Cys 355 360 365Ala Cys Cys
Ala Ala Thr Thr Cys Cys Ala Gly Thr Thr Gly Gly Thr 370 375 380Gly
Ala Thr Ala Thr Thr Thr Ala Thr Ala Ala Gly Ala Gly Ala Thr385 390
395 400Gly Gly Ala Thr Thr Ala Thr Thr Cys Thr Thr Gly Gly Thr Cys
Thr 405 410 415Thr Ala Ala Thr Ala Ala Gly Ala Thr Thr Gly Thr Thr
Ala Gly Ala 420 425 430Ala Thr Gly Thr Ala Thr Thr Cys Thr Cys Cys
Ala Gly Thr Thr Thr 435 440 445Cys Thr Ala Thr Thr Cys Thr Thr Gly
Ala Thr Ala Thr Thr Ala Gly 450 455 460Ala Cys Ala Ala Gly Gly Thr
Cys Cys Ala Ala Ala Gly Gly Ala Ala465 470 475 480Cys Cys Ala Thr
Thr Thr Ala Gly Ala Gly Ala Thr Thr Ala Thr Gly 485 490 495Thr Thr
Gly Ala Thr Ala Gly Ala Thr Thr Thr Thr Thr Thr Ala Ala 500 505
510Gly Ala Cys Thr Cys Thr Thr Ala Gly Ala Gly Cys Thr Gly Ala Ala
515 520 525Cys Ala Ala Gly Cys Thr Ala Cys Thr Cys Ala Ala Gly Ala
Ala Gly 530 535 540Thr Thr Ala Ala Gly Ala Ala Thr Thr Gly Gly Ala
Thr Gly Ala Cys545 550 555 560Thr Gly Ala Thr Ala Cys Thr Cys Thr
Thr Cys Thr Thr Gly Thr Thr 565 570 575Cys Ala Ala Ala Ala Thr Gly
Cys Thr Ala Ala Thr Cys Cys Ala Gly 580 585 590Ala Thr Thr Gly Thr
Ala Ala Gly Ala Cys Thr Ala Thr Thr Cys Thr 595 600 605Thr Ala Gly
Gly Gly Cys Thr Cys Thr Thr Gly Gly Thr Cys Cys Ala 610 615 620Gly
Gly Thr Gly Cys Thr Ala Cys Thr Cys Thr Thr Gly Ala Ala Gly625 630
635 640Ala Ala Ala Thr Gly Ala Thr Gly Ala Cys Thr Gly Cys Thr Thr
Gly 645 650 655Thr Cys Ala Ala Gly Gly Thr Gly Thr Thr Gly Gly Thr
Gly Gly Thr 660 665 670Cys Cys Ala Gly Gly Thr Cys Ala Thr Ala Ala
Gly Gly Cys Thr Ala 675 680 685Gly Ala Gly Thr Thr Cys Thr Thr Thr
Ala Ala 690 695111232PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE
111Met Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val His Gln Ala Ile1
5 10 15Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile Glu Glu Lys
Ala 20 25 30Phe Ser Pro Glu Val Ile Pro Met Phe Thr Ala Leu Ser Glu
Gly Ala 35 40 45Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly
Gly His Gln 50 55 60Ala Ala Met Gln Met Leu Lys Asp Thr Ile Asn Glu
Glu Ala Ala Glu65 70 75 80Trp Asp Arg Leu His Pro Val His Ala Gly
Pro Ile Ala Pro Gly Gln 85 90 95Met Arg Glu Pro Arg Gly Ser Asp Ile
Ala Gly Thr Thr Ser Thr Leu 100 105 110Gln Glu Gln Ile Ala Trp Met
Thr Ser Asn Pro Pro Ile Pro Val Gly 115 120 125Asp Ile Tyr Lys Arg
Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 130 135 140Met Tyr Ser
Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu145 150 155
160Pro Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu
165 170 175Gln Ala Thr Gln Glu Val Lys Asn Trp Met Thr Asp Thr Leu
Leu Val 180 185 190Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Arg
Ala Leu Gly Pro 195 200 205Gly Ala Thr Leu Glu Glu Met Met Thr Ala
Cys Gln Gly Val Gly Gly 210 215 220Pro Gly His Lys Ala Arg Val
Leu225 2301121731DNAARTIFICIAL SEQUENCESYNTHETIC SEQUENCE
112atgagggtgt tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc
cacctccacg 60catacaagcg gcggctgcgg ctgccagcca ccgccgccgg ttcatctacc
gccgccggtg 120catctgccac ctccggttca cctgccacct ccggtgcatc
tcccaccgcc ggtccacctg 180ccgccgccgg tccacctgcc accgccggtc
catgtgccgc cgccggttca tctgccgccg 240ccaccatgcc actaccctac
tcaaccgccc cggcctcagc ctcatcccca gccacaccca 300tgcccgtgcc
aacagccgca tccaagcccg tgccagacca tggacgacga tgataagtgc
360ggcaagaagg ccatcggcac cgtgctggtg ggccccaccc ccgtgaacat
catcggccgg 420aacatgctga cccagctggg ctgcaccctg aacttcccca
tcagccccat cgagaccgtg 480cccgtgaagc tgaagcccgg catggacggc
cccaaggtga agcagtggcc cctgaccgag 540gtgaagatca aggccctgac
cgccatctgc gaggagatgg agaaggaggg caagatcacc 600aagatcggcc
ccgagaaccc ctacaacacc cccatcttcg ccatcaagaa ggaggacagc
660accaagtggc ggaagctggt ggacttccgg gagctgaaca agcggaccca
ggacttctgg 720gaggtgcagc tgggcatccc ccaccccgcc ggcctgaaga
agaagaagag cgtgaccgtg 780ctggacgtgg gcgacgccta cttcagcgtg
cccctggacg agggcttccg gaagtacacc 840gccttcacca tccccagcat
caacaacgag acccccggca tccggtacca gtacaacgtg 900ctgccccagg
gctggaaggg cagccccgcc atcttccagg ccagcatgac caagatcctg
960gagcccttcc gggccaagaa ccccgagatc gtgatctacc agtacatggc
cgccctgtac 1020gtgggcagcg acctggagat cggccagcac cgggccaaga
tcgaggagct gcgggagcac 1080ctgctgaagt ggggcttcac cacccccgac
aagaagcacc agaaggagcc ccccttcctg 1140tggatgggct acgagctgca
ccccgacaag tggaccgtgc agcccatcca gctgcccgag 1200aaggacagct
ggaccgtgaa cgacatccag aagctggtgg gcaagctgaa ctggaccagc
1260cagatctacc ccggcatcaa ggtgcggcag ctgtgcaagc tgctgcgggg
caccaaggcc 1320ctgaccgaca tcgtgcccct gaccgaggag gccgagctgg
agctggccga gaaccgggag 1380atcctgaagg agcccgtgca cggcgtgtac
tacgacccca gcaaggacct gatcgccgag 1440atccagaagc agggcgacga
ccagtggacc taccagatct accaggagcc cttcaagaac 1500ctgaaaaccg
gcaagtacgc caagcggcgg accacccaca ccaacgacgt gaagcagctg
1560accgaggccg tgcagaagat cagcctggag agcatcgtga cctggggcaa
gacccccaag 1620ttccggctgc ccatccagaa ggagacctgg gagatctggt
ggaccgacta ctggcaggcc 1680acctggatcc ccgagtggga gttcgtgaac
agcggccgct ttcgaatcta g 1731113576PRTARTIFICIAL SEQUENCESYNTHETIC
SEQUENCE 113Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala
Ala Ser1 5 10 15Ala Thr Ser Thr His Thr Ser Gly Gly Cys Gly Cys Gln
Pro Pro Pro 20 25 30Pro Val His Leu Pro Pro Pro Val His Leu Pro Pro
Pro Val His Leu 35 40 45Pro Pro Pro Val His Leu Pro Pro Pro Val His
Leu Pro Pro Pro Val 50 55 60His Leu Pro Pro Pro Val His Val Pro Pro
Pro Val His Leu Pro Pro65 70 75 80Pro Pro Cys His Tyr Pro Thr Gln
Pro Pro Arg Pro Gln Pro His Pro 85 90 95Gln Pro His Pro Cys Pro Cys
Gln Gln Pro His Pro Ser Pro Cys Gln 100 105 110Thr Met Asp Asp Asp
Asp Lys Cys Gly Lys Lys Ala Ile Gly Thr Val 115 120 125Leu Val Gly
Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Met Leu Thr 130 135 140Gln
Leu Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu Thr Val145 150
155 160Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val Lys Gln
Trp 165 170 175Pro Leu Thr Glu Val Lys Ile Lys Ala Leu Thr Ala Ile
Cys Glu Glu 180 185 190Met Glu Lys Glu Gly Lys Ile Thr Lys Ile Gly
Pro Glu Asn Pro Tyr 195 200 205Asn Thr Pro Ile Phe Ala Ile Lys Lys
Glu Asp Ser Thr Lys Trp Arg 210 215 220Lys Leu Val Asp Phe Arg Glu
Leu Asn Lys Arg Thr Gln Asp Phe Trp225 230 235 240Glu Val Gln Leu
Gly Ile Pro His Pro Ala Gly Leu Lys Lys Lys Lys 245 250 255Ser Val
Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser Val Pro Leu 260 265
270Asp Glu Gly Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro Ser Ile Asn
275 280 285Asn Glu Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu Pro
Gln Gly 290 295 300Trp Lys Gly Ser Pro Ala Ile Phe Gln Ala Ser Met
Thr Lys Ile Leu305 310 315 320Glu Pro Phe Arg Ala Lys Asn Pro Glu
Ile Val Ile Tyr Gln Tyr Met 325 330 335Ala Ala Leu Tyr Val Gly Ser
Asp Leu Glu Ile Gly Gln His Arg Ala 340 345 350Lys Ile Glu Glu Leu
Arg Glu His Leu Leu Lys Trp Gly Phe Thr Thr 355 360 365Pro Asp Lys
Lys His Gln Lys Glu Pro Pro Phe Leu Trp Met Gly Tyr 370 375 380Glu
Leu His Pro Asp Lys Trp Thr Val Gln Pro Ile Gln Leu Pro Glu385 390
395 400Lys Asp Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val Gly Lys
Leu 405 410 415Asn Trp Thr Ser Gln Ile Tyr Pro Gly Ile Lys Val Arg
Gln Leu Cys 420 425 430Lys Leu Leu Arg Gly Thr Lys Ala Leu Thr Asp
Ile Val Pro Leu Thr 435 440 445Glu Glu Ala Glu Leu Glu Leu Ala Glu
Asn Arg Glu Ile Leu Lys Glu 450 455 460Pro Val His Gly Val Tyr Tyr
Asp Pro Ser Lys Asp Leu Ile Ala Glu465 470 475 480Ile Gln Lys Gln
Gly Asp Asp Gln Trp Thr Tyr Gln Ile Tyr Gln Glu 485 490 495Pro Phe
Lys Asn Leu Lys Thr Gly Lys Tyr Ala Lys Arg Arg Thr Thr 500 505
510His Thr Asn Asp Val Lys Gln Leu Thr Glu Ala Val Gln Lys Ile Ser
515 520 525Leu Glu Ser Ile Val Thr Trp Gly Lys Thr Pro Lys Phe Arg
Leu Pro 530 535 540Ile Gln Lys Glu Thr Trp Glu Ile Trp Trp Thr Asp
Tyr Trp Gln Ala545 550 555 560Thr Trp Ile Pro Glu Trp Glu Phe Val
Asn Ser Gly Arg Phe Arg Ile 565 570 5751141404PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 114Ala Thr Gly Thr Cys Thr Thr Cys Ala
Ala Gly Thr Gly Thr Thr Thr1 5 10 15Ala Thr Gly Ala Gly Thr Cys Gly
Ala Thr Cys Ala Thr Thr Cys Ala 20 25 30Gly Ala Cys Ala Ala Ala Ala
Gly Cys Thr Thr Cys Ala Gly Thr Cys 35 40 45Thr Gly Gly Gly Gly Ala
Thr Cys Ala Ala Cys Thr Gly Cys Ala Thr 50 55 60Cys Thr Gly Gly Thr
Ala Ala Ala Gly Cys Thr Gly Thr Thr Gly Thr65 70 75 80Ala Gly Ala
Thr Thr Cys Thr Thr Ala Cys Thr Gly Gly Ala Thr Thr 85 90 95Cys Ala
Thr Gly Ala Ala Cys Thr Thr Gly Gly Thr Ala Cys Thr Gly 100 105
110Gly Thr Thr Cys Thr Cys Cys Ala Cys Thr Ala Gly Thr Thr Cys Ala
115 120 125Ala Ala Cys Cys Cys Ala Gly Cys Thr Gly Thr Ala Thr Thr
Cys Thr 130 135 140Gly Ala Thr Thr Cys Ala Ala Gly Ala Ala Gly Cys
Ala Ala Ala Ala145 150 155 160Gly Thr Ala Gly Cys Thr Thr Thr Gly
Gly Cys Thr Ala Thr Ala Cys 165 170 175Thr Gly Cys Ala Ala Ala Gly
Gly Thr Ala Gly Gly Gly Ala Ala Thr 180 185 190Cys Thr Thr Cys Cys
Cys Thr Gly Thr Gly Ala Gly Gly Ala Ala Gly 195 200 205Ala Ala Gly
Ala Ala Ala Thr Thr Cys Thr Thr Thr Cys Thr Cys Ala 210 215 220Gly
Cys Ala Thr Gly Thr Gly Thr Ala Thr Ala Thr Cys Cys Cys Thr225 230
235 240Ala Thr Thr Thr Thr Thr Gly Ala Thr Gly Ala Thr Gly Thr Thr
Gly 245 250 255Ala Thr Thr Thr Thr Ala Gly Cys Ala Thr Cys Ala Ala
Thr Ala Thr 260 265 270Thr Gly Ala Thr Gly Ala Cys Thr Cys Thr Gly
Thr Thr Cys Thr Gly 275 280 285Gly Cys Ala Cys Thr Gly Thr Cys Thr
Gly Thr Thr Thr Gly Cys Thr 290 295 300Cys Cys Ala Ala Cys Ala Cys
Ala Gly Thr Cys Ala Ala Thr Ala Cys305
310 315 320Thr Ala Ala Cys Gly Gly Ala Gly Thr Gly Ala Ala Ala Cys
Ala Thr 325 330 335Cys Ala Ala Gly Gly Thr Cys Ala Thr Thr Thr Gly
Ala Ala Ala Gly 340 345 350Thr Thr Thr Thr Gly Thr Cys Thr Cys Cys
Thr Gly Cys Thr Cys Ala 355 360 365Gly Cys Thr Cys Cys Ala Cys Thr
Cys Thr Ala Thr Thr Gly Gly Ala 370 375 380Thr Cys Thr Ala Cys Cys
Ala Thr Gly Ala Ala Cys Gly Gly Ala Thr385 390 395 400Cys Thr Gly
Ala Thr Ala Thr Thr Ala Cys Ala Gly Ala Cys Cys Gly 405 410 415Ala
Thr Thr Cys Cys Ala Gly Cys Thr Cys Cys Ala Ala Gly Ala Ala 420 425
430Ala Ala Ala Gly Ala Thr Ala Thr Ala Ala Thr Thr Cys Cys Cys Ala
435 440 445Ala Thr Gly Ala Cys Ala Gly Gly Thr Ala Cys Ala Thr Thr
Gly Ala 450 455 460Ala Gly Cys Thr Gly Thr Ala Ala Ala Cys Ala Ala
Ala Gly Gly Cys465 470 475 480Thr Cys Thr Thr Thr Gly Thr Cys Thr
Thr Gly Thr Gly Thr Thr Ala 485 490 495Ala Ala Gly Ala Gly Cys Ala
Thr Ala Cys Cys Thr Ala Thr Ala Ala 500 505 510Gly Gly Thr Cys Gly
Ala Gly Ala Thr Gly Thr Gly Cys Thr Ala Cys 515 520 525Ala Ala Thr
Cys Ala Ala Gly Cys Thr Thr Thr Ala Gly Gly Cys Ala 530 535 540Ala
Ala Gly Thr Gly Ala Ala Thr Gly Thr Thr Cys Thr Ala Thr Cys545 550
555 560Cys Cys Cys Thr Ala Ala Cys Ala Gly Ala Ala Ala Thr Gly Thr
Cys 565 570 575Cys Ala Thr Gly Ala Ala Thr Gly Gly Cys Thr Gly Thr
Ala Cys Ala 580 585 590Gly Thr Thr Thr Cys Ala Ala Gly Cys Cys Ala
Ala Ala Thr Thr Thr 595 600 605Cys Ala Ala Thr Cys Ala Ala Gly Thr
Thr Gly Ala Ala Ala Gly Cys 610 615 620Ala Ala Cys Ala Ala Cys Ala
Gly Ala Ala Cys Thr Gly Thr Ala Ala625 630 635 640Ala Thr Thr Cys
Thr Cys Thr Thr Gly Cys Ala Gly Thr Gly Ala Ala 645 650 655Ala Thr
Cys Thr Cys Thr Gly Cys Thr Cys Ala Thr Gly Thr Cys Ala 660 665
670Gly Cys Ala Gly Gly Ala Ala Ala Thr Ala Ala Cys Ala Thr Cys Ala
675 680 685Thr Gly Cys Cys Thr Ala Ala Cys Thr Cys Thr Cys Ala Gly
Gly Cys 690 695 700Thr Thr Thr Thr Gly Thr Cys Ala Ala Ala Gly Cys
Thr Thr Cys Cys705 710 715 720Ala Cys Thr Gly Ala Thr Thr Cys Thr
Cys Ala Thr Thr Thr Cys Ala 725 730 735Ala Gly Cys Thr Gly Ala Gly
Cys Cys Thr Cys Thr Gly Gly Cys Thr 740 745 750Ala Ala Gly Ala Gly
Thr Thr Cys Cys Ala Ala Ala Gly Gly Thr Thr 755 760 765Thr Thr Gly
Ala Ala Gly Cys Ala Gly Ala Thr Thr Thr Cys Cys Ala 770 775 780Thr
Thr Cys Ala Gly Ala Ala Ala Thr Thr Gly Thr Thr Cys Ala Ala785 790
795 800Ala Gly Thr Thr Gly Cys Ala Gly Gly Ala Gly Ala Thr Gly Ala
Ala 805 810 815Ala Cys Thr Ala Ala Cys Ala Ala Ala Ala Cys Ala Thr
Thr Thr Thr 820 825 830Ala Thr Thr Thr Ala Thr Cys Thr Ala Thr Thr
Gly Cys Thr Thr Gly 835 840 845Cys Ala Thr Thr Cys Cys Ala Ala Ala
Cys Cys Ala Thr Ala Ala Cys 850 855 860Ala Gly Thr Gly Thr Thr Gly
Ala Gly Ala Cys Ala Gly Cys Thr Thr865 870 875 880Thr Ala Ala Ala
Cys Ala Thr Thr Thr Cys Thr Gly Thr Thr Ala Thr 885 890 895Thr Thr
Gly Cys Ala Ala Gly Cys Ala Thr Cys Ala Gly Cys Thr Cys 900 905
910Cys Cys Ala Ala Thr Cys Cys Gly Thr Ala Ala Ala Thr Thr Thr Ala
915 920 925Ala Ala Gly Cys Thr Cys Cys Thr Thr Thr Thr Gly Ala Ala
Thr Thr 930 935 940Ala Thr Cys Ala Ala Thr Gly Ala Thr Gly Thr Thr
Thr Thr Cys Thr945 950 955 960Gly Ala Thr Thr Thr Ala Ala Ala Gly
Gly Ala Gly Cys Cys Thr Thr 965 970 975Ala Cys Ala Ala Cys Ala Thr
Thr Gly Thr Thr Cys Ala Thr Gly Ala 980 985 990Thr Cys Cys Thr Thr
Cys Ala Thr Ala Thr Cys Cys Thr Cys Ala Gly 995 1000 1005Ala Gly
Gly Ala Thr Thr Gly Thr Thr Cys Ala Thr Gly Cys Thr 1010 1015
1020Cys Thr Gly Cys Thr Thr Gly Ala Ala Ala Cys Thr Cys Ala Cys
1025 1030 1035Ala Cys Gly Thr Cys Thr Thr Thr Thr Gly Cys Ala Cys
Ala Ala 1040 1045 1050Gly Thr Thr Cys Thr Thr Thr Gly Cys Ala Ala
Cys Ala Ala Cys 1055 1060 1065Thr Thr Gly Cys Ala Ala Gly Ala Ala
Gly Ala Cys Gly Thr Gly 1070 1075 1080Ala Thr Cys Ala Thr Cys Thr
Ala Cys Ala Cys Thr Thr Thr Gly 1085 1090 1095Ala Ala Cys Ala Ala
Cys Thr Ala Thr Gly Ala Gly Cys Thr Ala 1100 1105 1110Ala Cys Thr
Cys Cys Thr Gly Gly Ala Ala Ala Gly Thr Thr Ala 1115 1120 1125Gly
Ala Thr Cys Thr Ala Gly Gly Thr Gly Ala Ala Ala Gly Ala 1130 1135
1140Ala Cys Cys Thr Thr Ala Ala Ala Thr Thr Ala Cys Ala Gly Thr
1145 1150 1155Gly Ala Ala Gly Ala Thr Gly Thr Cys Thr Gly Cys Ala
Ala Ala 1160 1165 1170Ala Gly Gly Ala Ala Ala Thr Ala Thr Thr Thr
Cys Cys Thr Cys 1175 1180 1185Thr Cys Ala Ala Ala Ala Ala Cys Ala
Cys Thr Thr Gly Ala Ala 1190 1195 1200Thr Gly Thr Cys Thr Thr Cys
Cys Ala Thr Cys Thr Ala Ala Cys 1205 1210 1215Ala Cys Ala Cys Ala
Ala Ala Cys Thr Ala Thr Gly Thr Cys Thr 1220 1225 1230Thr Ala Cys
Thr Thr Ala Gly Ala Cys Ala Gly Cys Ala Thr Cys 1235 1240 1245Cys
Ala Ala Ala Thr Cys Cys Cys Thr Thr Cys Cys Thr Gly Gly 1250 1255
1260Ala Ala Gly Ala Thr Ala Gly Ala Cys Thr Thr Thr Gly Cys Thr
1265 1270 1275Ala Gly Gly Gly Gly Ala Gly Ala Ala Ala Thr Thr Ala
Ala Ala 1280 1285 1290Ala Thr Thr Thr Cys Thr Cys Cys Ala Cys Ala
Ala Thr Cys Thr 1295 1300 1305Gly Thr Thr Thr Cys Ala Gly Thr Thr
Gly Cys Ala Ala Ala Ala 1310 1315 1320Thr Cys Thr Thr Thr Gly Thr
Thr Ala Ala Ala Gly Cys Thr Thr 1325 1330 1335Gly Ala Thr Thr Thr
Ala Ala Gly Thr Gly Gly Gly Ala Thr Cys 1340 1345 1350Ala Ala Ala
Ala Ala Gly Ala Ala Ala Gly Ala Ala Thr Cys Thr 1355 1360 1365Ala
Ala Gly Ala Thr Thr Thr Cys Gly Gly Ala Ala Gly Cys Ala 1370 1375
1380Thr Gly Thr Gly Cys Thr Thr Cys Ala Gly Gly Ala Thr Cys Ala
1385 1390 1395Ala Ala Ala Thr Ala Ala 1400115467PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 115Met Ser Ser Ser Val Tyr Glu Ser Ile
Ile Gln Thr Lys Ala Ser Val1 5 10 15Trp Gly Ser Thr Ala Ser Gly Lys
Ala Val Val Asp Ser Tyr Trp Ile 20 25 30His Glu Leu Gly Thr Gly Ser
Pro Leu Val Gln Thr Gln Leu Tyr Ser 35 40 45Asp Ser Arg Ser Lys Ser
Ser Phe Gly Tyr Thr Ala Lys Val Gly Asn 50 55 60Leu Pro Cys Glu Glu
Glu Glu Ile Leu Ser Gln His Val Tyr Ile Pro65 70 75 80Ile Phe Asp
Asp Val Asp Phe Ser Ile Asn Ile Asp Asp Ser Val Leu 85 90 95Ala Leu
Ser Val Cys Ser Asn Thr Val Asn Thr Asn Gly Val Lys His 100 105
110Gln Gly His Leu Lys Val Leu Ser Pro Ala Gln Leu His Ser Ile Gly
115 120 125Ser Thr Met Asn Gly Ser Asp Ile Thr Asp Arg Phe Gln Leu
Gln Glu 130 135 140Lys Asp Ile Ile Pro Asn Asp Arg Tyr Ile Glu Ala
Val Asn Lys Gly145 150 155 160Ser Leu Ser Cys Val Lys Glu His Thr
Tyr Lys Val Glu Met Cys Tyr 165 170 175Asn Gln Ala Leu Gly Lys Val
Asn Val Leu Ser Pro Asn Arg Asn Val 180 185 190His Glu Trp Leu Tyr
Ser Phe Lys Pro Asn Phe Asn Gln Val Glu Ser 195 200 205Asn Asn Arg
Thr Val Asn Ser Leu Ala Val Lys Ser Leu Leu Met Ser 210 215 220Ala
Gly Asn Asn Ile Met Pro Asn Ser Gln Ala Phe Val Lys Ala Ser225 230
235 240Thr Asp Ser His Phe Lys Leu Ser Leu Trp Leu Arg Val Pro Lys
Val 245 250 255Leu Lys Gln Ile Ser Ile Gln Lys Leu Phe Lys Val Ala
Gly Asp Glu 260 265 270Thr Asn Lys Thr Phe Tyr Leu Ser Ile Ala Cys
Ile Pro Asn His Asn 275 280 285Ser Val Glu Thr Ala Leu Asn Ile Ser
Val Ile Cys Lys His Gln Leu 290 295 300Pro Ile Arg Lys Phe Lys Ala
Pro Phe Glu Leu Ser Met Met Phe Ser305 310 315 320Asp Leu Lys Glu
Pro Tyr Asn Ile Val His Asp Pro Ser Tyr Pro Gln 325 330 335Arg Ile
Val His Ala Leu Leu Glu Thr His Thr Ser Phe Ala Gln Val 340 345
350Leu Cys Asn Asn Leu Gln Glu Asp Val Ile Ile Tyr Thr Leu Asn Asn
355 360 365Tyr Glu Leu Thr Pro Gly Lys Leu Asp Leu Gly Glu Arg Thr
Leu Asn 370 375 380Tyr Ser Glu Asp Val Cys Lys Arg Lys Tyr Phe Leu
Ser Lys Thr Leu385 390 395 400Glu Cys Leu Pro Ser Asn Thr Gln Thr
Met Ser Tyr Leu Asp Ser Ile 405 410 415Gln Ile Pro Ser Trp Lys Ile
Asp Phe Ala Arg Gly Glu Ile Lys Ile 420 425 430Ser Pro Gln Ser Val
Ser Val Ala Lys Ser Leu Leu Lys Leu Asp Leu 435 440 445Ser Gly Ile
Lys Lys Lys Glu Ser Lys Ile Ser Glu Ala Cys Ala Ser 450 455 460Gly
Ser Lys4651169PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 116Ala Met
Gln Met Leu Lys Asp Thr Ile1 511719PRTARTIFICIAL SEQUENCESYNTHETIC
SEQUENCE 117Asn Pro Pro Ile Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile
Ile Gly1 5 10 15Leu Asn Lys11820PRTARTIFICIAL SEQUENCESYNTHETIC
SEQUENCE 118Phe Arg Asp Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala
Glu Gln1 5 10 15Ala Thr Gln Glu 2011919PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 119Pro Lys Val Lys Gln Trp Pro Leu Thr
Glu Val Lys Ile Lys Ala Leu1 5 10 15Thr Ala Ile12011PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 120Val Tyr Tyr Asp Pro Ser Lys Asp Leu
Ile Ala1 5 101219PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 121Thr
Ile His Asp Ile Ile Leu Glu Cys1 512210PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 122Phe Ala Phe Arg Asp Leu Cys Ile Val
Tyr1 5 1012310PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 123Tyr Met
Leu Asp Leu Gln Pro Glu Thr Thr1 5 101249PRTARTIFICIAL
SEQUENCESYNTHETIC SEQUENCE 124Leu Glu Asp Leu Leu Met Gly Thr Leu1
51259PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 125Asp Leu Tyr Cys
Tyr Glu Gln Leu Asn1 5126462DNAARTIFICIAL SEQUENCESYNTHETIC
SEQUENCE 126cccgccgcca ccatgcacgg cgacaccccc accctgcacg agtacatgct
ggacctgcag 60cccgagacca ccgacctgta ctgcatctgc agccagaaac ccaagtgcga
cagcaccctg 120cggctgtgcg tgcagagcac ccacgtggac atccggaccc
tggaggacct gctgatgggc 180accctgggca tcgtgtgccc ctacgagcag
ctgaacgaca gcagcgagga ggaggatgag 240atcgacggcc ccgccggcca
ggctgagccc gaccgggccc actacaacat cgtgaccttc 300tgctgccaac
cagagacaac tgatctctac tgttatgagc aattaaatga cagctcagag
360cattacaata ttgtaacctt ttgttgcaag tgtgactcta cgcttcggtt
gtgcatgggc 420acactaggaa ttgtgtgccc catctgttct cagaaaccat aa
4621274912DNAARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 127gacggatcgg
gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60ccgcatagtt
aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
120cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
aagaatctgc 180ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
cagatatacg cgttttgaga 240tttctgtcgc cgactaaatt catgtcgcgc
gatagtggtg tttatcgccg atagagatgg 300cgatattgga aaaatcgata
tttgaaaata tggcatattg aaaatgtcgc cgatgtgagt 360ttctgtgtaa
ctgatatcgc catttttcca aaagtgattt ttgggcatac gcgatatctg
420gcgatagcgc ttatatcgtt tacgggggat ggcgatagac gactttggtg
acttgggcga 480ttctgtgtgt cgcaaatatc gcagtttcga tataggtgac
agacgatatg aggctatatc 540gccgatagag gcgacatcaa gctggcacat
ggccaatgca tatcgatcta tacattgaat 600caatattggc cattagccat
attattcatt ggttatatag cataaatcaa tattggctat 660tggccattgc
atacgttgta tccatatcat aatatgtaca tttatattgg ctcatgtcca
720acattaccgc catgttgaca ttgattattg actagttatt aatagtaatc
aattacgggg 780tcattagttc atagcccata tatggagttc cgcgttacat
aacttacggt aaatggcccg 840cctggctgac cgcccaacga cccccgccca
ttgacgtcaa taatgacgta tgttcccata 900gtaacgccaa tagggacttt
ccattgacgt caatgggtgg agtatttacg gtaaactgcc 960cacttggcag
tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
1020ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
tcctacttgg 1080cagtacatct acgtattagt catcgctatt accatggtga
tgcggttttg gcagtacatc 1140aatgggcgtg gatagcggtt tgactcacgg
ggatttccaa gtctccaccc cattgacgtc 1200aatgggagtt tgttttggca
ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 1260gccccattga
cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
1320cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
cctccataga 1380agacaccggg accgatccag cctccgcggc cgggaacggt
gcattggaac gcggattccc 1440cgtgccaaga gtgacgtaag taccgcctat
agagtctata ggcccacccc cttggcttct 1500tatgcatgct atactgtttt
tggcttgggg tctatacacc cccgcttcct catgttatag 1560gtgatggtat
agcttagcct ataggtgtgg gttattgacc attattgacc actcccctat
1620tggtgacgat actttccatt actaatccat aacatggctc tttgccacaa
ctctctttat 1680tggctatatg ccaatacact gtccttcaga gactgacacg
gactctgtat ttttacagga 1740tggggtctca tttattattt acaaattcac
atatacaaca ccaccgtccc cagtgcccgc 1800agtttttatt aaacataacg
tgggatctcc acgcgaatct cgggtacgtg ttccggacat 1860gggctcttct
ccggtagcgg cggagcttct acatccgagc cctgctccca tgcctccagc
1920gactcatggt cgctcggcag ctccttgctc ctaacagtgg aggccagact
taggcacagc 1980acgatgccca ccaccaccag tgtgccgcac aaggccgtgg
cggtagggta tgtgtctgaa 2040aatgagctcg gggagcgggc ttgcaccgct
gacgcatttg gaagacttaa ggcagcggca 2100gaagaagatg caggcagctg
agttgttgtg ttctgataag agtcagaggt aactcccgtt 2160gcggtgctgt
taacggtgga gggcagtgta gtctgagcag tactcgttgc tgccgcgcgc
2220gccaccagac ataatagctg acagactaac agactgttcc tttccatggg
tcttttctgc 2280agtcaccgtc cttgacacga agcttggtac cgagctcgga
tccactagta acggccgcca 2340gtgtgctgga attctgcaga tatccatcac
actggcggcc gctcgagcat gcatctagag 2400ggccctattc tatagtgtca
cctaaatgct agagctcgct gatcagcctc gactgtgcct 2460tctagttgcc
agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
2520gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
tctgagtagg 2580tgtcattcta ttctgggggg tggggtgggg caggacagca
agggggagga ttgggaagac 2640aatagcaggc atgctgggga tgcggtgggc
tctatggctt ctgaggcgga aagaaccagc 2700tggggctcga ggggggatcg
atcccgtcga cctcgagagc ttggcgtaat catggtcata 2760gctgtttcct
gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
2820cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
ttgcgttgcg 2880ctcactgccc gctttccagt cgggaaacct gtcgtgccag
ctgcattaat gaatcggcca 2940acgcgcgggg agaggcggtt tgcgtattgg
gcgctcttcc gcttcctcgc tcactgactc 3000gctgcgctcg gtcgttcggc
tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 3060gttatccaca
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
3120ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
gcccccctga 3180cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
aacccgacag gactataaag 3240ataccaggcg tttccccctg gaagctccct
cgtgcgctct cctgttccga ccctgccgct 3300taccggatac ctgtccgcct
ttctcccttc gggaagcgtg gcgctttctc aatgctcacg 3360ctgtaggtat
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
3420ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
ccaacccggt 3480aagacacgac ttatcgccac tggcagcagc cactggtaac
aggattagca gagcgaggta 3540tgtaggcggt gctacagagt tcttgaagtg
gtggcctaac tacggctaca ctagaaggac 3600agtatttggt atctgcgctc
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 3660ttgatccggc
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
3720tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
ttttctacgg ggtctgacgc 3780tcagtggaac gaaaactcac gttaagggat
tttggtcatg agattatcaa aaaggatctt 3840cacctagatc cttttaaatt
aaaaatgaag ttttaaatca atctaaagta tatatgagta 3900aacttggtct
gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
3960atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
tacgggaggg 4020cttaccatct ggccccagtg ctgcaatgat accgcgagac
ccacgctcac cggctccaga 4080tttatcagca ataaaccagc cagccggaag
ggccgagcgc agaagtggtc ctgcaacttt 4140atccgcctcc atccagtcta
ttaattgttg ccgggaagct agagtaagta gttcgccagt 4200taatagtttg
cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
4260tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
gatcccccat 4320gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
gttgtcagaa gtaagttggc 4380cgcagtgtta tcactcatgg ttatggcagc
actgcataat tctcttactg tcatgccatc 4440cgtaagatgc ttttctgtga
ctggtgagta ctcaaccaag tcattctgag aatagtgtat 4500gcggcgaccg
agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
4560aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
caaggatctt 4620accgctgttg agatccagtt cgatgtaacc cactcgtgca
cccaactgat cttcagcatc 4680ttttactttc accagcgttt ctgggtgagc
aaaaacagga aggcaaaatg ccgcaaaaaa 4740gggaataagg gcgacacgga
aatgttgaat actcatactc ttcctttttc aatattattg 4800aagcatttat
cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
4860taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
4912128386PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 128Met Gly Ser
Ile Gly Ala Ala Ser Met Glu Phe Cys Phe Asp Val Phe1 5 10 15Lys Glu
Leu Lys Val His His Ala Asn Glu Asn Ile Phe Tyr Cys Pro 20 25 30Ile
Ala Ile Met Ser Ala Leu Ala Met Val Tyr Leu Gly Ala Lys Asp 35 40
45Ser Thr Arg Thr Gln Ile Asn Lys Val Val Arg Phe Asp Lys Leu Pro
50 55 60Gly Phe Gly Asp Ser Ile Glu Ala Gln Cys Gly Thr Ser Val Asn
Val65 70 75 80His Ser Ser Leu Arg Asp Ile Leu Asn Gln Ile Thr Lys
Pro Asn Asp 85 90 95Val Tyr Ser Phe Ser Leu Ala Ser Arg Leu Tyr Ala
Glu Glu Arg Tyr 100 105 110Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val
Lys Glu Leu Tyr Arg Gly 115 120 125Gly Leu Glu Pro Ile Asn Phe Gln
Thr Ala Ala Asp Gln Ala Arg Glu 130 135 140Leu Ile Asn Ser Trp Val
Glu Ser Gln Thr Asn Gly Ile Ile Arg Asn145 150 155 160Val Leu Gln
Pro Ser Ser Val Asp Ser Gln Thr Ala Met Val Leu Val 165 170 175Asn
Ala Ile Val Phe Lys Gly Leu Trp Glu Lys Thr Phe Lys Asp Glu 180 185
190Asp Thr Gln Ala Met Pro Phe Arg Val Thr Glu Gln Glu Ser Lys Pro
195 200 205Val Gln Met Met Tyr Gln Ile Gly Leu Phe Arg Val Ala Ser
Met Ala 210 215 220Ser Glu Lys Met Lys Ile Leu Glu Leu Pro Phe Ala
Ser Gly Thr Met225 230 235 240Ser Met Leu Val Leu Leu Pro Asp Glu
Val Ser Gly Leu Glu Gln Leu 245 250 255Glu Ser Ile Ile Asn Phe Glu
Lys Leu Thr Glu Trp Thr Ser Ser Asn 260 265 270Val Met Glu Glu Arg
Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met 275 280 285Glu Glu Lys
Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr 290 295 300Asp
Val Phe Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Glu305 310
315 320Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile
Asn 325 330 335Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly
Val Asp Ala 340 345 350Ala Ser Val Ser Glu Glu Phe Arg Ala Asp His
Pro Phe Leu Phe Cys 355 360 365Ile Lys His Ile Ala Thr Asn Ala Val
Leu Phe Phe Gly Arg Cys Val 370 375 380Ser
Pro385129807PRTARTIFICIAL SEQUENCESYNTHETIC SEQUENCE 129Ala Thr Gly
Ala Gly Gly Gly Thr Gly Thr Thr Gly Cys Thr Cys Gly1 5 10 15Thr Thr
Gly Cys Cys Cys Thr Cys Gly Cys Thr Cys Thr Cys Cys Thr 20 25 30Gly
Gly Cys Thr Cys Thr Cys Gly Cys Thr Gly Cys Gly Ala Gly Cys 35 40
45Gly Cys Cys Ala Cys Cys Thr Cys Cys Ala Cys Gly Cys Ala Thr Ala
50 55 60Cys Ala Ala Gly Cys Gly Gly Cys Gly Gly Cys Thr Gly Cys Gly
Gly65 70 75 80Cys Thr Gly Cys Cys Ala Gly Cys Cys Ala Cys Cys Gly
Cys Cys Gly 85 90 95Cys Cys Gly Gly Thr Thr Cys Ala Thr Cys Thr Ala
Cys Cys Gly Cys 100 105 110Cys Gly Cys Cys Gly Gly Thr Gly Cys Ala
Thr Cys Thr Gly Cys Cys 115 120 125Ala Cys Cys Thr Cys Cys Gly Gly
Thr Thr Cys Ala Cys Cys Thr Gly 130 135 140Cys Cys Ala Cys Cys Thr
Cys Cys Gly Gly Thr Gly Cys Ala Thr Cys145 150 155 160Thr Cys Cys
Cys Ala Cys Cys Gly Cys Cys Gly Gly Thr Cys Cys Ala 165 170 175Cys
Cys Thr Gly Cys Cys Gly Cys Cys Gly Cys Cys Gly Gly Thr Cys 180 185
190Cys Ala Cys Cys Thr Gly Cys Cys Ala Cys Cys Gly Cys Cys Gly Gly
195 200 205Thr Cys Cys Ala Thr Gly Thr Gly Cys Cys Gly Cys Cys Gly
Cys Cys 210 215 220Gly Gly Thr Thr Cys Ala Thr Cys Thr Gly Cys Cys
Gly Cys Cys Gly225 230 235 240Cys Cys Ala Cys Cys Ala Thr Gly Cys
Cys Ala Cys Thr Ala Cys Cys 245 250 255Cys Thr Ala Cys Thr Cys Ala
Ala Cys Cys Gly Cys Cys Cys Cys Gly 260 265 270Gly Cys Cys Thr Cys
Ala Gly Cys Cys Thr Cys Ala Thr Cys Cys Cys 275 280 285Cys Ala Gly
Cys Cys Ala Cys Ala Cys Cys Cys Ala Thr Gly Cys Cys 290 295 300Cys
Gly Thr Gly Cys Cys Ala Ala Cys Ala Gly Cys Cys Gly Cys Ala305 310
315 320Thr Cys Cys Ala Ala Gly Cys Cys Cys Gly Thr Gly Cys Cys Ala
Gly 325 330 335Ala Cys Cys Ala Thr Gly Gly Ala Cys Gly Ala Cys Gly
Ala Thr Gly 340 345 350Ala Thr Ala Ala Gly Ala Thr Gly Cys Ala Cys
Gly Gly Cys Gly Ala 355 360 365Cys Ala Cys Cys Cys Cys Cys Ala Cys
Cys Cys Thr Gly Cys Ala Cys 370 375 380Gly Ala Gly Thr Ala Cys Ala
Thr Gly Cys Thr Gly Gly Ala Cys Cys385 390 395 400Thr Gly Cys Ala
Gly Cys Cys Cys Gly Ala Gly Ala Cys Cys Ala Cys 405 410 415Cys Gly
Ala Cys Cys Thr Gly Thr Ala Cys Thr Gly Cys Ala Thr Cys 420 425
430Thr Gly Cys Ala Gly Cys Cys Ala Gly Ala Ala Ala Cys Cys Cys Ala
435 440 445Ala Gly Thr Gly Cys Gly Ala Cys Ala Gly Cys Ala Cys Cys
Cys Thr 450 455 460Gly Cys Gly Gly Cys Thr Gly Thr Gly Cys Gly Thr
Gly Cys Ala Gly465 470 475 480Ala Gly Cys Ala Cys Cys Cys Ala Cys
Gly Thr Gly Gly Ala Cys Ala 485 490 495Thr Cys Cys Gly Gly Ala Cys
Cys Cys Thr Gly Gly Ala Gly Gly Ala 500 505 510Cys Cys Thr Gly Cys
Thr Gly Ala Thr Gly Gly Gly Cys Ala Cys Cys 515 520 525Cys Thr Gly
Gly Gly Cys Ala Thr Cys Gly Thr Gly Thr Gly Cys Cys 530 535 540Cys
Cys Thr Ala Cys Gly Ala Gly Cys Ala Gly Cys Thr Gly Ala Ala545 550
555 560Cys Gly Ala Cys Ala Gly Cys Ala Gly Cys Gly Ala Gly Gly Ala
Gly 565 570 575Gly Ala Gly Gly Ala Thr Gly Ala Gly Ala Thr Cys Gly
Ala Cys Gly 580 585 590Gly Cys Cys Cys Cys Gly Cys Cys Gly Gly Cys
Cys Ala Gly Gly Cys 595 600 605Thr Gly Ala Gly Cys Cys Cys Gly Ala
Cys Cys Gly Gly Gly Cys Cys 610 615 620Cys Ala Cys Thr Ala Cys Ala
Ala Cys Ala Thr Cys Gly Thr Gly Ala625 630 635 640Cys Cys Thr Thr
Cys Thr Gly Cys Thr Gly Cys Cys Ala Ala Cys Cys 645 650 655Ala Gly
Ala Gly Ala Cys Ala Ala Cys Thr Gly Ala Thr Cys Thr Cys 660 665
670Thr Ala Cys Thr Gly Thr Thr Ala Thr Gly Ala Gly Cys Ala Ala Thr
675 680 685Thr Ala Ala Ala Thr Gly Ala Cys Ala Gly Cys Thr Cys Ala
Gly Ala 690 695 700Gly Cys Ala Thr Thr Ala Cys Ala Ala Thr Ala Thr
Thr Gly Thr Ala705 710 715 720Ala Cys Cys Thr Thr Thr Thr Gly Thr
Thr Gly Cys Ala Ala Gly Thr 725 730 735Gly Thr Gly Ala Cys Thr Cys
Thr Ala Cys Gly Cys Thr Thr Cys Gly 740 745 750Gly Thr Thr Gly Thr
Gly Cys Ala Thr Gly Gly Gly Cys Ala Cys Ala 755 760 765Cys Thr Ala
Gly Gly Ala Ala Thr Thr Gly Thr Gly Thr Gly Cys Cys 770 775 780Cys
Cys Ala Thr Cys Thr Gly Thr Thr Cys Thr Cys Ala Gly Ala Ala785 790
795 800Ala Cys Cys Ala Thr Ala Ala 805
* * * * *