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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 IdentifierPublication Date
US 20110262478 A1October 27, 2011
Family ID: 1000001930244
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

Application NumberFiling DatePatent Number
11709527Feb 22, 20078163880
13123510
60776391Feb 23, 2006

Current U.S. Class: 1/1
Current CPC Class: A61K 39/21 20130101; A61K 2039/585 20130101; C12N 7/00 20130101; A61K 39/0011 20130101; A61K 39/12 20130101; A61K 2039/54 20130101; C12N 2740/16034 20130101; A61K 2039/53 20130101; C12N 2740/16022 20130101; A61K 2039/6031 20130101; C12N 2740/16234 20130101; C12N 2740/16222 20130101; C12N 2710/20034 20130101; C12N 2710/20071 20130101; C12N 2710/20022 20130101; A61K 2039/575 20130101; A61K 2039/572 20130101; A61K 39/385 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

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[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

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