US20030044936A1

US20030044936A1 - Peptide repeat immunogens

Info

Publication number: US20030044936A1
Application number: US10/122,483
Authority: US
Inventors: Jaulang Hwang; Chia-Tse Hsu; Chun-Jen Ting
Original assignee: Individual
Current assignee: Academia Sinica
Priority date: 1999-10-05
Filing date: 2002-04-11
Publication date: 2003-03-06
Also published as: EP1090994A3; CA2304377A1; TW528760B; AU6250000A; DE60037626D1; EP1090994B1; NZ507368A; EP1090994A2; US6838553B1; AU772387B2

Abstract

A polypeptide containing a carrier peptide sequence and a plurality of epitope peptide sequences.

Description

RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No. 09/412,558, filed Oct. 5, 1999, incorporated herein in its entirety.[0001]

BACKGROUND

The successful development of a protein-based vaccine often requires a delicate balance between enhancing immunogenicity of a particular antigen and minimizing any toxicity elicited by the enhancement. For example, an effective adjuvant used in animal studies (e.g., complete Freund's) may be too toxic to be used in vaccines prepared for humans.

SUMMARY

The invention is based on the discovery of a new means of generating an immune response to an epitope peptide by concatemerizing the epitope peptide and fusing the concatemer to a receptor binding domain (e.g., the receptor binding domain of a Pseudomonas exotoxin) or a carrier peptide with low immunogenicity (e.g., glutathione-S-transferase). Such a fusion protein elicits antigen-specific antibodies in mammals, with little or no toxicity observed.

Accordingly, the invention features a fusion protein, more specifically, the fusion protein is a polypeptide including a carrier peptide sequence, and a plurality (e.g., 2, 5, 10, 20, or 30 copies) of epitope peptide sequences. One class of the carrier peptides each bears a receptor binding domain for better intake of the epitope peptide by antigen presenting cells and, thus, more effective activation of helper T-cells. The second class of the carrier peptides each has weak immunogenicity and helps to induce humoral immunity dominantly against the epitope peptide by increasing the size of the fusion protein.

The epitope peptide sequence must be at least two amino acids in length (e.g., at least 3, 5, 7, 9, or 10 amino acids in length). In a preferred embodiment, the epitope peptide sequence can be less than 1000 amino acids in length (e.g., less than 500, 100, 50, or 20 amino acids in length). It can include any antigen in which an immune response against it is beneficial, such as gonadotropin releasing hormone or GnRH, e.g., EHWSYGLRPG (SEQ ID NO:1), a fragment of a vaccinia virus coat protein, e.g., LIGICVAVTVAI, (SEQ ID NO:2), or a fragment of the human topoisomerase IIβ, e.g., NEGDYNPGRKTS (SEQ ID NO:10).

A receptor binding domain is an amino acid sequence within a polypeptide which specifically binds to an LDL or α ₂-macroglobulin cell receptor. An example of a receptor binding domain is amino acids 1-252 (domain Ia) of the P. aeruginosa exotoxin A (PE):


MHLIPHWIPLVASLGLLAGGSSASAAEEAFDL	(SEQ ID NO:3)

WNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLH

YSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEP

NKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIH

ELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRA

HESNEMQPTLAISHAGVSVVMAQTQPRREKRWSEWAS

GKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRV.

Variants of the sequence immediately above, including substitutions, deletions, or additions are permissible, provided that the receptor binding domain specifically binds to a LDL/α ₂-macroglobulin cell receptor.

The position of the various elements within the polypeptide can vary, as long as the polypeptide is able to elicit an immune response in a mammal. In one example, all copies of the epitope peptide sequence are in a consecutive series, meaning that all copies occur as a single block of peptide repeats without intervening amino acids in between any two copies. In another example, a spacer (e.g., 2-4 amino acids such as glycine, proline, or a combination of the two) is placed between two copies of the epitope peptide sequence. In addition, the carrier peptide can occur anywhere in the polypeptide (e.g., at the N-terminus, at the C-terminus, internal, or between two copies of the epitope peptide sequence), as long as the intended function of the polypeptide as an antigen is not disrupted.

The invention also features a nucleic acid (e.g., a recombinant expression vector) encoding one of the fusion proteins described above. The nucleic acid can be used for production of the fusion protein.

The polypeptides of the invention can be used to provide new antigens and vaccines for eliciting an immune response against specific epitope peptide sequences. These antigens can be formulated as safe and effective vaccines in mammals (including humans).

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a representation of a GnRH peptide sequence (SEQ ID NO:1) useful in the polypeptides of the invention and primer sequences (SEQ ID NO:4 for Oligo A; SEQ ID NO:5 for Oligo B) which encode it or portions thereof. [0012]
FIG. 2 is a representation of how successive extension, denaturation, and annealing (i.e., PCR) of the primers shown in FIG. 1 produce a nucleic acid encoding concatamers of the peptide sequence also shown in FIG. 1. [0013]
FIG. 3 is a representation of a final PCR amplification of the concatemeric nucleic acid shown in FIG. 2 to introduce at the ends of the concatemer a stop codon and suitable restriction sites for cloning. Adaptor primer A is designated SEQ ID NO:6. Adaptor primer B is designated SEQ ID NO:7. The 5′ overhang sequence containing the EcoRI site is designated SEQ ID NO:8. The 5′ overhang sequence containing the SacII site is designated SEQ ID NO:9.[0014]

DETAILED DESCRIPTION

The invention relates to new polypeptides having multiple copies of an epitope peptide fused to a receptor binding domain (e.g., the receptor binding domain of a Pseudomonas exotoxin) or to a carrier peptide with low immunogenicity (e.g., glutathione-S-transferase). Multiple copies of a peptide antigen increase immunologic presentation of epitopes present in each monomeric peptide sequence. In addition, a receptor binding domain increases binding between the polypeptide and antigen presenting cells, thereby facilitating uptake and presentation of peptide sequences. A carrier peptide with low immunogenicity facilitates antigen recognition by increasing the size of the protein, thereby activating the antibody generating system. [0015]
The invention also features a nucleic acid (e.g., a recombinant expression vector) encoding a fusion protein described above. The nucleic acid can be used for production of the fusion protein. The recombinant expression vectors can be designed for expression of polypeptides in prokaryotic (e.g., [0016] E. coli) or eukaryotic cells (e.g., insect cells, yeast cells, or mammalian cells). Production of recombinant proteins in cells is well known in the art. See Goeddel (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. Alternatively, the nucleic acids of the invention can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.
I. Generation of Antibodies [0017]
The polypeptides of the invention can be used to generate antibodies that specifically bind a monomeric peptide sequence. Such antibodies can be used in diagnostic and/or therapeutic procedures that require the enhancement, inhibition, or detection of any molecule which contains the epitope presented by the peptide sequence. [0018]
In particular, various host animals can be immunized by injection of a composition containing a polypeptide of the invention. Host animals can include rabbits, mice, guinea pigs, and rats. Various adjuvants can be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and [0019] Corynebacterium parvum.
Antibodies include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)[0020] ₂fragments, and molecules produced using a Fab expression library.
Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be prepared using the polypeptides described above and standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling et al., In: Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981; U.S. Pat. No. 4,376,110; Kosbor et al., Immunology Today 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA 80:2026, 1983; and Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1983). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the Mab may be cultivated in vitro or in vivo. The ability to produce high titers of mAbs in vivo makes this an excellent method of production. [0021]
The antibodies can be used, for example, to detect the presence of an antigen in a biological sample as part of a diagnostic assay, and also to evaluate the effectiveness of medical treatments by other therapeutic approaches. [0022]
In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1984; Neuberger et al., Nature 312:604, 1984; Takeda et al., Nature 314:452, 1984) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine Mab and a human immunoglobulin constant region. [0023]
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692) can be adapted to produce single chain antibodies against a particular peptide antigen. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. [0024]
Antibody fragments that recognize and bind to specific epitopes can be generated by known techniques. For example, such fragments include, but are not limited to, F(ab′)[0025] ₂fragments that can be produced by pepsin digestion of the antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab′)₂fragments.
II. Production and Use of Vaccine Compositions [0026]
The invention includes vaccine compositions (e.g., parenteral injectable vaccines) containing at least one polypeptide of the invention and, optionally, a pharmaceutically acceptable carrier, such as the diluents phosphate buffered saline or a bicarbonate solution (e.g., 0.24 M NaHCO[0027] ₃). The carriers used in the composition are selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. An adjuvant, e.g., a cholera toxin, Escherichia coli heat-labile enterotoxin (LT), liposome, or immune-stimulating complex (ISCOM), can also be included in the new vaccine composition, if necessary.
The amount of vaccine administered will depend, for example, on the particular peptide antigen in the polypeptide, whether an adjuvant is co-administered with the antigen, the type of adjuvant co-administered, the mode and frequency of administration, and the desired effect (e.g., protection or treatment), as can be determined by one skilled in the art. In general, the new vaccine antigens are administered in amounts ranging between 1 μg and 100 mg polypeptide per adult human dose. If adjuvants are administered with the vaccines, amounts ranging between 1 ng and 1 mg per adult human dose can generally be used. Administration is repeated as necessary, as can be determined by one skilled in the art. For example, a priming dose can be followed by three booster doses at weekly intervals. A booster shot can be given at 8 to 12 weeks after the first immunization, and a second booster can be given at 16 to 20 weeks, using the same formulation. Sera or T-cells can be taken from the individual for testing the immune response elicited by the vaccine against the neurotoxin. Methods of assaying antibodies or cytotoxic T cells against a specific antigen are well known in the art. Additional boosters can be given as needed. By varying the amount of polypeptide, the copy number of peptide antigen in the polypeptide, and frequency of administration, the immunization protocol can be optimized for eliciting a maximal immune response. [0028]
Before administering the above compositions in humans, toxicity and efficacy testing in animals are desirable. In an example of efficacy testing, mice (e.g., Swiss-Webster mice) can be vaccinated via an oral or parenteral route with a composition containing a polypeptide of the invention. For vaccines against an infectious agent, after the initial vaccination or after optional booster vaccinations, the mice (and corresponding control mice receiving mock vaccinations) are challenged with a LD[0029] ₉₅dose of the infectious agent. End points other than lethality can also be used. Efficacy is determined if mice receiving the vaccine die at a rate lower than the mock-vaccinated mice. Preferably, the difference in death rates should be statistically significant. Rabbits can be used in the above testing procedure instead of mice.
Alternatively, the new vaccine compositions can be administered as ISCOMs. Protective immunity has been generated in a variety of experimental models of infection, including toxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS as the delivery vehicle for antigens (Mowat et al., Immunology Today 12:383-385, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMs have been found to produce class I mediated cytotoxic T cell responses (Takahashi et al., Nature 344:873-875, 1990). [0030]
The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety. [0031]

EXAMPLE 1

A Multimeric Vaccine Against Gonadotropin Releasing Hormone

Gonadotropin releasing hormone (GnRH) is a decapeptide produced by the arcuate nuclei of the hypothalamus and regulates expression of luteinizing hormone and follicle-stimulating hormone, which in turn regulates gonad development in humans. In addition, increased expression of GnRH and its receptor has been correlated with a variety of tumors, including cancer of the breast, ovary, endometrium, and prostate. See, e.g., Imai et al., Cancer 74:2555-2561, 1994; Eidne et al., J. Clin. Endocrinol. Metab. 64:425-432, 1987; and Irmer et al., Cancer Res. 55:817-822, 1995. Therefore, antibodies against GnRH may provide a means to modulate reproductive hormone activity and/or cancer development and progression. [0032]
To produce an antigen containing concatamers of GnRH fused to a receptor binding domain, the following approach, as outlined in FIGS. [0033] 1-3, was used. This procedure was designated template repeat PCR (TRPCR).
Two oligonucleotides were designed for TRPCR (FIG. 1). Oligo A encoded target antigen GnRH. Oligo B was complementary to oligo A. Oligo A is dissected into 5′ half A1 and 3′ half A2, both halves being of equal lengths. Oligo B is dissected into 5′ [0034] half B 1 and 3′ half B2, again both halves being of equal lengths. A1 was complementary to B1, while A2 was complementary to B2.
The thermal cycler was programmed for denaturation at 94° C. for 30 seconds, annealing at 37° C. for 30 seconds, and extension at 72° C. for 30 seconds. Using oligos A and B, PCR was performed for 30 cycles, followed by a final extension at 72° C. for 10 minutes. This PCR should produced DNA species containing repeated sequences encoding GnRH, as illustrated in FIG. 2. [0035]
Two adapter primers (Adaptor A and Adaptor B) were designed to add an appropriate stop codon at the end of the repeated open reading frame (ORF) and two suitable restriction sites flanking the ORF (FIG. 3). For this step (which was designated adaptor PCR), the template for the PCR was a 100-fold dilution of the TRPCR product produced as described above. The thermal cycler was programmed as described above, except that the denaturation was set for 1 minute instead of 30 seconds. The resulting PCR product contained a SacII site at the 5′ end, an EcoRI site at the 3′ end, and a stop codon at the end of the ORF. [0036]
The products of TRPCR and adaptor-PCR were then examined on a polyacrylamide gel. The majority of the TRPCR products were 500 bp to 700 bp in length. After adaptor PCR, products were distributed in a ladder, the lowest band containing the dimer of the GnRH DNA repeat and the slower migrating bands containing higher order multimers. The number of repeats present ranged from 3 to at least 12. One clone containing 12 repeats of GnRH coding sequence was chosen for further study. [0037]
The DNA fragment encoding 12 repeats of GnRH was subcloned into plasmid pPEDI, which was produced by subcloning the sequence encoding domain Ia of PE in pJH14 (Hwang et al., J. Biol. Chem. 264:2379-2384, 1989) into pET (Novagen). This plasmid expresses a polypeptide containing domain Ia of PE, which includes the toxin receptor binding domain of the toxin, and contains a His[0038] ₆tag at its N-terminus. The GnRH repeats were inserted at the 3′ end of PE structure gene to produce pPEDIG12. The protein produced by this expression plasmid was designated PEIa-GnRH12.
pPEDIG12 was transformed into BL21(DE3)lysS. The transformants were cultured at 37° C. in LB medium containing ampicillin (50 μg/ml), chloramphenicol (25 μg/ml) and tetracycline (10 μg/ml). When the A[0039] ₆₀₀of the culture reached 0.2, IPTG was added to the culture to medium to achieve a final concentration of 0.1 mM. Cells were cultured for another 90 minutes and then harvested. Since PEIa-GnRH12 is overexpressed in the form of inclusion bodies, the cells were extracted with 6 M urea.
The extracts were then purified using the Novagen pET His-Tag System. The fractions containing PEIa-GnRH12 were collected and dialysed against 50 mM ammonium acetate (pH 4.0). PEIa-GnRH12 was stable for at least 6 months when stored at 4° C. After nickel-agarose affinity column chromatography, PEIa-GnRH12 was isolated to about 95% purity, as determined by SDS-PAGE. [0040]
To test this new immunogen, six week old New Zealand female rabbits were immunized with PEIa-GnRH12. A 0.5 ml bolus containing 100 μg of PEIa-GnRH12 and 125 μg aluminum phosphate (pH 7.0) was injected into the rabbit at the first time point (six weeks after birth). One week after the first immunization, an identical 0.5 ml bolus was injected into the rabbits for a second immunization, followed by an identical injection two weeks after the first immunization. After the three immunizations, sera were collected for ELISA and immunoblotting analysis. To test whether antibodies against the multimeric antigen were directed to monomeric epitopes or to epitopes spanning the junction between monomers, several GST fusion proteins containing various multimers of GnRH were produced as targets for ELISA. [0041]
Unexpectedly, the antibodies produced in the immunized rabbits failed to recognize GST alone, while the sera at 5,000-fold dilution recognized all GST-GnRH fusion proteins to the same extent, irrespectively of how many GnRH repeats the GST fusion proteins contained. These results suggested that most of the antibodies produced in response to the immunogen recognized monomeric GnRH epitopes rather than any hybrid epitopes created by concatemerization. [0042]
GST-GnRH1 and GST-GnRH5 were specifically used as ELISA targets. Sera from thrice immunized rabbits, diluted 10,000-fold, recognized GST-GnRH1 and GST-GnRH5 to the same extent, thereby confirming that the immune response against the immunogen was directed to GnRH monomer-specific epitopes. [0043]
To confirm that the antibodies elicited by the new immunogen were physiologically active, immunized rabbits were monitored for ovary development. As a control, rabbits were also immunized with PEIa-TopN8, a polypeptide containing domain Ia of PE fused to 8 repeats of a 10-amino acid topoisomerase N-terminal peptide. The PEIa-TopN8-immunized rabbits showed normal ovary development, while the ovaries of PEIa-GnRH12-immunized rabbits were significantly decreased in size. Thus, the GnRH immunogen produced can be used as an immunogen to induce autoantibodies useful for treating GnRH-associated diseases. [0044]
To further confirm the utility of the new GnRH immunogen, mice and a pig were also immunized with PEIa-GnRH12. The mice immunization regimen was identical to the rabbit immunization described above, except that each mouse received a 100 μl bolus containing 10 μg PEIa-GnRH12 and 25 μg aluminum phosphate (pH 7.0) for each injection. In addition, a 24 day-old pig was injection once with a 1 ml bolus containing 10 mg PEIa-GnRH12 and 250 μg aluminum phosphate (pH 7.0). GnRH-specific antibodies were readily elicited in the mice and pig, as well as in rabbits, indicating that the antigens can elicit an immune response in a variety of animals. Further, a single immunization was sufficient to elicit an immune response. [0045]

EXAMPLE 2

A Multimeric Vaccine Against Vaccinia Virus

Instead of a GnRH peptide sequence, a DNA sequence containing a repeat of the vaccinia virus coat protein peptide LIGICVAVTVAI (SEQ ID NO:2) was constructed using PCR as described in Example 1 above. A PE fusion protein containing 16 repeats of the virus coat protein peptide was produced and purified according to the procedures in Example 1. [0046]
Six-week old rabbits were injected with a 1 ml bolus containing 100 μg viral peptide repeat antigen and 100 μg complete Freund's adjuvant. Four weeks later, the rabbits received a second 1 ml injection containing 100 μg antigen and 100 μg incomplete Freund's adjuvant. At 8 weeks after the first immunization, the rabbits were injected with a 1 ml bolus containing 100 μg antigen (no adjuvant). Unexpectedly, rabbits thrice immunized with the virus coat protein immunogen produced vaccinia virus-specific antibodies. [0047]
Thus, the general procedure of linking a peptide repeat to a PE receptor binding domain was shown to be successful for a second peptide. [0048]

EXAMPLE 3

A Multimeric Immunogen Against Human DNA Topoisomerase IIβ

A DNA sequence containing a repeat of a human DNA topoisomerase IIβ peptide NEGDYNPGRKTS (SEQ ID NO:10) was constructed using PCR as described in Example 1 above. A glutathione-S-transferase (GST) fusion protein containing 7 repeats of the human DNA topoisomerase IIβ peptide (GST-TOP2β-7M) was produced and purified according to the procedures described in Example 1. [0049]
Four New Zealand female rabbits were immunized with the purified GST-TOP2β-7M fusion protein. Sera were collected for Western blot analysis. Twenty micrograms of HeLa nuclear extract was separated by 5% SDS-PAGE. Proteins were transferred to a PVDF membrane which was subsequently blotted with a 5000X dilution of the anti-TOP2β antiserum. Unexpectedly, the results indicate that rabbits immunized with the GST-TOP2β-7M immunogen produced high titer antibodies specific for human DNA topoisomerase IIβ. [0050]

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. [0051]
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims. [0052]

Claims

What is claimed is:

1. A polypeptide comprising:

a carrier peptide sequence, and

a plurality of epitope peptide sequences.

2. The polypeptide of claim 1, wherein the carrier peptide contains a receptor binding domain.

3. The polypeptide of claim 2, wherein the polypeptide contains 2 to 30 copies of the epitope peptide sequence.

4. The polypeptide of claim 3, wherein all copies of the epitope peptide sequence are in a consecutive series.

5. The polypeptide of claim 2, wherein all copies of the epitope peptide sequence are in a consecutive series.

6. The polypeptide of claim 2, wherein the receptor binding domain is that of Pseudomonas exotoxin A.

7. The polypeptide of claim 6, wherein the polypeptide contains 2 to 30 copies of the epitope peptide sequence.

8. The polypeptide of claim 7, wherein all copies of the epitope peptide sequence are in a consecutive series.

9. The polypeptide of claim 6, wherein all copies of the epitope peptide sequence are in a consecutive series.

10. The polypeptide of claim 1, wherein the carrier peptide is a peptide with low immunogenicity.

11. The polypeptide of claim 10, wherein the polypeptide contains 2 to 30 copies of the epitope peptide sequence.

12. The polypeptide of claim 11, wherein all copies of the epitope peptide sequence are in a consecutive series.

13. The polypeptide of claim 10, wherein all copies of the epitope peptide sequence are in a consecutive series.

14. The polypeptide of claim 10, wherein the carrier peptide is glutathione-S-transferase.

15. The polypeptide of claim 14, wherein the polypeptide contains 2 to 30 copies of the epitope peptide sequence.

16. The polypeptide of claim 15, wherein all copies of the epitope peptide sequence are in a consecutive series.

17. The polypeptide of claim 14, wherein all copies of the epitope peptide sequence are in a consecutive series.

18. The polypeptide of claim 1, wherein the polypeptide contains 2 to 30 copies of the epitope peptide sequence.

19. The polypeptide of claim 18, wherein all copies of the epitope peptide sequence are in a consecutive series.

20. The polypeptide of claim 1, wherein all copies of the epitope peptide sequence are in a consecutive series.