AU2010241379A1

AU2010241379A1 - Immunogenic lipopeptides

Info

Publication number: AU2010241379A1
Application number: AU2010241379A
Authority: AU
Inventors: David Jackson; Weiguang Zeng
Original assignee: Queensland Institute of Medical Research QIMR
Current assignee: QIMR Berghofer Medical Research Institute
Priority date: 2002-08-12
Filing date: 2010-11-11
Publication date: 2010-12-02
Also published as: AU2006202423A1

Description

3295672 I.DOC Australian Patents Act 1990 - Regulation 3.2A ORIGINAL COMPLETE SPECIFICATION 5 STANDARD PATENT Invention Title "Immunogenic lipopeptides" 10 The following statement is a full description of this invention, including the best method of performing it known to us:- 3295672 I.DOC 2 Immunogenic Lipopeptides This is a divisional of Australian patent application No. 2006202423, the entire contents of which are incorporated herein by reference. FIELD OF THE INVENTION 5 The present invention relates generally to the field of immunology, and more particularly to reagents for generating antibody and/or cellular responses to a peptide immunogen, and methods for using said reagents for enhancing the immune response of a subject, or for the vaccination of a subject. Even more specifically, the present invention relates to novel lipopeptides having enhanced immunogenic activity, 10 formulations and vaccine compositions comprising said lipopeptides, such as, for example, in combination with a pharmaceutically acceptable carrier or excipient, and to methods for making and using the formulations and vaccine compositions of the invention. BACKGROUND TO THE INVENTION 15 General This specification contains amino acid sequence information prepared using Patentln Version 3.1, presented herein after the Abstract. Each sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, etc). The length of each sequence and source organism are 20 indicated by information provided in the numeric indicator fields <211> and <213>, respectively. Sequences referred to in the specification are defined by the term "SEQ ID NO:", followed by the sequence identifier (eg. SEQ ID NO: 1 refers to the sequence designated as <400>1). As used herein the term "derived from" shall be taken to indicate that a specified 25 integer may be obtained from a particular source albeit not necessarily directly from that source.

3295672_.DOC 3 Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of 5 elements or integers. All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of 10 these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be 15 understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present invention is not to be limited in scope by the specific examples described 20 herein. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein. All the references cited in this application are specifically incorporated by reference herein. The present invention is performed without undue experimentation using, unless 25 otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase 3295672 I.DOC 4 peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated by reference: 1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and 5 III; 2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text; 3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; 10 Atkinson et al., pp35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; 5. Animal Cell Culture: Practical Approach, Third Edition (John R.W. Masters, ed., 2000), ISBN 0199637970, whole of text; 15 6. Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; 7. Perbal, B., A Practical Guide to Molecular Cloning (1984); 8. Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series; 20 9. J.F. Ramalho Ortigio, "The Chemistry of Peptide Synthesis" In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); 10. Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R.L. (1976). Biochem. Biophys. Res. Commun. 73 336-342 3295672 I.DOC 5 11. Merrifield, R.B. (1963). J. Am. Chem. Soc. 85, 2149-2154. 12. Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York. 13. WUnsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Methoden der 5 Organischen Chemie (Mller, E., ed.), vol. 15, 4th edn., Parts I and 2, Thieme, Stuttgart. 14. Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg. 15. Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, 10 Springer-Verlag, Heidelberg. 16. Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474. 17. Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications). Description of the Related Art 15 Immunotherapy or vaccination are attractive for the prophylaxis or therapy of a wide range of disorders, such as, for example, certain infectious diseases, or cancers. However, the application and success of such treatments are limited in part by the poor immunogenicity of the target antigen. Many peptides, glycopeptides, lipids, lipopeptides, carbohydrates etc., are poorly immunogenic. For example, synthetic 20 peptides, representing T cell immunogens elicit only weak immunity when delivered in isolation and as a consequence, are not effective in vaccine compositions. Full-length proteins containing CTL epitopes do not efficiently enter the MHC class I processing pathway. Additionally, CTL epitopes are HLA-restricted and the large degree of HLA 3295672 I.DOC 6 polymorphism in human populations means that CTL-based vaccines may not provide broad coverage to all genotypes within a population. Several techniques are used to enhance the immune response of a subject to a peptide immunogen. 5 It is known that utilization of an adjuvant formulation that is extrinsic to the peptide immunogen (i.e. it is mixed with the immunogen prior to use), such as, for example, complete Freund's adjuvant (CFA), will enhance the immune response of a subject to a peptide immunogen. However, many of the adjuvants currently available are too toxic for use in humans, or simply ineffective. Moreover, adjuvants of this type require prior 10 formulation with the peptide immunogen immediately before administration, such formulations often having a low solubility or being insoluble. Lipopeptides, wherein a lipid moiety that is known to act as an adjuvant is covalently coupled to a peptide immunogen, may be capable of enhancing the immunogenicity of an otherwise weakly immunogenic peptide in the absence of an extrinsic adjuvant 15 [Jung et al., Angew Chem, Int Ed Engl 10, 872, (1985); Martinon et al., J Immunol 149, 3416, (1992); Toyokuni et al., J Am Chem Soc 116, 395, (1994); Deprez, et al., J Med Chem 38, 459, (1995); and Sauzet et al., Vaccine 13, 1339, (1995); BenMohamed et al., Eur. J. Immunol. 27, 1242, (1997); Wiesmuller et al., Vaccine 7, 29, (1989); Nardin et al., Vaccine 16, 590, (1998); Benmohamed, et al. Vaccine 18, 2843, (2000); 20 and Obert, et al., Vaccine 16, 161, (1998)]. Suitable lipopeptides show none of the harmful side effects associated with adjuvant formulations, and both antibody and cellular responses have been observed against lipopeptides. Several different fatty acids are known for use in lipid moieties. Exemplary fatty acids include, but are not limited to, palmitoyl, myristoyl, stearoyl and decanoyl groups or, 25 more generally, any C 2 to C 30 saturated, monounsaturated, or polyunsaturated fatty acyl group is thought to be useful.

3295672_.DOC 7 The lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteine, also known as Pam 3 Cys or Pam 3 Cys-OH (Wiesmuller et al., Z. Physiol.Chem. 364 (1983), p593), is a synthetic version of the N-terminal moiety of Braun's lipoprotein that spans the inner and outer membranes of Gram negative bacteria. Pam 3 Cys has the structure of 5 Formula (I): H3C - (CH2)14 -CC Nl I CH COOH

CH

2 I S

CH

2

H

3

C-(CH

2

)

14 - CO-O- CH

H

3

C-(CH

2

)

14 -CO- O- CH 2 United States Patent No. 5, 700, 910 to Metzger et al (December 23, 1997) describes 10 several N-acyl-S-(2-hydroxyalkyl)cysteines for use as intermediates in the preparation of lipopeptides that are used as synthetic adjuvants, B lymphocyte stimulants, macrophage stimulants, or synthetic vaccines. Metzger et al. also teach the use of such compounds as intermediates in the synthesis of Pam3Cys-OH (Wiesmuller et al., Z. Physiol. Chem. 364 (1983), p593), and of lipopeptides that comprise this lipoamino 15 acid or an analog thereof at the N-terminus. The lipopeptides are prepared by coupling a lipoamino acid moiety to the peptide moiety during the synthesis process.

3295672 I.DOC 8 Pam 3 Cys has been shown to be capable of stimulating virus-specific cytotoxic T lymphocyte (CTL) responses against influenza virus-infected cells (Deres et al., Nature 342, 561, 1989) and to elicit protective antibodies against foot-and-mouth disease (Wiesmuller et al., Vaccine 7, 29, 1989; United States Patent No. 6,024,964 to Jung et 5 al., February 15, 2000) when coupled to the appropriate epitopes. For example, Pam 3 Cys when coupled to a CTL epitope peptide has been shown to be capable of stimulating CTL responses against influenza virus-infected cells (Deres et al., Nature 342, 561, 1989) and to elicit protective antibodies against foot-and-mouth disease (Wiesmuller et al., Vaccine 7, 29, 1989; United States Patent No. 6,024,964 to Jung et 10 al., February 15, 2000) when coupled to the N-terminus of an appropriate synthetic B cell epitope. Recently, Pam 2 Cys (also known as dipalmitoyl-S-glyceryl-cysteine or S-[2,3 bis(palmitoyloxy)propyl]cysteine), an analogue of Pam 3 Cys, has been synthesised (Metzger, J. W., A. G. Beck-Sickinger, M. Loleit, M. Eckert, W. G. Bessler, and G. 15 Jung. 1995. J Pept Sci 1:184.) and been shown to correspond to the lipid moiety of MALP-2, a macrophage-activating lipopeptide isolated from mycoplasma (Sacht, G., A. Marten, U. Deiters, R. Sussmuth, G. Jung, E. Wingender, and P. F. Muhlradt. 1998. Eur J Immunol 28:4207: Muhlradt, P. F., M. Kiess, H. Meyer, R. Sussmuth, and G. Jung. 1998. Infect Immun 66:4804: Muhlradt, P. F., M. Kiess, H. Meyer, R. Sussmuth, 20 and G. Jung. 1997. J Exp Med 185:1951). Pam2Cys has the structure of Formula (II): 3295672 I.DOC 9 Formula (II) H- NH- C COOH

CH

2 S

CH

2

H

3

C-(CH

2

)

1 4 -CO- 0- CH

H

3

C-(CH

2

)

1 4 - CO-0-CH 2 Pam 2 Cys is reported to be a more potent stimulator of splenocytes and macrophages 5 than Pam 3 Cys (Metzger et al., J Pept. Sci 1, 184, 1995; Muhlradt et al., J Exp Med 185, 1951, 1997; and Muhlradt et al., Infect Immun 66, 4804, 1998). Generation of an antibody response against a given antigen requires the generation of a strong T helper cell response. Accordingly, it is desirable to administer an antigen in conjunction with at least one T-helper cell epitope (Vitiello et al., J. Clin. Invest. 95, 10 341-349, 1995; Livingston et al., J. Immunol. 159, 1383-1392, 1997). However, because T helper cell responses are provided by CD4+ T-cells that recognize fragments of peptide antigens in context of MHC class II molecules on the surface of antigen presenting cells (APCs), most of the processed forms of peptide antigens are only presented by one or a few alleles of MHC haplotypes. This causes the T helper 15 response to a given antigenic peptide to be strictly under genetic control of an individual.

3295672 I.DOC 10 Generation of a strong CD8+ T cell response against a given CTL epitope requires the generation of a strong T helper cell response. CD4+ T-helper cells function in cell mediated immunity (CMI) by secreting sufficient cytokines, such as, for example IL-2, to thereby facilitate the expansion of CD8+ T cells or by interacting with the antigen 5 presenting cell (APC) thereby rendering it more competent to activate CD8+ T cells. Accordingly, it is desirable to administer a CTL epitope in conjunction with at least one T-helper cell epitope (Vitiello et al., J. Clin. Invest. 95, 341-349, 1995; Livingston et al., J. Immunol. 159, 1383-1392, 1997). These epitopes are recognized by T-helper cells in the context of MHC class II molecules on the surface of the APC. 10 To avoid large genetic variation in the immune responses of a given population of individuals to an antigen, an antigen is administered in conjunction with a large protein having a range of T helper epitopes. For example, the CTL epitope or isolated epitope can be administered in conjunction with a large protein having a range of T helper epitopes in order to accommodate the diversity of class II alleles within a population of 15 individuals Alternatively, promiscuous or permissive T-helper epitope-containing peptides are administered in conjunction with an antigen (e.g. CTL epitope or epitopes). Promiscuous or permissive T-helper epitope-containing peptides are presented in the context of a vast majority of MHC class II haplotypes, such that they induce strong CD4+ T helper responses in the majority of an outbred human 20 population. Examples of promiscuous or permissive T-helper epitopes are tetanus toxoid peptide, Plasmodium falciparum pfg27, lactate dehydrogenase, and HlVgp 120 (Contreas et al., Infect. Immun, 66, 3579-3590, 1998; Gaudebout et al., J. A.I.D.S. Human Retrovirol 14, 91-101, 1997; Kaumaya et al., J. Mol. Recog. 6, 81-94, 1993; and Fern and Good J. Immunol. 148, 907-913, 1992). Ghosh et al., Immunol 104, 58 25 66, 2001 and International Patent Application No. PCT/AUOO/00070 (WO 00/46390) also describe promiscuous T-helper epitopes from the fusion protein of Canine Distemper Virus (CDV-F). Certain promiscuous T-helper epitopes promote strong CTL responses to a given CTL epitope, or strong B cell responses to a given antigen, 3295672 I.DOC 11 and can bypass certain haplotype restricted immune responses (Kaumaya et al., J. Mol. Recog. 6, 81-94, 1993). Routinely, a vaccine preparation will comprise a mixture of polypeptides comprising the T-helper cell epitope and antigenic (e.g. CTL) epitope, however it is also known to 5 consist of a single polypeptide comprising both the T-helper epitope and the antigenic epitope. SUMMARY OF THE INVENTION In work leading up to the present invention, the inventors sought to improve methods for producing highly immunogenic lipopeptides having a lipid moiety and a 10 polypeptide moiety comprising both a T helper epitope and a CTL epitope or a B-cell epitope against which an immune response is desired. The inventors showed that a highly immunogenic lipopeptide comprising both a T-helper cell (Th) epitope and a B cell epitope or a CTL epitope can be produced by synthesizing a single polypeptide molecule comprising said epitopes with an internal lysine residue or internal lysine 15 analog residue and then coupling the lipid moiety to the side-chain amino group of said internal lysine residue or said internal lysine analog residue, as opposed to the N terminal attachments described previously. Examples of internal lysine analogs include, but are not limited to, ornithine, diaminopropionic acid and diaminobutryic acid. Coupling the lipid moiety to the side-chain amino group of said internal lysine 20 residue or said internal lysine analog residue enables the lipopeptide of the present invention to be synthesized conveniently using a single amino acid chain, thereby requiring no post-synthesis modification to incorporate both epitopes. Thus in a first aspect, the present invention provides a lipopeptide comprising a T helper cell (Th) epitope and a B cell epitope or a CTL epitope, wherein the amino acid 25 sequences of the Th epitope is different from the amino acid sequence of the B cell or CTL epitope; one or more internal lysine residues or internal lysine analog residues and 3295672 I.DOC 12 one or more lipid moieties wherein said lipid moieties are covalently attached to said internal lysine residues or internal lysine analog residues. In a second aspect the present invention provides a lipopeptide comprising a T helper cell (Th) epitope and a B cell epitope or a CTL epitope, wherein the amino acid 5 sequences of the Th epitope is different from the amino acid sequence of the B cell or CTL epitope; one or more internal lysine residues or internal lysine analog residues and one or more lipid moieties wherein said lipid moieties are covalently attached to said internal lysine residues or internal lysine analog residues and said lipopeptide is of the general Formula (VI): H epotipe- A-HN-C----CO- A- epitope

(CH

2 )n I 10 Z wherein: one of the epitopes is a T-helper epitope and the other is a B cell epitope or a CTL epitope; A is either present or absent and consists of an amino acid spacer of about 1 to 15 about 6 amino acids in length; n is an integer having a value of 0, 1, 2, 3, or 4; X is a terminal side-chain group selected from the group consisting of NH, 0 and

S;

3295672 I.DOC 13 Y is either present of absent and consists of an amino acid spacer of about 1 to about 6 amino acids in length; and Z is a lipid moiety. The lipid moiety is preferably PamiCys, Pam 2 Cys, Pam 3 Cys, Chol 2 Lys, Ste 2 Cys, 5 Lau 2 Cys, and Oct 2 Cys. By positioning said one or more lysine residue(s) or one or more internal lysine analog residue(s) at predetermined locations within the polypeptide during peptide synthesis, the attachment site of the lipid is readily specified. Positioning of the lipid moiety in the lipopeptide can thus be targeted to enhance the utility of the end-product for 10 vaccine or adjuvant formulations. The inventors have found that attachment of the lipid moiety via the side-chain epsilon amino group of an internal lysine residue or the terminal side-chain group of an internal lysine analog residue positioned between the amino acid sequences of the T helper epitope and the B cell epitope or CTL epitope, provides an enhancement of dendritic 15 cell maturation when compared to linear structures obtained in which lipid is attached to the N-terminus of the peptide. One advantage provided by the lipopetides of the present invention is that they are sufficiently immunogenic such that it is generally not necessary to include an extrinsic adjuvant in vaccine formulations comprising these lipopeptides. 20 The present invention clearly encompasses the attachment of a lipid moiety via the epsilon-amino group of an internal lysine residue or the terminal side-chain group of an internal lysine analog residue present in the amino acid sequence of the T helper epitope or the amino acid sequence of the B cell epitope or the CTL epitope, the only requirement being that the lipid moiety is not attached to the N-terminus or the C 25 terminus of the peptide. As exemplified herein, the inventors have clearly shown that, 3295672 I.DOC 14 for example, the lipid may be attached to the epsilon amino group of an internal lysine residue within the T-helper epitope without loss of the ability of the subject lipopeptides in generating an immune response, compared to a lipopeptide wherein the lipid is added to the epsilon amino group of a lysine positioned between the T-helper 5 epitope and the B-cell epitope. By "internal" means at a location other than the N-terminus or the C-terminus of a polypeptide comprising a T helper epitope and a B cell epitope or a CTL epitope. As will be known to the skilled person, solubility of an antigen is highly desirable for producing vaccine formulations on a commercial basis. In this respect, the inventors 10 have found that the most effective lipopeptides of the invention are highly soluble. The relative ability of the lipopeptides of the invention to induce an antibody response in the absence of external adjuvant was reflected by their ability to upregulate the surface expression of MHC class II molecules on immature dendritic cells (DC). Preferably, the lipid moiety is attached via the epsilon-amino group of a lysine residue 15 or via the terminal side-chain group of a lysine analog residue positioned between the amino acid.sequences of the T helper epitope and the antigenic B cell epitope or CTL epitope. Optionally, one or more amino acid spacers is added between the T-helper epitope and the B cell epitope or CTL epitope, such as, for example, at either side of an internal 20 lysine or lysine analog positioned between said epitopes. A spacer of any conventional type can also be added between the lipid moiety and the polypeptide moiety. Particularly preferred spacers in this context consist of serine dimers, trimers, teramers, etc. Alternative spacers, such as, for example, arginine dimers, trimers, tetramers, or 6-aminohexanoic acid, are also contemplated for use in 25 this context.

3295672_.DOC 15 As exemplified herein, the present inventors produced the lipopeptide of the invention by coupling the lipid moiety to an exposed epsilon-amino group of an internal lysine residue in the synthetic peptide moiety. Optionally, a spacer may be added to the exposed epsilon amino group before addition of the lipid moiety. 5 As exemplified herein, the structure of the lipid moiety is not essential to activity of the resulting lipopeptide, as lipid moieties comprising palmitic acid, lauric acid, stearic acid or octanoic acid can be used without loss of immunogenicity. Accordingly, the present invention is not to be limited by the structure of the lipid moiety, unless specified otherwise, or the context requires otherwise. 10 Similarly, the addition of multiple lipid moieties to the peptide moiety, although generally not required, is also encompassed by the invention, unless specified otherwise or the context requires otherwise. As exemplified herein, the addition of multiple lipid moieties to the peptide moiety, such as, for example, to a position within the T-helper epitope, and to a position between the T-helper epitope and the B-cell epitope, does not 15 adversely affect the ability of the lipopeptide to stimulate IgG production compared to a peptide having only a single lipid moiety attached. It will be apparent from the preceding that the polypeptide is synthesized conveniently as a single amino acid chain, thereby requiring no post-synthesis modification to incorporate both epitopes. 20 As will be apparent from the disclosure herein, a lipoamino acid of Formula (III) or (IV) may be added directly to the epsilon amino group of the internal lysine residue or to the terminal side-chain group of an internal lysine analog residue. Lipoamino acids selected from the group consisting of: (i) Pam 2 Cys (also known as dipalmitoyl-S glyceryl-cysteine or S-[2,3-bis(palmitoyloxy)propyl]cysteine), (ii) Ste 2 Cys (also known 25 as distearoyl-S-glyceryl-cysteine or S-[2,3-bis(stearoyloxy)propyl]cysteine), Lau 2 Cys (also known as dilauroyl-S-glyceryl-cysteine or S-[2,3-bis(lauroyloxy)propyl]cysteine), 3295672 .DOC 16 and Oct 2 Cys (also known as dioctanoyl-S-glyceryl-cysteine or S-[2,3 bis(octanoyloxy)propyl]cysteine) are also useful. In a third aspect the present invention provides a composition comprising a lipopeptide as defined herein. 5 As exemplified herein, lipopeptides of the invention comprising a CTL epitope directed against influenza virus induced a virus-specific CTL response in the absence of external adjuvant, as reflected by their ability to induce potent CTL-mediated virus clearing responses, to induce CD8+ T cell migration to the lungs and to upregulate the surface expression of MHC class II molecules on immature dendritic cells (DC). The 10 enhanced maturation of dendritic cells following administration of the subject lipopeptides is consistent with enhanced T-helper epitope presentation compared to lipopeptides having N-terminally coupled lipid. Also exemplified herein, the lipopeptide of the present invention induces the production of a high titer antibody against the B cell epitope moiety when administered 15 to an animal subject, without any requirement for an adjuvant to achieve a similar antibody titer. This utility is supported by the enhanced maturation of dendritic cells following administration of the subject lipopeptides (i.e. enhanced antigen presentation compared to lipopeptides having N-terminally coupled lipid). Also exemplified herein, a lipopeptide of the present invention comprising an antigenic 20 B cell epitope of LHRH is capable of inducing infertility in a mouse model representative of other mammals in which infertility is to be induced. The sustained production of antibodies against LHRH achieved by the lipopeptides of the invention demonstrates the general utility of the subject lipopeptides in inducing humoral immunity and as an active agent in a vaccine preparation. 25 Also exemplified herein, a lipopeptide of the present invention comprising an antigenic B cell epitope of the M protein of Group A Streptococcus (herein "GAS") is capable of 3295672 I.DOC 17 inducing protection in a mouse model representative of humans and other mammals in which vaccination against GAS is indicated. The data provided herein indicate that the lipopeptides of the present invention are capable of inducing a sustained production of antibodies against GAS (both serum IgG, and salivary and faecal IgA), and the 5 opsonization of GAS, and the survival of animals against a subsequent GAS challenge. These data demonstrate the general utility of the subject lipopeptides in inducing humoral immunity and as an active agent in a vaccine preparation against GAS. Also exemplified herein, a lipopeptide of the present invention comprising an antigenic B cell epitope of gastrin ("pentagastrin") is capable of inducing the sustained 10 production of antibodies against gastrin and/or cholecystekinin in a mouse model of other mammals in which inhibition of gastric acid secretion is indicated. The data provided herein demonstrate the general utility of the subject lipopeptides in inducing humoral immunity against gastrin and immunoneutralization of gastrin, to thereby block secretion of gastric acid, in an animal suffering from hypergastrinemia, 15 Zollinger-Ellison syndrome, gastric ulceration or duodenal ulceration due to excessive and unregulated secretion of gastric acid, or to reduce or prevent the formation of gastrin-dependent tumours in the pancreas or duodenum (i.e. the prophylaxis and/or therapy of gastrinoma). As will be clear to those skilled in the art, the nature of the T-helper epitope and B cell 20 epitope or CTL epitope is not critical in the context of the present invention. The novel approach of attaching the lipid moiety to the epsilon amino group of one or more internal lysine residues or lysine analogue residues within the polypeptide portion of the construct has broad application. Accordingly, based on the results presented herein, it will be understood that a wide range of T-helper, B cell and CTL epitopes can be 25 used in the lipopeptide constructs. In fact, the broad range of applications exemplified herein indicate the generality of the lipopeptides of the present invention in the prophylaxis and therapy of a number of 3295672_.DOC 18 different conditions in humans and other mammals in which the generation of an immune response against an antigenic B cell epitope or CTL epitope is indicated. Accordingly, the present invention is not to be limited to the treatment of any specific condition, ailment or disease state. 5 BRIEF DESCRIPTION OF THE FIGURES Figure I is a representation of the structures of synthetic peptides and lipopeptides comprising T-helper and B-cell epitopes (left), and the relative solubilities of a sample of those peptides and lipopeptides in saline solution (right). Peptides were designated as follows: 10 (i) [Th] consisting of a CD4+ T-helper epitope from the light chain of influenza virus haemagglutinin (SEQ ID NO: 1) or peptide P25 from CDV-F (SEQ ID NO: 24); (ii) [B] consisting of a B cell epitope consisting of residues 1-10 of LHRH (SEQ ID NO: 2) or residues 2-10 of LHRH (SEQ ID NO: 3) or residues 6-10 of LHRH (SEQ ID NO: 4), a B cell epitope of the M protein of Group A Streptococcus ("peptide J14"; 15 SEQ ID NO: 10 1); or a B cell epitope of gastrin contained within the C-terminal 5 residues of gastrin (i.e., "pentagastrin"; SEQ ID NO: 102); (iii) [Th]-[B] consisting of a polypeptide having (i) and (ii) (e.g., SEQ ID NOs: 5, 103, 104, 105, 107, 109 or 111); and (iv) [Th]-Lys-[B] consisting of a polypeptide having (i) and (ii) separated by a 20 lysine residue (e.g., SEQ ID NOs: 7, 9, 13, 106, 108, 110, or 112). Lipopeptides were designated as follows: (i) Pam 3 Cys-[Th]-[B] consisting of a lipid of the Formula (I) conjugated to the N terminus of peptide [Th]-[B] supra (i.e. to the N-terminus of, for example, any one of SEQ ID NOs: 5, 103, 104, 105, 107, 109 or I 11); 3295672 I.DOC 19 (ii) Pam 3 Cys-Ser-Ser-[Th]-[B] consisting of a lipoamino acid of the Formula (III) conjugated to the N-terminus of peptide [Th]-[B] supra (i.e. to the N-terminus of, for example, any one of SEQ ID NOs: 5, 103, 104, 105, 107, 109 or 111); (iii) Pam 2 Cys-[Th]-[B] consisting of a lipid of the Formula (II) conjugated to the N 5 terminus of peptide [Th]-[B] supra (i.e. to the N-terminus of, for example, any one of SEQ ID NOs: 5, 103, 104, 105, 107, 109 or 111); (iv) Pam 2 Cys-Ser-Ser-[Th]-[B] consisting of a lipid of the Formula (IV) conjugated to the N-terminus of peptide [Th]-[B] supra (i.e. to the N-terminus of, for example, any one of SEQ ID NOs: 5, 103, 104, 105, 107, 109 or 111); 10 (v) [Th]-Lys(Pam 3 Cys)-[B] consisting of peptide [Th]-Lys-[B] (e.g., any one of SEQ ID NOs: 7, 9, 13, 106, 108, 110, or 112) and a lipid of the Formula (I) conjugated to the epsilon-amino group of the internal lysine (Lys) of said peptide; (vi) [Th]-Lys(Pam 2 Cys)-[B] consisting of peptide [Th]-Lys-[B] (e.g., any one of SEQ ID NOs: 7, 9, 13, 106, 108, 110, or 112) and a lipid of the Formula (II) conjugated 15 to the epsilon-amino group of the internal lysine (Lys) of said peptide; and (vii) [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] consisting of peptide [Th]-Lys-[B] (e.g., any one of SEQ ID NOs: 7, 9, 13, 106, 108, 110, or 112) conjugated serially via the epsilon amino group of the internal lysine (Lys) to a serine homodimer (i.e. Ser-Ser) and then a lipid of the Formula (II). Thus, to produce this branched lipopeptide, the two serine 20 residues were added to the epsilon-amino group of the lysine residue before the lipid moiety was attached. Relative solubility of the peptides and lipopeptides based upon the influenza virus haemagglutinin T-helper epitope (SEQ ID NO: 1) and the LHRH 1-10 B-cell epitope (SEQ ID NO: 2) is indicated at the right of the figure, ranging from low solubility (-) to 25 high solubility (++++.+).

3295672 I.DOC 20 Figure 2 is a photographic representation showing the solubilities of lipopeptides designated [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] (left) and Pam 2 Cys-Ser-Ser-[Th]-[B] (right) in Figure 1, wherein the polypeptide moieties have the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 5, respectively. Both solutions are 5 approximately 1 mg/mI lipopeptide in saline solution. The enhanced clarity of the solution comprising lipopeptide [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] is indicative of its higher solubility compared to lipopeptide Pam 2 Cys-Ser-Ser-[Th]-[B]. Figure 3 is a graphical representation showing the anti-LHRH antibody titers obtained using each of the peptides and lipopeptides shown in Figure 1, wherein the polypeptide 10 moieties have the amino acid sequences set forth in SEQ ID NO: 5 or SEQ ID NO: 7. A negative control lipopeptide designated Pam 3 Cys-Ser-Lys 4 consisted of the lipid of Formula (I) conjugated to the N-terminus of a peptide having the amino acid sequence Ser-Lys-Lys-Lys-Lys (SEQ ID NO: 17). All peptides and lipopeptides were administered sub-cutaneously (s.c.) in saline for both primary inoculation (open circles) 15 and secondary inoculations (closed circles). The two non-lipidated peptides [Th]-Lys [B] and [Th]-[B] were administered in complete Freund's adjuvant (CFA) for the primary inoculations, and in incomplete Freund's adjuvant (IFA) for the secondary inoculations. For administration of the peptide [Th]-[B] in combination with the lipopeptide Pam3Cys-S-Lys4, peptide was dissolved in saline and mixed with the 20 lipopeptide in 1:1 or 1:5 molar ratio as indicated. The dose of peptide and lipopeptide immunogens administered was 20 nmole. In all cases, control groups of animals received saline emulsified in CFA for priming and saline emulsified in IFA for the secondary inoculation. Figure 4 is a graphical representation showing anti-LHRH antibody titers (log10) on 25 the ordinate for each anti-LHRH antibody isotype (i.e. IgM, IgA, IgG1, IgG2a, IgG2b, IgG3, and total Ig) (abscissa) obtained or elicited during secondary antibody responses following inoculation with the lipopeptide [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] (SEQ ID NO: 7). Mice were bled 2 weeks after receiving the second dose of the lipopeptide 3295672 I.DOC 21 vaccine administered in saline either subcutaneously (open squares) or intranasally (closed squares) in saline. Figure 5 is a graphical representation showing the relative abilities of peptides and lipopeptides shown in Figure 1 (i.e. SEQ ID NO: 5 or SEQ ID NO: 7) to enhance the 5 expression of MHC class II molecules on the surface of dendritic cells. Peptides and lipopeptides are indicated in each panel according to the nomenclature of Figure 1. For each peptide or lipopeptide , 8x10 4 DI cells were exposed to 4.5 fmole of peptide or lipopeptide and incubated overnight. The cells were collected and the MHC class II molecules expression was determined by flow cytometry after staining with FITC 10 conjugated anti-I-Ek,d monoclonal antibody. About 3x10 4 Dl cells were analyzed for each sample. Data shown are for a representative of four independent experiments, and indicate enhanced staining with monoclonal antibody (i.e. enhanced DI cell maturation) following administration of lipopeptides, particularly lipopeptide [Th] Lys(Pam 2 Cys-Ser-Ser)-[B] which induced a DI maturation rate approaching the level 15 observed for DI cells challenged with lipopolysaccharide (LPS). Data obtained using the non-lipidated peptide [Th]-Lys-[B] are substantially the same as for DI cells incubated in medium without any added peptide, lipopeptide or LPS, indicating a spontaneous maturation rate of about 26%. Figure 6 is a graphical representation showing anti-LHRH antibody responses elicited 20 by lipidated [Th]-Lys(Pam 2 Cys)-[B] in which [Th] consists of CD4+ T cell epitope from the light chain of influenza haemagglutinin (SEQ ID NO: 1) and [B] is LHRH I 10 (SEQ ID NO: 2) or LHRH 6-10 (i.e. the C-terminal 5 residues of LHRH; SEQ ID NO: 4), with or without a serine spacer (Ser-Ser) positioned between the lipid and peptide moieties. Lipopeptide [Th]-Lys(Pam 2 Cys)-GlyLeuArgProGly is structurally 25 similar to [Th]-Lys(Pam 2 Cys)-[B], however this lipopeptide comprises SEQ ID NO: 4 in place of SEQ ID NO: 2.

3295672_.DOC 22 Figure 7 is a representation showing structural data, HPLC and mass spectra data for different lipopeptide constructs based on the T helper epitope P25 (SEQ ID NO: 24) and LHRH 2-10 (SEQ ID NO: 3), wherein the peptide moiety has the amino acid sequence set forth in SEQ ID NO: 9 and the lipid moiety is selected from the group 5 consisting of: (i) Pam 2 Cys; (ii) Ste 2 Cys; (iii) Lau 2 Cys; and (iv) Oct 2 Cys. Different spacers were also positioned between the lipid moiety and the peptide moiety, as follows: (i) Ser-Ser, two serine residues; (ii) Arg-Arg, two arginine residues; and (iii) Ahx, 6-aminohexanoic acid. Structures of the lipopeptides are indicated in the left column; HPLC chromatograms for each lipopeptide are indicated in the middle 10 column; and mass spectra are shown in the right column of the figure. Figure 8 is a graphical representation showing the immunogenicity of those lipopeptides indicated in the legend to Figure 7 having a Ser-Ser spacer between the peptide and the lipid moiety and wherein the lipid moiety is selected from the group consisting of: (i) Pam 2 Cys; (ii) Ste 2 Cys; (iii) Lau 2 Cys; and (iv) Oct 2 Cys. Groups of 15 BALB/c mice (6-8 weeks old) were inoculated subcutaneously with 20 nmoles of peptide immunogens for both primary and secondary vaccinations. All lipopeptides were administered in saline. The non lipidated peptide [Th]-Lys-[B] was administered in CFA as a control. Sera were obtained from blood taken at 4 weeks following the primary vaccination (open circles) and 2 weeks following the secondary vaccination 20 (closed circles). Figure 9 is a graphical representation showing immunogenicity of lipopeptide immunogens from Figure 7 having different spacers positioned between the lipid and peptide moieties, in particular spacers consisting of serine homodimers (Ser-Ser), arginine homodimers (Arg-Arg), or 6-aminohexanoic acid (Ahx). Groups of BALB/c 25 mice (6-8 weeks old) were inoculated subcutaneously with 20 nmoles of peptide immunogens for both primary and secondary vaccinations. All lipopeptides were administered in saline. The non lipidated peptide [Th]-Lys-[B] was administered in CFA as a control. Sera were obtained from blood taken at 4 weeks following the 3295672 I.DOC 23 primary vaccination (open circles) and 2 weeks following the secondary vaccination (closed circles). Figure 10 is a graphical representation showing quality control data for a lipopeptide construct [Th](Pam 2 Cys-Ser-Ser)-[B] in which the lipid moiety is pendant from the 5 epsilon-amino group of an internal lysine residue (Lys-14) within the helper T cell epitope of the peptide set forth in SEQ ID NO: 103. The structures of the lipopeptide is indicated in the left column; an HPLC chromatogram for the lipopeptide is indicated in the middle column; and mass spectra data are shown in the right column of the figure. Figure 11 is a graphical representation showing immunogenicity of the lipopeptide 10 immunogen described in the legend to Figure 10, compared to a lipopeptide immunogen having the lipid moiety added to an internal lysine residue positioned between the T-helper epitope and the B-cell epitope (i.e., the lipid moiety is added to the amino acid sequence set forth in SEQ ID NO: 9, which differs from SEQ ID NO: 103 in having an internal lysine added between the T-helper and B-cell epitopes). A 15 control non-lipidated peptide having the amino acid sequence set forth in SEQ ID NO: 9 (i.e., [Th]-Lys-[B]) was also used as a control. Groups of BALB/c mice (6-8 weeks old) were inoculated subcutaneously with 20 nmoles of peptide immunogens for both primary and secondary vaccinations. All lipopeptides were administered in saline. The non lipidated control peptide [Th]-Lys-[B] was administered in CFA. Sera were 20 obtained from blood taken at 4 weeks following the primary vaccination (open circles) and 2 weeks following the secondary vaccination (closed circles). The lipopeptide construct [Th](Pam 2 Cys-Ser-Ser)-[B] has the lipid moiety attached to the epsilon amino group of a lysine residue (Lys-14) within the helper T cell epitope. The lipopeptide construct [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] has the lipid attached to the 25 epsilon-amino group of a lysine residue placed between the two peptide epitopes. Figure 12 is a graphical representation showing the ability of a lipopeptide comprising the T-helper epitope P25 (SEQ ID NO: 24) and a Group A Streptococcus B cell epitope 3295672_.DOC 24 ("J14"; SEQ ID NO: 101) and having the amino acid sequence of SEQ ID NO: 106, and one or two lipid moieties to elicit serum IgG in mice. The lipoamino acid moiety Pam 2 Cys-Ser-Ser was added to an internal lysine positioned between the T-helper epitope and the B-cell epitope in all lipopeptides tested. In the lipopeptide [Th] 5 Lys(Pam 2 Cys-Ser-Ser)-[J 14], this is the only lipid moiety, whereas in the lipopeptide Pam 2 Cys-Ser-Ser-[Th]-Lys(Pam 2 Cys-Ser-Ser)-[J14], an additional lipoamino acid moiety Pam 2 Cys-Ser-Ser was added to the N-terminal amino group of the T-helper epitope. Other immunogens were as follows: J14, non-lipidated peptide consisting of the J14 B-cell epitope-containing peptide (SEQ ID NO: 101); [Th]-[J14], a non 10 lipidated peptide consisting of the T-helper epitope (SEQ ID NO: 24) and the J14 peptide (SEQ ID NO: 101) and having the amino acid sequence of SEQ ID NO: 106; a lipidated peptide consisting of the T-helper epitope (SEQ ID NO: 24) and the LHRH B cell epitope-containing peptide (SEQ ID NO: 3) and having the amino acid sequence of SEQ ID NO: 9; and phosphate-buffered saline (PBS). Female outbred Quackenbush 15 mice 4-6 weeks old (15/group) were inoculated intranasally with 60pg of peptide-based vaccine in a total volume of 30pl PBS. Mice received three doses of vaccine at 21-day intervals. Seven days following the final dose mice were bled from the tail vein and J14-specific serum IgG was determined. Mice that received either J14-containing lipopeptides had significantly higher (P<0.05) serum IgG titres than did the control 20 groups. Figure 13 is a graphical representation showing the opsonisation capability of antisera elicited by the non-lipidated peptides and lipopeptides indicated in the legend to Figure 12. Female outbred Quackenbush mice 4-6 weeks old (15/group) were inoculated intranasally with 60pg of peptide-based vaccine in a total volume of 30 1 PBS. Mice 25 received three doses of vaccine at 21-day intervals. Indirect bacteriacidal assays were performed to determine the ability of sera from immunized mice to opsonise or "kill" the Ml GAS strain in vitro. Sera collected from mice immunized with either J14- 3295672 I.DOC 25 containing lipopeptides were capable of significant (P<0.05) killing of GAS compared to sera collected from animals immunized with control peptides or lipopeptides or PBS. Figure 14 is a graphical representation showing the ability of the non-lipidated peptides and lipopeptides indicated in the legend to Figure 12 to elicit salivary IgA in 5 mice. Female outbred Quackenbush mice 4-6 weeks old (15/group) were inoculated intranasally with 60ptg of each peptide-based vaccine in a total volume of 30pIl PBS. Mice received three doses of vaccine at 21-day intervals. Eight days following the final dose saliva was collected from individual mice and the average J14-specific salivary IgA antibody titres were determined by standard ELISA. The mice inoculated with 10 either JI4-containing lipopeptides had significantly (P<0.05) higher titres than the control groups that were immunized with control peptides or control lipopeptides or PBS. Figure 15 is a graphical representation showing the ability of the non-lipidated J14 containing peptides and J14-containing lipopeptides indicated in the legend to Figure 15 12 to elicit fecal IgA in mice. Female outbred Quackenbush mice 4-6 weeks old (I 5/group) were inoculated intranasally with 60pg of peptide-based vaccine in a total volume of 30pl PBS. Mice received three doses of vaccine at 21-day intervals. Fecal IgA was determined 6 days following the last dose of antigen. Only mice inoculated with mono-lipidated J14-containing peptide, wherein the lipid moiety was positioned 20 between the T-helper epitope and the B-cell epitope (i.e., [Th]-Lys(Pam 2 Cys-Ser-Ser) [J14]) had significant (P<0.05) faecal IgA titres. Figure 16 is a graphical representation showing the ability of mice to survive challenge with bacteria following inoculation with the non-lipidated peptides and lipopeptides indicated in the legend to Figure 12. Two weeks after the last dose of antigen, mice 25 were challenged intranasally with M l GAS strain and survival determined at various time points afterwards. Mice inoculated with mono-lipidated J14-containing peptide, wherein the lipid moiety was positioned between the T-helper epitope and the B-cell 3295672 I.DOC 26 epitope (i.e., [Th]-Lys(Pam 2 Cys-Ser-Ser)-[J14]) demonstrated the best survival following challenge. Figure 17 is a graphical representation showing the immunogenicity of lipopeptide immunogens based on gastrin. Groups (5 animals/group) of BALB/c mice (6-8 weeks 5 of age) were inoculated subcutaneously in the base of tail with 20nmoles of peptide immunogens. The peptides used were Gastrin-17 (SEQ ID NO: 113); [P251-Lys [PentaGastrin] (SEQ ID NO: 110) in which PentaGastrin is the C-terminal sequence GWMDF of gastrin as set forth in (SEQ ID NO: 102); and [P25]-Lys(Pam 2 Cys-Ser Ser)-[PentaGastrin] (SEQ ID NO: 110 with lipid added to an internal lysine residue). 10 All lipopeptides were administered in PBS and the non-lipidated peptides were administered in CFA. The negative control was saline emulsified with CFA. Sera were obtained from animals 4 weeks after immunisation and at the same time the animals received a second similar dose of antigen. Mice were bled a second time 2 weeks after receiving the second dose of antigen and antibodies capable of reacting 15 with the peptide gastrin-17 sequence detected by ELISA. The results are expressed as the titre of anti-gastrin-17 antibodies. Figure 18 is a representation of the structures of synthetic peptides and lipopeptides comprising T-helper and CTL cell epitopes (left), and the relative solubilities of a sample of those peptides and lipopeptides in saline solution (right). Peptide structures 20 consisted of a CD4+ helper T cell epitope [Th] and a CTL cell epitope [CTL] assembled as tandem linear sequences with a linking internal lysine residue (i.e. [Th] Lys-[CTL]) or without any internal lysine (i.e. [Th]-[CTL]). Lipopeptides were branched structures wherein a lipid moiety was attached through the epsilon-amino group of a lysine residue, Lys, situated between the two epitopes at the approximate 25 centre of the molecule (i.e. [Th]-Lys(Pam 3 Cys-Ser-Ser)-[CTL]; [Th]-Lys(Pam 2 Cys Ser-Ser)-[CTL]; or [Th]-Lys(PamiCys-Ser-Ser)-[CTL]. In the case of branched constructs, the centrally located lysine residue to which the lipid is attached is denoted in italics, Lys. In some cases two serine residues (Ser-Ser) were added between the 3295672_I.DOC 27 peptide and lipid moiety. For the lipopeptide Pal 2 LysLys[Th]-[CTL], two palmitic acid residues were attached to the alpha and epsilon-amino groups of the N-terminal lysine residue and [Th] was attached to the epsilon-amino group of the penultimate lysine in the amino acid sequence. In the case of [Th]-Lys(Chol 2 Lys-Ser-Ser)-[CTL), two 5 residues of cholesterol were attached to an N-terminal lysine residue. Figure 19 is a representation of the primary amino acid sequences of the peptide moieties attached to the lipid moieties for the structures shown in Figure 18. Non lipidated peptides comprising these amino acid sequences were designated as follows: (i) [Th] consisting of a CD4+ T-helper epitope from the light chain of influenza 10 virus hemagglutinin as set forth in SEQ ID NO: 18; (ii) [CTL] consisting of an immunodominant H-2d-restricted CTL epitope consisting of amino acid residues 147-155 of the nucleoprotein of influenza virus strain A/Puerto Rico/8/34 (PR8;HNl1) as set forth in SEQ ID NO: 113; (iii) [Th]-[CTL] consisting of a polypeptide having (i) and (ii). The sequence of the 15 assembled peptide is shown in SEQ ID NO: 114; (iv) [Th]-Lys-[CTL] consisting of a polypeptide having (i) and (ii) separated by a lysine residue (bold underlined residue). The sequence of the assembled peptide is shown in SEQ ID NO: 113; (v) [P25]-Lys-[SIINFEKL] consisting of a T-helper epitope from CDV-F protein 20 designated P25 (SEQ ID NO: 24) and a CTL epitope from ovalbumin (SEQ ID NO: 246) separated by a lysine residue (bold underlined residue). The sequence of the assembled peptide is shown in SEQ ID NO: 247; (vi) [P25]-Lys-[LLO91-99] consisting of a T-helper epitope from CDV-F protein designated P25 (SEQ ID NO: 24) and a CTL epitope from Listeria monocytogenes 3295672 I.DOC 28 (SEQ ID NO: 245) separated by a lysine residue (bold underlined residue). The sequence of the assembled peptide is shown in SEQ ID NO: 248. and (vii) [P25]-Lys-[HCV] consisting of a T-helper epitope from CDV-F protein designated P25 (SEQ ID NO: 24) and a CTL epitope from the core protein of hepatitic 5 C virus (SEQ ID NO: 249) separated by a lysine residue (bold underlined residue). The sequence of the assembled peptide is shown in SEQ ID NO: 250. Lipopeptides comprising these amino acid sequences were designated as follows: (i) [Th]-Lys(Pam 3 Cys-Ser-Ser)-[CTL] consisting of peptide [Th]-Lys-[CTL] (i.e. SEQ ID NO: 116) and a lipid of the Formula (III) conjugated to the epsilon-amino 10 group of the internal lysine (bold underlined residue); (ii) [Th]-Lys(Pam 2 Cys-Ser-Ser)-[CTL] consisting of peptide [Th]-Lys-[CTL] (i.e. SEQ ID NO: 116) and a lipid of the Formula (IV) conjugated to the epsilon-amino group of the internal lysine (bold underlined residue); (iii) [P25]-Lys(Pam 2 Cys-Ser-Ser)-[LLO91-99] consisting of peptide [P25]-Lys 15 [LLO91-99] and a lipid of the Formula (IV) conjugated to the epsilon-amino group of the internal lysine (bold underlined residue) of said peptide; (iv) [P25]-Lys(Pam 2 Cys-Ser-Ser)-[SIINFEKL] consisting of peptide [P25]-Lys [SIINFEKL] and a lipid of the Formula (IV) conjugated to the epsilon-amino group of the internal lysine (bold underlined residue) of said peptide;and 20 (v) [P25]-Lys(Pam 2 Cys-Ser-Ser)-[HCV] consisting of peptide [P25]-Lys-[HCV] and a lipid of the Formula (IV) conjugated to the epsilon-amino group of the internal lysine (bold underlined residue) of said peptide. Figure 20 is a graphical representation showing the reduced viral load of mice primed with lipopeptides referred to in the legend to Figure 18 and subsequently challenged 3295672 I.DOC 29 with influenza virus. Mice were inoculated intranasally with 9 nmol of the lipopeptides [Th]-Lys(Pam 3 Cys-Ser-Ser)-[CTL] and [Th]-Lys(Pam 2 Cys-Ser-Ser)-[CTL] in 50 Pl PBS (columns 2 and 3, respectively), or for the [Th]-Lys-[CTL] peptide in 50 Pl PBS (column 1), or with PBS alone (column 4). Peptide and lipopeptide designations are as 5 for the legend to Figure 19. On day 9 post immunization, mice were anesthetized using penthrane and challenged intranasally with 30,000 plaque forming units of influenza virus subtype H3NI known as A/Memphis/1/71 (Mem 71). Five days later, their lungs were removed and assayed for the presence of infectious virus by plaque assay on MDCK cells. Each bar represents the geometric mean titre of viral titres from a group 10 of 5 BALB/c mice and error bars represent the standard deviation of the mean. Numbers above the bars represent the percentage reduction in lung viral titre relative to the PBS control. Figure 21a is a graphical representation showing enhanced lipopeptide-induced viral clearance in immunized mice receiving the lipopeptides referred to in the legend to 15 Figure 19. Mice were inoculated with 9 nmoles of the lipopeptides [Th]-Lys(Pam 3 Cys Ser-Ser)-[CTL] and [Th]-Lys(Pam 2 Cys-Ser-Ser)-[CTL] in 50 pl PBS (columns 2 and 3, respectively), or for the [Th]-Lys-[CTL] peptide in 50 pd PBS (column 1), or with PBS alone (column 4). On day 28 post immunization, mice were challenged with 30,000 plaque forming units of Mem 71 virus. Peptide and lipopeptide designations are as for 20 the legend to Figure 19. Data are expressed as the percentage reduction in lung viral titre on day 5 post challenge. Data show enhanced reduction in infectious virus in the lungs of mice immunized with the lipopeptides [Th]-Lys(Pam 3 Cys-Ser-Ser)-[CTL] (column 2) or [Th]-Lys(Pam 2 Cys-Ser-Ser)-[CTL] (column 3) compared to peptide alone (column 1) or PBS alone (column 4) at 5 days post-challenge. 25 Figure 21b is a graphical representation showing enhanced T cell activation in immunized mice receiving the lipopeptides referred to in the legend to Figure 19. Mice were inoculated with 9 nmoles of the lipopeptides [Th]-Lys(Pam 3 Cys-Ser-Ser)-[CTL] and [Th]-Lys(Pam 2 Cys-Ser-Ser)-[CTL] in 50 pl PBS (columns 2 and 3, respectively), 3295672 I.DOC 30 or for the [Th]-Lys-[CTL] peptide in 50 l PBS (column 1), or with PBS alone (column 4). Immunized mice were killed 9 days post-immunization and a bronchio-alviolar lavage (BAL) performed. Adherent cells were removed by incubation of the BAL sample in a petri dish at 37 0 C for 1 hour. The non-adherent cells were recovered and 5 stained for CD8 and CD4 expression. The cells were analyzed by flow cytometry. The lymphocyte population was identified based on the forward and side scatter profile and 10,000 lymphocytes were analysed. Data are expressed as the percentage of non adherent cells in the BAL fluid that are CD8+ lymphocytes. Data show enhanced activation of virus-specific CD8+ T cells in the BAL samples from mice immunized 10 with the lipopeptides [Th]-Lys(Pam 3 Cys-Ser-Ser)-[CTL] (column 2) or [Th] Lys(Pam 2 Cys-Ser-Ser)-[CTL] (column 3) compared to peptide alone (column 1) or PBS alone (column 4) at 5 days post-challenge. Peptide and lipopeptide designations are as for the legend to Figure 19. Figure 21c is a graphical representation showing enhanced maturation of dendritic 15 cells in response to the lipopeptides referred to in the legend to Figure 19. A line of BALB/c splenic-derived dendritic cells (DI cells) were incubated overnight with 0.45 nmoles/mL of the peptide [Th]-Lys-[CTL] (column 1) or the lipopeptides lipopeptides [Th]-Lys(Pam 3 Cys-Ser-Ser)-[CTL] (column 2) or [Th]-Lys(Pam 2 Cys-Ser-Ser)-[CTL] (column 3) or with medium alone as a negative control (column 4) or 20 lipopolysaccharide as a positive control (LPS; column 5). The percentage of Dl cells expressing high levels of surface MHC class II molecules, and therefore in a mature state, were determined by flow cytometry. Peptide and lipopeptide designations are as for the legend to Figure 19. Data show enhanced expression of MHC class II molecules on the surface of dendritic cells (ie. enhanced dendritic cell maturation) 25 following exposure to the peptides [Th]-Lys(Pam 3 Cys-Ser-Ser)-[CTL] or [Th] Lys(Pam 2 Cys-Ser-Ser)-[CTL] compared to peptide alone or medium alone. Figure 22 is a graphical representation showing the induction of pulmonary viral clearing responses in mice inoculated with synthetic immunogens indicated on the x- 3295672_.DOC 31 axis, that each include the CD4+ T-helper epitope set forth in SEQ ID NO: 18 and the H-2d-restricted CTL epitope set forth in SEQ ID NO: 114. Groups of 5 mice were immunised intranasally with 9 nmoles of the specified lipopeptides in PBS. Mice were challenged 28 days after priming with 104.5 PFU of Mem7l influenza virus 5 intranasally. Titres of infectious virus in lung homogenates sampled 5 days following challenge were determined by plaque formation on MDCK cell monolayers. Each circle respresents the virus titre of an individual mouse and the line represents the geometric mean titre of the group. The percentage reduction in mean viral titre relative to the PBS control group is shown above each column of data. 10 Figure 23 is a graphical representation showing accelerated influx of CTL derminant specific CD8+-T cells into the lungs of lipopeptide-vaccinated mice during virus challenge. Lipopeptides comprised the CD4+ T-helper epitope set forth in SEQ ID NO: 18 and the H-2d-restricted CTL epitope set forth in SEQ ID NO: 114. Groups of three mice were inoculated intranasally with 9 nmole of the indicated lipopeptides 15 intranasally. On day 28 post priming, they were challenged intranasally with 104.5 PFU of Mem7l influenza virus intranasally. CTL determinant-specific IFN-gamma secreting cells were enumerated in the lungs of mice on day 5 post-challenge by an intracellular cytokine production assay. 10,000 CD8+ cells were analysed for each sample. Data represent the mean and standard deviation for each group of mice. 20 Figure 24 is a graphical representation showing show accelerated influx of CTL determinant-specific CD8 T cells into the lungs in mice inoculated with lipopeptides following viral challenge. Lipopeptides comprised the CD4+ T-helper epitope set forth in SEQ ID NO: 18 and the H-2d-restricted CTL epitope set forth in SEQ ID NO: 114. Mice were inoculated intranasally with 9 nmole of the specified lipopeptides in PBS. 25 Nine days after inoculation mice were challenged intranasally with 104.5 PFU of Mem7I influenza virus. On day 5 post infection, CTL-determinant-specific CD8 T cells in the lungs were enumerated by staining the lymphocytes from the lungs with 3295672 I.DOC 32 anti-CD8 antibody and with tetrameric MHC class I complexes loaded with the CTL epitope. A total of 30,000 CD8 T cells were analysed. Figure 25 is a graphical representation showing cytotoxic T cell activity in naive mice. CTL determinant specific cytotoxicity in vivo was measured using syngeneic spleen 5 cells pulsed with the CTL determinant and labelled with high intensity CFSE. Non pulsed spleen cells labelled with low intensity CFSE were used as a control. A mixture of 15 x 106 cells of each target cell population was injected intravenously on day 4 post-infection into nafve mice. The mice were killed 16 hr later and spleens were analysed for the presence of CFSE-high and CFSE-low cell populations by flow 10 cytometry. A total of 1 x 106 lymphocytes were analysed for each sample. Figure 26 is a graphical representation showing cytotoxic T cell activity in lipopeptide primed mice. A mouse was inoculated intranasally with 9 nmoles [Th]-Lys(Pam 2 Cys Ser-Ser)-[CTL] comprising the CD4+ T-helper epitope set forth in SEQ ID NO: 18 and the H-2d-restricted CTL epitope set forth in SEQ ID NO: 114, in PBS. Mice were 15 challenged with Mem71 on day 28. CTL determinant specific cytotoxicity in vivo was measured using syngeneic spleen cells pulsed with the CTL determinant and labelled with high intensity CFSE. Non-pulsed spleen cells labelled with low intensity CFSE were used as a control. A mixture of 15 x 106 cells of each target cell population was injected intravenously on day 4 post-infection into the lipopeptide-primed and 20 challenged mice. The mice were killed 16 hr later and spleens were analysed for the presence of CFSE-high and CFSE-low cell populations by flow cytometry. A total of I x 106 lymphocytes were analysed for each sample. Figure 27 is a graphical representation showing the ability of various peptide-based immunogens to induce epitope-specific CTL. Lipopeptides comprised the CD4+ T 25 helper epitope set forth in SEQ ID NO: 18 and the H-2d-restricted CTL epitope set forth in SEQ ID NO: 114. Groups of three mice were inoculated intranasally with various lipopeptides in PBS and challenged with Mem71 on day 28. In order to 3295672 I.DOC 33 analyze CTL determinant specific cytotoxicity in vivo, syngeneic spleen cells were pulsed with the CTL determinant and labelled with high intensity CFSE. Antigen specific lysis was controlled by co-injecting syngeneic spleen cells labelled with low intensity CFSE. A mixture of 15 x 106 cells of each target cell population was injected 5 intraveniously on day 4 post-infection. The mice were killed 16 hr later and spleens were analysed for the presence of CFSE-high and CFSE-low cell populations by flow cytometry. A total of 1 x 106 lymphocytes were analysed for each sample. Individual mice are represented by the closed squares and the bars represent the geometric mean titre. 10 Figure 28 is a graphical representation showing induction of interferon-gamma producing cells by lipopeptide. Peptide comprising a T-helper epitope and a CTL epitope of Listeria monocytogenes linked via the epsilon amino group of an internal lysine residue positioned between said epitopes to Pam 2 Cys (i.e. the peptide [P25] Lys(Pam 2 Cys-Ser-Ser)-[LLO91-99] listed in Figure 19 and based upon SEQ ID NO: 15 248), or lipopeptide(s) based on this structure in which Pam 2 Cys was linked through the epsilon amino group of said lysine, were used to inoculate mice. Five BALB/c mice were inoculated intravenously with bacteria, or subcutaneously with either 9 nmoles of lipidated peptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[LLO91-99) or 9 nmoles of non-lipidated peptide [P25]-Lys-[LLO91-99] (SEQ ID NO: 248; Figure 19) or 20 phosphate buffered saline (PBS), as indicated on the x-axis. Splenocytes were obtained from the immunized animals and stimulated in vitro with either the isolated CTL epitope having the sequence set forth in SEQ ID NO: 245 (open bars) or no antigen (filled bars), and the number of (IFN-gamma) producing cells present was measured 28 days later. The ordinate indicates the number of IFN-gamma producing cells per 25 1,000,000 splenocytes. Data show enhanced numbers of IFN-gamma producing cells for mice immunized with lipopeptide, indicating an enhanced ability of the lipopeptides to activate T cells relative to non-lipidated peptide.

3295672_.DOC 34 Figure 29 is a graphical representation showing enhanced protection against L. monocytogenes infection for mice immunized with the lipopeptide designated [P25] Lys(Pam 2 Cys-Ser-Ser)-[LLO91-99] in Figure 19 (based upon SEQ ID NO: 248). Five BALB/c mice were inoculated intravenously with 1,000 bacteria (column 1), or 5 immunized subcutaneously with PBS (column 2) or 9 nmol [P25]-Lys(Pam 2 Cys-Ser Ser)-[LLO91-99] peptide (column 3) or 9 nmol non-lipidated [P25]-Lys-[LLO91-99] peptide (SEQ ID NO: 248; column 4), as indicated on the x-axis. Mice were challenged with whole bacteria and the number of colony forming units present in liver was measured 28 days post-challenge (ordinate). 10 Figure 30 is a graphical representation showing protection against B16 melanoma with lipopeptide vaccination. C57BL/6 mice were vaccinated with 20 nmoles lipidated peptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[SIINFEKL] (open circles), non-lipidated peptide [P25]-Lys-[SIINFEKL] (open triangles) or with PBS (open squares) subcutaneously in the base of the tail. Mice were then challenged s.c. on the back 14 days later with 15 2x10 5 B 16-OVA cells (n=6 per group) and tumour growth monitored as described (Anraku, et al., J Virol. 76; 3791-3799, 2002). Figure 31 is a graphical representation showing therapeutic treatment of Lewis Lung tumour with a lipopeptide immunogen, as determinbed by the percentage of animals that remain tumor free following immunization. Mice were injected with 3x]0 4 Lewis 20 Lung tumour cells that had been transfected with ovalbumin and therefore expressed the CTL epitope SIINFEKL [Nelson et al., J Immunol. 166: 5557-5566, 2001]. Four days after receiving tumour cells, animals were inoculated with 20 nmoles lipidated peptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[SIrNFEKL] (open circles), non-lipidated peptide [P25]-Lys-[SIINFEKL] (open triangles) or with PBS (open squares) subcutaneously in 25 the base of the tail. A second and similar dose of immunogen was administered eleven days after receiving the tumour cells. Animals were monitored for tumour incidence; animals were euthanased when tumour area exceeded 100 mm 2 3295672_.DOC 35 Figure 32 is a graphical representation showing therapeutic treatment of Lewis Lung tumour with a lipopeptide immunogen, as determined by measuring survival of animals following immunization. Mice were injected with 3x10 4 Lewis Lung tumour cells that had been transfected with ovalbumin and therefore expressed the CTL epitope 5 SIINFEKL [Nelson et al., J Immunol. 166: 5557-5566, 2001]. Four days after receiving tumour cells, animals were inoculated with 20nmoles lipidated peptide [P25] Lys(Pam 2 Cys-Ser-Ser)-[SIINFEKL] (open circles), non-lipidated peptide [P25]-Lys [SIINFEKL] (open triangles) or with PBS (open squares) subcutaneously in the base of the tail. A second and similar dose of immunogen was administered eleven days after 10 receiving the tumour cells. Animals were monitored for survival; animals were euthanased when tumour area exceeded 100 mm 2 Figure 33 is a graphical representation showing the ability of peptide and lipopeptide based immunogens to up-regulate the expression of MHC class II, CD83 and CD86 on human dendritic cells. Human monocyte-derived dendritic cells were incubated with 15 media alone, LPS (5ptg/mL), non-lipidated peptide [P25]-Lys-[HCV] (5pg/mL) or lipopeptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[HCV] (5pg/mL) for 48 hours before staining with FITC-conjugated antibodies for HLA-DR, CD83 and CD86 before analysis by flow cytometry. Histograms are representative of live large granular cells gated on the forward and side scatter dot plot. Regions of histograms shaded in grey and the given 20 values correspond to the percentage of cells that express high levels of antigen within the analysed populations. The T helper cell epitope was identified from Mobillivirus and has the amino acid sequence KLIPNASLIENCTKAEL (SEQ ID NO: 24); the CTL epitope with the amino acid sequence DLMGYIPLV (SEQ ID NO: 249) is an HLA A2-restricted CTL epitope from the core protein of hepatitis C virus.

3295672 I.DOC 36 DETAILED DESCRIPTION OF THE INVENTION Lipopeptides One aspect of the invention provides a lipopeptide comprising a T helper cell (Th) epitope and a B cell epitope or a CTL epitope, wherein the amino acid sequences of the 5 Th epitope is different from the amino acid sequence of the B cell or CTL epitope; one or more internal lysine residues or internal lysine analog residues and one or more lipid moieties wherein said lipid moieties are covalently attached to said internal lysine residues or internal lysine analog residues. As used herein, the term "lipopeptide" means any non-naturally occurring composition 10 of matter comprising one or more lipid moieties and one or more amino acid sequences that are directly or indirectly conjugated, said composition of matter being substantially free of conspecific non-conjugated lipid or protein. By "directly" means that a lipid moiety and an amino acid sequence are juxtaposed in said lipopeptide (i.e. they are not separated by a spacer molecule). 1 5 By "indirectly" means that a lipid moiety and an amino acid sequence are separated by a spacer comprising one or more carbon-containing molecules, such as, for example, one or more amino acid residues. The amino acid sequence may be of any length, constrained by the requirement for functionality of both the T-helper epitope and the B cell epitope or CTL epitope. 20 As used herein, the term "internal lysine residue" means a lysine residue in the polypeptide comprising both the T-helper epitope and the B cell epitope or the CTL epitope, wherein said lysine is not the N-terminal amino acid residue or the C-terminal residue of said polypeptide. Accordingly, the internal lysine residue may be a C terminal or N-terminal residue of either the T-helper epitope, B cell epitope or CTL 25 epitope, provided that it is internalized in the polypeptide. This means that the internal 3295672 I.DOC 37 lysine residue to which the lipid moiety is attached is a residue that is present in the amino acid sequence of the T helper cell epitope or the amino acid sequence of the B cell epitope or CTL epitope. The internal lysine residue may also be distinct from the T-helper epitope, B cell epitope or CTL epitope, in which case it must link these two 5 epitopes of the polypeptide. Similarly, the term "internal lysine analog residue" means a lysine analog residue in the polypeptide comprising both the T-helper epitope and the B cell epitope or CTL epitope, wherein said lysine analog is not the N-terminal amino acid residue or the C terminal residue of said polypeptide. The crtieria for establishing whether or not a 10 lysine residue is "internal" shall apply mutatis mutandis to determing whether or not a lysine analog is internal. By "lysine analog" is meant a synthetic compound capable of being incorporated into the internal part of a peptide that has a suitable side-group to which the lipid moiety can be coupled, including an amino acid analog or non-naturally occurring amino acid 15 having such an amino side group. Preferred lysine analogs include compounds of the following general Formula (V):

H

2 N-C-COOH

(CH

2 )n X wherein n is an integer from 0 to 3 and wherein X is a terminal side-chain group of said internal lysine analog residue selected from the group consisting of NH, 0 and S. More 20 preferably, n is an integer having a value from 1 to 3. More preferably, X is an amino group. In a particularly preferred embodiment, the lysine analog is selected from the group consisting of 2,3 diaminopropionic acid (Dpr), 2,4-diaminobutyric acid (Dab) and 2,5-diaminovaleric acid [i.e. ornithine (Orn)].

3295672 i.DOC 38 Those skilled in the art will know the meaning of the term "epsilon-amino group". The term "terminal side-chain group" means a substituent on the side chain of a lysine analog the is distal to the alpha-carbon of said analog, such as, for example, a beta amino of Dpr, gamma-amino of Dab, or delta-amino of Orn. 5 Preferably, the lipid moiety is attached via the epsilon amino group of a lysine residue or to a terminal side-chain group of said internal lysine analog residue that is positioned between the amino acid sequences of the T helper epitope and the B cell or CTL epitope. The enhanced ability of the lipopeptides of the invention to elicit a T cell response is 10 reflected by their ability to upregulate the surface expression of MHC class II molecules on immature dendritic cells (DC), particularly Dl cells, and by the enhanced number of CD8+ T cells in tissue samples of immunized animals. In the case of animals immunized using CTLs of a viral pathogen, the enhanced ability of the lipopeptides of the invention to elicit a T cell response is also indicated by the enhanced 15 viral clearance following immunization of animals. Preferably, the lipopeptides are soluble, more preferably highly soluble. As will be known to those skilled in the art, the epsilon amino group of lysine is the terminal amino group of the side chain of this amino acid. Use of the terminal side chain group of the internal lysine or internal lysine analog for cross-linkage to the lipid 20 moiety facilitates the synthesis of the polypeptide moiety as a co-linear amino acid sequence incorporating both the T-helper epitope and the B cell epitope or CTL epitope. There is a clear structural distinction between a lipopeptide having lipid attached via the epsilon amino group of a lysine residue or the terminal side-chain group of a lysine analog, and a lipopeptide having the lipid attached via an alpha amino 25 group of a lysine in the peptide.

3295672 I.DOC 39 Accordingly, it is particularly preferred for at least one internal lysine residue or internal lysine analog to which the lipid moiety is attached to be positioned within the polypeptide moiety so as to separate the immunologically-functional epitopes. For example, the internal lysine residue or internal lysine analog may act as a spacer and/or 5 linking residue between the epitopes. Naturally, wherein the internal lysine or internal lysine analog is positioned between the T-helper epitope and the B cell epitope or CTL epitope, the lipid moiety will be attached at a position that is also between these epitopes, albeit forming a branch from the amino acid sequence of the polypeptide. Preferably, a single internal lysine residue or internal lysine analog is used to separate 10 B cell and T-helper epitopes (e.g., any one of SEQ ID NOs: 7, 9, 13, 106, 108, 110, or 112), or CTL and T-helper epitopes (e.g. SEQ ID NO: 117), in which case the lipid moiety is attached via the epsilon amino group of a lysine residue or the terminal side chain group of a lysine analog positioned between the amino acid sequences of the T helper epitope and the antigenic B cell epitope or CTL epitope. 15 The present invention clearly contemplates the nesting of the internal lysine residue or internal lysine analog residue within a third amino acid sequence that does not function as a CTL epitope, B cell epitope or T-helper epitope. For example, the internal lysine or internal lysine analog may be conjugated to one or more different amino acid residues. 20 The epsilon amino group of the internal lysine or terminal side-chain group of an internal lysine analog can be protected by chemical groups which are orthogonal to those used to protect the alpha-amino and side-chain functional groups of other amino acids. In this way, the epsilon amino group or other side-chain group of an internal lysine or lysine analog can be selectively exposed to allow attachment of chemical 25 groups, such as lipid-containing moieties, specifically to the epsilon amino group or side-chain amino group, as appropriate.

3295672 I.DOC 40 For peptide syntheses using using Fmoc chemistry, a suitable orthogonally protected epsilon group of lysine is provided by the modified amino acid residue Fmoc-Lys(Mtt) OH (Na-Fmoc-Ne-4-methyltriiyl-L-lysine). Similar suitable orthogonally-protected side-chain groups are available for various lysine anlogs contemplated herein, eg. 5 Fmoc-Om(Mtt)-OH (Na-Fmoc-No-4-methyltrityl-L-Ornithine), Fmoc-Dab(Mtt)-OH (Na-Fmoc-Ny-4-methyltrityl-L-diaminobutyric acid) and Fmoc-Dpr(Mtt)-OH (Na Fmoc-Nfi-4-methyltriiyl-L-diaminopropionic acid). The side-chain protecting group Mtt is stable to conditions under which the Fmoc group present on the alpha amino group of lysine or a lysine analog is removed but can be selectively removed with 1% 10 trifluoroacetic acid in dichloromethane. Fmoc-Lys(Dde)-OH (NaFmoc-N- 1-(4,4 dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-lysine) or Fmoc-Lys(ivDde)-OH (Na Fmoc-Ne-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine) can also be used in this context, wherein the Dde side-chain protecting groups is selectively removed during peptide synthesis by treatment with hydrazine. 15 For peptide syntheses using Boc chemistry, Boc-Lys(Fmoc)-OH can be used. The side chain protecting group Fmoc can be selectively removed by treatment with piperidine or DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene) but remain in place when the Boc group is removed from the alpha terminus using trifluoroacetic acid. The optimum distance between the T-helper epitope and the B cell epitope or CTL 20 epitope, and consequently, the precise positioning and number of internal lysine or lysine analog residues in the lipopeptide of the invention, is readily determined empirically, for each combination of T helper, B cell, CTL epitopes and lipids. In the case of synthetic peptides and polypeptides, the limitations of the synthesis methodology used to prepare the polypeptides may, in part, determine the separation 25 between the T-helper epitope and the B cell epitope or CTL epitope that is achievable, and the number and positioning of internal lysine or lysine analog residue(s).

3295672 I.DOC 41 Preferably, the T helper epitope and CTL epitope are separated by at least one or two or three or four or five amino acid residues including a single internal lysine residue or internal lysine analog residue. The present invention clearly contemplates the addition of multiple lipid moieties to the 5 polypeptide moiety. For example, the polypeptide may include multiple internal lysine residues and/or multiple internal lysine analogs. Steric hindrance may occur in the addition of lipid if multiple internal lysines or multiple lysine analogs are positioned more closely together, thereby producing a mixture of end-products, or a reduced yield. Relevant to this consideration is the fact that it is not necessary for the entire amino 10 acid sequence comprising the T-helper epitope or the entire amino acid sequence comprising the B cell epitope or CTL epitope to have an immune function. Accordingly, the said amino acid sequences, whilst comprising said epitopes may have additional sequence not possessing T-helper cell activity, a B cell epitope or a CTL epitope. Where such additional sequences include one or more internal lysine or lysine 15 analog residues, the terminal side-chain groups of such residues may serve as attachment sites for the lipid moiety. Naturally, it is essential to retain both T-helper function and the B cell or CTL epitope function. The positioning of the internal lysine residue or internal lysine analog for attachment of the lipid moiety should also be selected such that attachment of the lipid moiety does 20 not interfere with the immune function of the T-helper epitope, the B cell epitope or the CTL epitope in a subject to whom the lipopeptide is administered. For example, depending upon the selection of lipid moiety, the attachment of said lipid within the B cell or CTL epitope may sterically hinder the epitope presentation. A generalized preferred form of the lipopeptide of the invention, wherein the internal 25 lysine or internal lysine analog is positioned between the T-helper epitope and B cell or CTL epitopes is provided by the general Formula (VI).

3295672 I.DOC 42 Formula (VI) H epotipe- A - HN---C-CO- A- epitope

(CH

2 )n I X z wherein: epitopeis a T-helper epitope or CTL epitope; 5 A is either present or absent and consists of an amino acid spacer of about 1 to about 6 amino acids in length; n is an integer having a value of 1, 2, 3, or 4; X is a terminal side-chain group selected from the group consisting of NH, 0 and S and preferably consisting of NH; 10 Y is either present of absent and consists of an amino acid spacer of about 1 to about 6 amino acids in length, wherein it is preferred for said amino acid to be serine; and Z is a lipid moiety, preferably selected from the group consisting of Pam 2 Cys or Pam 3 Cys, Ste 2 Cys, Lau 2 Cys and Oct 2 Cys. 15 Those skilled in the art will be aware that Ste 2 Cys is also known as S-[2,3 bis(stearoyloxy)propyl]cysteine or distearoyl-S-glyceryl-cysteine; that Lau 2 Cys is also known as S-[2,3-bis(lauroyloxy)propyl]cysteine or dilauroyl-S-glyceryl-cysteine); and 3295672 IDOC 43 that Oct 2 Cys is also known as S-[2,3-bis(octanoyloxy)propyl]cysteine or dioctanoyl-S glyceryl-cysteine). The T-helper epitope is any T-helper epitope known to the skilled artisan for enhancing an immune response in a particular target subject (i.e. a human subject, or a specific 5 non-human animal subject such as, for example, a rat, mouse, guinea pig, dog, horse, pig, or goat). Preferred T-helper epitopes comprise at least about 10-24 amino acids in length, more generally about 15 to about 20 amino acids in length. Promiscuous or permissive T-helper epitopes are particularly preferred as these are readily synthesized chemically and obviate the need to use longer polypeptides 10 comprising multiple T-helper epitopes. Examples of promiscuous or permissive T-helper epitopes suitable for use in the lipopeptides of the present invention are selected from the group consisting of: (i) a rodent or human T-helper epitope of tetanus toxoid peptide (TTP), such as, for example amino acids 830-843 of TTP (Panina-Bordignon et al., Eur. J. Immun. 19, 15 2237-2242, 1989); (ii) a rodent or human T-helper epitope of Plasmodium falciparum pfg27; (iii) a rodent or human T-helper epitope of lactate dehydrogenase; (iv) a rodent or human T-helper epitope of the envelope protein of HIV or HlVgp 120 (Berzofsky et al., J. Clin. Invest. 88, 876-884, 1991); 20 (v) a synthetic human T-helper epitope (PADRE) predicted from the amino acid sequence of known anchor proteins (Alexander et al., Immunity 1, 751-761, 1994); 3295672 I.DOC 44 (vi) a rodent or human T-helper epitope of measles virus fusion protein (MV-F; Muller et al., Mol. Immunol. 32, 37-47, 1995; Partidos et al., J. Gen. Virol., 71, 2099 2105, 1990); (vii) a T-helper epitope comprising at least about 10 amino acid residues of canine 5 distemper virus fusion protein (CDV-F) such as, for example, from amino acid positions 148-283 of CDV-F (Ghosh et al., Immunol. 104, 58-66, 2001; International Patent Publication No. WO 00/46390); (viii) a human T-helper epitope derived from the peptide sequence of extracellular tandem repeat domain of MUCI mucin (US Patent Application No. 0020018806); 10 (ix) a rodent or human T-helper epitope of influenza virus haemagglutinin (IV-H) (Jackson et al. Virol. 198, 613-623, 1994; ; and (x) a bovine or camel T-helper epitope of the VP3 protein of foot and mouth disease virus (FMDV-01 Kaufbeuren strain), comprising residues 173 to 176 of VP3 or the corresponding amino acids of another strain of FMDV. 15 As will be known to those skilled in the art, a T-helper epitope may be recognised by one or more mammals of different species. Accordingly, the designation of any T helper epitope herein is not to be considered restrictive with respect to the immune system of the species in which the epitope is recognised. For example, a rodent T helper epitope can be recognised by the immune system of a mouse, rat, rabbit, guinea 20 pig, or other rodent, or a human or dog. More preferably, the T-helper epitope will comprise an amino acid sequence selected from the group consisting of: (i) GALNNRFQIKGVELKS from IV-H (SEQ ID NO: 1); (ii) ALNNRFQIKGVELKS from IV-H (SEQ ID NO: 18); 3295672 I.DOC 45 (iii) LSEIKGVIVHRLEGV from MV-F (SEQ ID NO: 19); (iv) TAAQITAGIALHQSNLN from CDV-F (SEQ ID NO: 20); (v) IGTDNVHYKIMTRPSHQ from CDV-F (SEQ ID NO: 21); (vi) YKIMTRPSHQYLVIKLI from CDV-F (SEQ ID NO: 22); 5 (vii) SHQYLVIKLIPNASLIE from CDV-F (SEQ ID NO: 23); (viii) KLIPNASLIENCTKAEL from CDV-F (SEQ ID NO: 24); (ix) LIENCTKAELGEYEKLL from CDV-F (SEQ ID NO: 25); (x) AELGEYEKLLNSVLEPI from CDV-F (SEQ ID NO: 26); (xi) KLLNSVLEPINQALTLM from CDV-F (SEQ ID NO: 27); 10 (xii) EPINQALTLMTKNVKPL from CDV-F (SEQ ID NO: 28); (xiii) TLMTKNVKPLQSLGSGR from CDV-F (SEQ ID NO: 29); (xiv) KPLQSLGSGRRQRRFAG from CDV-F (SEQ ID NO: 30); (xv) SGRRQRRFAGVVLAGVA from CDV-F (SEQ ID NO: 31); (xvi) FAGVVLAGVALGVATAA from CDV-F (SEQ ID NO: 32); 15 (xvii) GVALGVATAAQITAGIA from CDV-F (SEQ ID NO: 33); (xviii) G IALHQSNLNAQAIQSL from CDV-F (SEQ ID NO: 34); (xix) NLNAQAIQSLRTSLEQS from CDV-F (SEQ ID NO: 35); (xx) QSLRTSLEQSNKAIEEI from CDV-F (SEQ ID NO: 36); 3295672 I.DOC 46 (xxi) EQSNKAIEEIREATQET from CDV-F (SEQ ID NO: 37); (xxii) SSKTQTHTQQDRPPQPS from CDV-F (SEQ ID NO: 38); (xxiii) QPSTE LEETRTSRARHS from CDV-F (SEQ ID NO: 39); (xxiv) RHSTTSAQRSTHYDPRT from CDV-F (SEQ ID NO: 40); 5 (xxv) PRTSDRPVSYTMNRTRS from CDV-F (SEQ ID NO: 41); (xxvi) TRSRKQTSHRLKNIPVH from CDV-F (SEQ ID NO: 42); (xxvii) TELLSIFGPSLRDPISA from CDV-F (SEQ ID NO: 43); (xxviii)PRYIATNGYLISNFDES from CDV-F (SEQ ID NO: 44); (xxix) CIRGDTSSCARTLVSGT from CDV-F (SEQ ID NO: 45); 10 (xxx) DESSCVFVSESAICSQN from CDV-F (SEQ ID NO: 46); (xxxi) TSTIINQSPDKLLTFIA from CDV-F (SEQ ID NO: 47); (xxxii) SPDKLLTFIASDTCPLV from CDV-F (SEQ ID NO: 48); (xxxiii)STAPPAHGVTSAPDTRAPGSTAPP from MUC-1 (SEQ ID NO: 49); (xxxiv)GVTSAPDTRPAPGSTASSL from MUC-1 (SEQ ID NO: 50); 15 (xxxv) GVTSAPDTRPAPGSTASL from MUC-1(SEQ ID NO: 51); (xxxvi)TAPPAHGVTSAPDTRPAPGSTAPPKKG from MUC-I (SEQ ID NO: 52); (xxxvii) STAPPAHGVTSAPDTRPAPGSTAPPK from MUC-1 (SEQ ID NO: 53); (xxxviii) GVAE from FMDV-VP3 protein (SEQ ID NO: 54); 3295672_.DOC 47 (xxxix)TASGVAETTN from FMDV-VP3 protein (residues 170 to 179) (SEQ ID NO: 55); and (xl) TAKSKKFPSYTATYQF from FMDV (SEQ ID NO: 56). The T-helper epitopes disclosed herein are included for the purposes of exemplification 5 only. Using standard peptide synthesis techniques known to the skilled artisan, the T helper epitopes referred to herein are readily substituted for a different T-helper epitope to adapt the lipopeptide of the invention for use in a different species. Accordingly, additional T-helper epitopes known to the skilled person to be useful in eliciting or enhancing an immune response in a target species are not to be excluded. 10 Additional T-helper epitopes may be identified by a detailed analysis, using in vitro T cell stimulation techniques of component proteins, protein fragments and peptides to identify appropriate sequences (Goodman and Sercarz, Ann. Rev. Immunol., 1, 465, (1983); Berzofsky, In: "The Year in Immunology, Vol. 2" page 151, Karger, Basel, 1986; and Livingstone and Fathman, Ann. Rev. Immunol., 5, 477, 1987). 15 The B cell epitope is conveniently derived from the amino acid sequence of an immunogenic protein, lipoprotein, or glycoprotein of a virus, prokaryotic or eukaryotic organism, including but not limited to an antigen derived from a mammalian subject or a bacterium, fungus, protozoan, or parasite that infects said subject. Idiotypic and anti idiotypic B cell epitopes against which an immune response is desired are specifically 20 included, as are lipid-modified B cell epitopes. Alternatively, the B cell epitope may be a carbohydrate antigen, such as, for example, an ABH blood group antigen, transplantation antigen (eg. Gal alphal-3Gal betal -4GIcNAc; Sandrin et al., Proc. Natl. Acad. Sci. USA 90, 11391-11395, 1993; Galili et al., Proc. Nati. Acad. Sci. USA 84, 1369-1373, 1987; Schofield et al., Nature 418: 785-789, 2002) or a conjugate thereof.

3295672I.DOC 48 The B-cell epitope will be capable of eliciting the production of antibodies when administered to a mammal, preferably neutralizing antibody, and more preferably, a high titer neutralizing antibody. Shorter B cell epitopes are preferred, to facilitate peptide synthesis. 5 Preferably, the length of the B cell epitope will not exceed about 30 amino acids in length. More preferably, the B cell epitope sequence consists of about 25 amino acid residues or less, and more preferably less than 20 amino acid residues, and even more preferably about 5-20 amino acid residues in length. Preferably, peptides will assume a conformation that mimics the conformation of the 10 native polypeptide from which the B cell epitope is derived. Preferred B cell epitopes from parasites are those associated with leishmania, malaria, trypanosomiasis, babesiosis, or schistosomiasis, such as, for example a B cell epitope selected from the group consisting of: (i) a B cell epitope of Plasmodium falciparum (NANP) 3 (Good et al., J. Exp. Med. 15 164, 655 1986); (ii) a B cell epitope of Circumsporozoa (Good et al., Protein Sci., 235, 1059, 1987); (iii) a B cell epitope comprising amino acid residues 326-343 of Leishmania donovani Repetitive Peptide (Liew et al., J. Exp. Med. 172, 1359 (1990)); (iv) a B cell epitope of Toxoplasma gondii P30 surface protein (Darcy et al., J. 20 Immunol. 149, 3636 (1992)); and (v) a B cell epitope of Schistosoma mansoni Sm-28GST antigen (Wolowxzuk et al., J. Immunol 146:1987 (1991)).

3295672 I.DOC 49 Preferred virus-specific B cell epitopes are derived from and/or capable of generating antibodies against Rotaviruses, Herpes viruses, Corona viruses, Picornaviruses (eg. Apthovirus), Respiratory Synctial virus, Influenza Virus, Parainfluenza virus, Adenovirus, Pox viruses, Bovine herpes virus Type I, Bovine viral diarrhea virus, 5 Bovine rotaviruses, Canine Distemper Virus (CDV), Equine Rhinitis A Virus (ERAV); Equine Rhinitis B Virus (ERBV); Foot and Mouth Disease Virus (FMDV), Measles Virus (MV), Human Immunodeficiency Viruses (HIV), Feline Immunodeficiency Viruses (FIV), Epstein-Barr virus (EBV), or hepatitis virus, and the like. Suitable viral B cell epitopes include, but are not limited to epitopes selected from the group 10 consisting of: (i) HIV gp120 V3 loop, amino acid residues 308-331 (Jatsushita et al., J. Virol. 62, 2107 (1988)); (ii) HIV gp120 amino acid residues 428-443 (Ratner et al., Nature 313:277 (1985)); (iii) HIV gpl20 amino acid residues 112-124 (Berzofsky et al., Nature 334, 706 15 (1988)); (iv) a B cell epitope of HIV Reverse transcriptase (Hosmalin et al. Proc. Natl Acad. Sci.(USA) 87, 2344 (1990)); (v) Influenza virus nucleoprotein amino acid residues 335-349 (Townsend et al. Cell 44, 959 (1986)); 20 (vi) Influenza virus nucleoprotein amino acid residues 366-379 (Townsend et al. Cell 44, 959 (1986)); (vii) Influenza virus hemagglutinin amino acid residues 48-66 (Mills et al., J. Exp. Med. 163, 1477 (1986)); 3295672 IDOC 50 (viii) Influenza virus hemagglutinin amino acid residues 111-120 (Hackett et al., J. Exp. Med 158, 294 (1983)); (ix) Influenza virus hemagglutinin amino acids 114-131 (Lamb and Green, Immunology 50, 659 (1983)); 5 (x) Epstein-Barr LMP amino acid residues 43-53 (Thorley-Lawson et al., Proc. Natl Acad. Sci. (USA) 84, 5384 (1987)); (xi) Hepatitis B virus surface antigen amino acid residues 95-109 (Milich et al., J. Immunol. 134, 4203 (1985)); (xii) Hepatitis B virus surface antigen amino acid residues 140-154; 10 (xiii) Hepatitis B virus Pre-S antigen amino acid residues 120-132 (Milich et al., J. Exp. Med. 164, 532 (1986)); (xiv) Herpes simplex virus gD protein amino acid residues 5-23 (Jayaraman et al., J. Immunol. 151, 5777 (1993)); (xv) Herpes simplex virus gD protein amino acid residues 241-260 (Wyckoff et al., 15 Immunobiol., 177, 134 (1988)); (xvi) Rabies glycoprotein amino acid residues 32-44 (MacFarlan et al., J. Immunol. 133, 2748 (1984)); (xvii) The major FMDV epitope comprising at least amino acid residues 134-168 or 137-160 or residues 142-160 or residues 137-162 or residues 145-150 of the VPI 20 capsid protein of FMDV serotype 01. or the corresponding amino acid residues of another serotype, such as, for example, serotypes A, C, SATI, SAT2, SAT3, or ASIA 1 (US Patent Nos. 5,864,008 and 6,107,021); and (xviii) The h ypervariable region-I (HVRJ) of the E2 protein of hepatitis C virus 3295672_.DOC 51 (HCV) variant AD78 (Zibert et al., J. Virol. 71, 4123-4127, 1997). Preferred bacteria-specific B cell epitopes are derived from and/or capable of generating antibodies against Pasteurella, Actinobacillus, Haemophilus, Listeria monocytogenes, Mycobacterium, Staphylococcus, E. coli, Shigella, and the like. 5 Suitable bacterial B cell epitopes include, but are not limited to epitopes selected from the group consisting of: (i) Mycobacterium tuberculosis 65Kd protein amino acid residues 112-126 (Lamb et al., EMBO J., 6, 1245 (1987)); (ii) M. tuberculosis 65Kd protein amino acid residues 163-184 (Lamb et al., EMBO 10 J., 6, 1245 (1987)); (iii) M. tuberculosis 65Kd protein amino acid residues 227-243 (Lamb et al., EMBO J., 6, 1245 (1987)); (iv) M. tuberculosis 65Kd protein amino acid residues 242-266 (Lamb et al., EMBO J., 6, 1245 (1987)); 15 (v) M. tuberculosis 65Kd protein amino acid residues 437-459 (Lamb et al., EMBO J., 6, 1245 (1987)); (vi) M. tuberculosis ESAT-6 protein residues 3-15 (Morten et al., Infect. Immun. 66, 717-723, 1998); (vii) M. tuberculosis ESAT-6 protein residues 40-62 (Morten et al., Infect. Immun. 20 66,717-723, 1998); (viii) Mycobacterium scrofulaceum alpha-antigen residues 279-290 (Mikiko et al., Microb. Path. 23, 95-100, 1997); 3295672_.DOC 52 (ix) Staphylococcus aureus nuclease protein amino acid residues 61-80 (Finnegan et al., J. Exp. Med. 164, 897 (1986)); (x) a B cell epitope of Escherichia coli heat stable enterotoxin (Cardenas et al., Infect. Immunity 61, 4629 (1993)); 5 (xi) a B cell epitope of Escherichia coli heat labile enterotoxin (Clements et al., Infect. Immunity 53, 685 (1986)); (xii) a B cell epitope of Shigella sonnei form I antigen (Formal et al., Infect. Immunity 34, 746 (1981)); (xiii) a B cell epitope from Group A Streptococcus , preferably derived from the M 10 protein, more preferably from the C-terminal half of the M protein and more preferably a minimum, helical, non-host-cross-reactive peptide derived from the conserved C terminal half of the M protein and comprising a non-M-protein peptide designed to maintain helical folding and antigenicity displayed within said minimum, helical, non host-cross-reactive peptide. For example, the non-M-protein peptide (eg peptide J1 4) 15 can be linked to one or more serotypic M protein peptides using chemistry that enables the immunogen to display all the individual peptides pendant from an alkane backbone, thereby conferring excellent immunogenicity and protection (US Pat. No. 6,174,528; Brandt et al., Nat. Med. 6: 455-459, 2000); (xiv) a B cell epitope of the Cholera toxin B subunit (CTB), such as, for example 20 described by Kazemi and Finkelstein Mol. Immunol. 28, 865-876, 1991; (xv) a B cell epitope of a protein of Bacillus anthracis (anthrax), such as, for example, a B cell epitope derived from a protein of the outer exosporium of anthrax such as the 250 kDa glycoprotein (Sylvestre et al., In: Proc. 4th Int. Conf. Anthrax, St John's College Annapolid, Mayland, CA June 10-13, 2001, Abstract 31B; and 3295672 I.DOC 53 (xvi) a B cell epitope from a protein of tetanus, such as, for example, the tetanus toxoid protein. Preferred B cell epitopes from mammalian subjects are derived from and/or capable of generating antibodies against a tumor antigen. Tumor antigens are usually native or 5 foreign antigens, the expression of which is correlated with the development, growth, presence or recurrence of a tumor. In as much as tumor antigens are useful in differentiating abnormal from normal tissue, they are useful as a target for therapeutic intervention. Tumor antigens are well known in the art. Indeed, several examples are well-characterized and are currently the focus of great interest in the generation of 10 tumor-specific therapies. Non-limiting examples of tumor antigens are carcinoembryonic antigen (CEA), prostate specific antigen (PSA), melanoma antigens (MAGE, BAGE, GAGE), and mucins, such as MUC-1. Alternatively, a preferred B cell epitope from a mammalian subject is derived from zona pellucida protein such as ZP3 (Chamberlin and Dean Proc. Natl. Acad. Sci.( USA) 15 87, 6014-6018, 1990) or ZP3a (Yurewicz et al., Biochim. Biophys. Acta 1174, 211 214, 1993)] of humans or other mammals such as pigs. Particuarly preferred B cell epitopes within this category include amino acid residues 323-341 of human ZP3 (Chamberlin and Dean Proc. Natl. Acad. Sci.(USA) 87, 6014-6018, 1990); amino acid residues 8-18 or residues 272-283 or residues 319-330 of porcine ZP3a (Yurewicz et 20 al., Biochim. Biophys. Acta 1174, 211-214, 1993). Further preferred B cell epitopes from a mammalian subject are derived from and/or capable of generating antibodies against a peptide hormone, such as, for example, a satiety hormone (eg. leptin), a digestive hormone (eg. gastrin), or a reproductive peptide hormone [eg. luteinising hormone-releasing hormone (LHRH), follicle 25 stimulating hormone (FSH), luteinising hormone (LH), human chorionic gonadotropin (hCG; Carlsen et al., J. Biol. Chem. 248, 6810-6827, 1973), or alternatively, a hormone receptor such as, for example, the FSH receptor (Kraaij et al., J. Endocrinol. 158, 127- 3295672 I.DOC 54 136, 1998). Particuarly preferred B cell epitopes within this category include the C terminal portion (CTP) of b-hCG that is antigenically non cross-reactive with LH (Carlsen et al., J. Biol. Chem. 248, 6810-6827, 1973). In a particularly preferred embodiment, a peptide comprising a B-cell epitope will 5 comprise an amino acid sequence selected from the group consisting of: (i) EHWSYGLRPG derived from LHRH (herein referred to as "LHRH 1-10"; SEQ ID NO: 2); (ii) HWSYGLRPG derived from LHRH (herein referred to as "LHRH 2-10"; SEQ ID NO: 3); 10 (ii) GLRPG derived from LHRH ((herein referred to as "LHRH 6-10"; SEQ ID NO: 4); (iii) EAEEAARLQA from Leishmani major (SEQ ID NO: 57); (ii)(iv) a sequence from a non-structural protein 3A, 3B, or 3C of FMDV (US Patent No. 6,048,538) selected from the group consisting of: FRERTLTGQRACNDVNSE 15 (SEQ ID NO: 58), NPLETSGASTVGFRERTL (SEQ ID NO: 59), IRETRKRQKMVDDAVNEY (SEQ ID NO: 60), AKAPVVKEGPYEGPVKKPV (SEQ ID NO: 61), AGPLERQKPLKVKAKAPVV (SEQ ID NO: 62), 20 KVRAKLPQQEGPYAGPLER (SEQ ID NO: 63), GPYTGPLERQRPLKVRAKL (SEQ ID NO: 64), 3295672 I.DOC 55 VGRLIFSGEALTYKDIVV (SEQ ID NO: 65), TKHFRDTARMKKGTPVVGV (SEQ ID NO: 66), and SGAPPTDLQKMVMGNTKPV (SEQ ID NO: 67); (iii)(v) NKYSASGSGVRGDFGSLAPRVARQLPASFNYGAIK from the FMDV VPI 5 major epitope (US Patent No. 6,107,021; SEQ ID NO: 68); (iv)(vi)A sequence from prostate specific antigen (US Patent No. 6,326,471) selected from the group consisting of: LYTKVVHYRKWIKDTIVANP (SEQ ID NO: 69), AVKVMDLPQEPALGTTCYA (SEQ ID NO: 70), IVGGWECEKHSQPWQVLVAS (SEQ ID NO: 71), CAQVHPQKVTKFML (SEQ ID NO: 72), 10 YLMLLRLSEPAELTDDAVKVM (SEQ ID NO: 73), LLKNRFLRPGDDSSHDLMLLY (SEQ ID NO: 74), and ILLGRHSLFHPEDTGQVFQVY (SEQ ID NO: 75); (v)(vii) TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ from b-hCG (SEQ ID NO: 76); 15 (vi)(viii) CQDSKVTEIPTLPRNAI from the FSH receptor (SEQ ID NO: 77); (vii)(ix) NKGDCGTPSHSRRQPHVMS from human ZP3 protein (SEQ ID NO: 78); (viii)(x) a sequence from porcine ZP3a protein selected from the group consisting of: WLCFPLCLALP (SEQ ID NO: 79) LGGLYCGPSSF (SEQ ID NO: 80), 20 GSITRDSIFRLR (SEQ ID NO: 81), SALPVNIQVFTL (SEQ ID NO: 82), ELQIAKDERYGS (SEQ ID NO: 83), and VKLLREPIYVEV (SEQ ID NO: 84); (ix)(xi)PPAQYSWLIDGN from carcinoembryonic antigen (CEA; SEQ ID NO: 85); 3295672 I.DOC 56 (x)(xii)a sequence from Staphylococcal nuclease (Cone et al., J. Biol. Chem. 246, 3103-3110. 1971) selected from the group consisting of: ANASQTDNGVNRSGSEDPTV (SEQ ID NO: 86) and PETKHPKKGVEKYGPEASAF (SEQ ID NO: 87); 5 (xi)(xiii) a sequence of Hepatitis B virus Surface antigen (Kobayashi and Koike, Gene 30, 227-232, 1984) selected from the group consisting of: LVLLDYQGMLPVCPL (SEQ ID NO: 88) and TKPSDGNCTCIPIPS (SEQ ID NO: 89); (xii)(xiv) MQWNSTTFHQALL from Hepatitis B virus precursor Surface antigen 10 (SEQ ID NO: 90); (xiii)(xv) A sequence from Influenza virus nucleoprotein (Gregory et al., J. Gen. Virol. 82, 1397-1406, 2001) selected from the group consisting of: AAFEDLRVSSFIRGT (SEQ ID NO: 91) and SNENMETMDSSTLE (SEQ ID NO: 92); 15 (xiv)(xvi) A sequence from Influenza virus hemagglutinin selected from the group consisting of: HPLILDTCTIEGLIYGNPS (SEQ ID NO: 93), YQRIQIFPDT (SEQ ID NO: 94), and IQIFPDTIWNVSYSGTSK (SEQ ID NO: 95); (xv)(xvii) CKYSASGSGVRGDFGSLAPRVARCLPASFNTGAIKNKY from the FMDV envelope glycoprotein VP1 (SEQ ID NO: 96); 20 (xvi)(xviii) A sequence from the M. tuberculosis ESAT-6 protein selected from the group consisting of: EQQWNFAGIEAAA (SEQ ID NO: 97) and AAAWGGSGSEAYQGVQQKWDATA (SEQ ID NO: 98). (xvii)(xix) GGPTRTIGGSQAQTASGLVSMFSVGPSQK (SEQ ID NO: 99) from

HCV;

3295672 I.DOC 57 (xx) KFQDAYNAAGGH (SEQ ID NO: 100) from M. scrofulaceum alpha antigen; (xxi) KQAEDKVKASREAKKQVEKALEQLEDKVK (SEQ ID NO: 101) from the M protein of group A Streptococcus (i.e., peptide designated herein as "J14"); and (xxii) GWMDF (SEQ ID NO: 102) from gastrin (i.e., pentagastrin consisting of the C 5 terminal five amino acid residues of gastrin). It will be apparent from the preceding description that the polypeptide moiety of the subject lipopeptide is synthesized conveniently as a single amino acid chain, thereby requiring no post-synthesis modification to incorporate both epitopes. A polypeptide moiety which comprises a highly immunogenic B cell epitope of LHRH 10 (eg. SEQ ID NO: 2 or 3 or 4) linked either to a T-helper epitope of influenza virus hemagglutinin (eg. SEQ ID NO: 1) or a T-helper epitope of CDV-F (eg. SEQ ID NO: 20, 24, 26, or 44) is particularly preferred, such as, for example, a polypeptide comprising an amino acid sequence selected from the group consisting of: (i) GALNNRFQIKGVELKSEHWSYGLRPG (SEQ ID NO:5); 15 (ii) EHWSYGLRPGGALNNRFQIKGVELKS (SEQ ID NO: 6); (iii) GALNNRFQIKGVELKSKEHWSYGLRPG (SEQ ID NO: 7); (iv) EHWSYGLRPGKGALNNRFQIKGVELKS (SEQ ID NO: 8); (v) KLIPNASLIENCTKAELKHWSYGLRPG (SEQ ID NO: 9); (vi) AELGEYEKLLNSVLEPIKEHWSYGLRPG (SEQ ID NO: 10); 20 (vii) TAAQITAGIALHQSNLNKEHWSYGLRPG (SEQ ID NO: 11); (viii) PRYIATNGYLISNFDESKEHWSYGLRPG (SEQ ID NO: 12); 3295672 I.DOC 58 (ix) KLIPNASLIENCTKAELKGLRPG (SEQ ID NO: 13); (x) AELGEYEKLLNSVLEPIKGLRPG (SEQ ID NO: 14); (xi) TAAQITAGIALHQSNLNKGLRPG (SEQ ID NO: 15); (xii) PRYIATNGYLISNFDESKGLRPG (SEQ ID NO: 16); 5 (xiii) KLIPNASLIENCTKAELHWSYGLRPG (SEQ ID NO: 103); and (xiv) KLIPNASLIENCTKAELGLRPG (SEQ ID NO: 104). In a particularly preferred embodiment, the LHRH epitope (i.e. LHRH 1-10 as set forth in SEQ ID NO: 2; LHRH 2-10 as set forth in SEQ ID NO: 3; or LHR-H.6-10 as set forth in SEQ ID NO: 4) is positioned such that the C-terminal glycine residue is exposed or 10 not internal. Accordingly, the configuration set forth in any one of SEQ ID Nos: 5, 7, or 9-16 is particularly preferred. In one exemplified embodiment, LHRH 1-10 is conjugated to the T-helper epitope of influenza virus haemagglutinin (i.e., SEQ ID NO: 1) as described by the sequence set forth in SEQ ID NO: 5 or 7, and LHRH 2-10 or LHRH 6-10 is conjugated to a T-helper 15 epitope of CDV-F (i.e., SEQ ID NO: 24) as described by the sequence set forth in SEQ ID NO: 9, 13, 103 or 104. Other combinations are clearly possible and encompassed by the present invention. In an alternative embodiment, a polypeptide moiety which comprises a highly immunogenic B cell epitope of the M protein of Group A streptococcus (eg. the J14 20 peptide set forth in SEQ ID NO: 101) linked to a T-helper epitope of CDV-F (eg. SEQ ID NO: 24) or influenza virus haemagglutinin (e.g., SEQ ID NO: 1) is particularly preferred, such as, for example, a polypeptide comprising an amino acid sequence selected from the group consisting of: 3295672 I.DOC 59 (i) KLIPNASLIENCTKAELKQAEDKVKASREAKKQVEKALEQLEDKVK (SEQ ID NO: 105); (ii) KLIPNASLIENCTKAELKKQAEDKVKASREAKKQVEKALEQLEDKVK (SEQ ID NO: 106); 5 (iii) GALNNRFQIKGVELKSKQAEDKVKASREAKKQVEKALEQLEDKVK (SEQ ID NO: 107); and (iv) GALNNRFQIKGVELKSKKQAEDKVKASREAKKQVEKALEQLEDKVK (SEQ ID NO: 108). In a further alternative embodiment, a polypeptide moiety which comprises a highly 10 immunogenic B cell epitope of pentagastrin (eg. SEQ ID NO: 102) linked to a T-helper epitope of CDV-F (eg. SEQ ID NO: 24) or influenza virus haemagglutinin (e.g., SEQ ID NO: 1) is particularly preferred, such as, for example, a polypeptide comprising an amino acid sequence selected from the group consisting of: (i) KLIPNASLIENCTKAELGWMDF (SEQ ID NO: 109); 15 (ii) KLIPNASLIENCTKAELKGWMDF (SEQ ID NO: 1 10); (iii) GALNNRFQIKGVELKSGWMDF (SEQ ID NO: 111); and (iv) GALNNRFQIKGVELKSKGWMDF (SEQ ID NO: 112). The skilled artisan will readily be able to synthesize additional polypeptide moieties to those exemplified herein for use in the subject lipopeptides, by substituting the T-helper 20 epitope and/or the B cell epitope of any one of SEQ ID Nos: 5-16 or any one of SEQ ID Nos: 103-112 with another T-helper epitope or B cell epitope, such as, for example a T-helper epitope set forth in any one of SEQ ID Nos: 18-56, or a B cell epitope set forth in any one of SEQ ID Nos: 57-102. Moreover, the selection of appropriate T- 3295672 I.DOC 60 helper epitope and B cell combinations will be apparent to the skilled artisan from the disclosure provided herein, according to the target species and the antigen against which an immune response is sought. The amino acid sequences of the polypeptide moities described herein, including those 5 exemplified polypeptides set forth in SEQ ID Nos: 5-16 and SEQ ID Nos: 103-112, may be modified for particular purposes according to methods well known to those of skill in the art without adversely affecting their immune function. For example, particular peptide residues may be derivatized or chemically modified in order to enhance the immune response or to permit coupling of the peptide to other agents, 10 particularly lipids. It also is possible to change particular amino acids within the peptides without disturbing the overall structure or antigenicity of the peptide. Such changes are therefore termed "conservative" changes and tend to rely on the hydrophilicity or polarity of the residue. The size and/or charge of the side chains also are relevant factors in determining which substitutions are conservative. 15 The skilled artisan will readily be able to synthesize additional polypeptide moieties to those exemplified herein for use in the subject lipopeptides, by substituting the T-helper epitope and/or the B cell epitope of any one of SEQ ID Nos: 5-16 or any one of SEQ ID Nos: 103-112 with another T-helper epitope or B cell epitope, such as, for example a T-helper epitope set forth in any one of SEQ ID Nos: 18-56, or a B cell epitope set 20 forth in any one of SEQ ID Nos: 57-102. Moreover, the selection of appropriate T helper epitope and B cell combinations will be apparent to the skilled artisan from the disclosure provided herein, according to the target species and the antigen against which an immune response is sought. The CTL epitope is conveniently derived from the amino acid sequence of an 25 immunogenic protein, lipoprotein, or glycoprotein of a virus, prokaryotic or eukaryotic organism, including but not limited to a CTL epitope derived from a mammalian subject or a bacterium, fungus, protozoan, or parasite that infects said subject.

3295672 I.DOC 61 Mimotopes of the CTL epitopes are specifically included within the scope of the invention. The CTL epitope will be capable of eliciting a T cell response when administered to a mammal, preferably by activating CD8+ T cells specific for the epitope or antigen from 5 which the epitope was derived, and more preferably, by inducing cell mediated immunity against the pathogen or tumour cell from which the epitope is derived. Shorter CTL epitopes are preferred, to facilitate peptide synthesis. Preferably, the length of the CTL epitope will not exceed about 30 amino acids in length. More preferably, the CTL epitope sequence consists of about 25 amino acid residues or less, 10 and more preferably less than 20 amino acid residues, and even more preferably about 8-12 amino acid residues in length. Preferred CTL epitopes from parasites are those associated with leishmania, malaria, trypanosomiasis, babesiosis, or schistosomiasis, such as, for example a CTL epitope of an antigen of a parasite selected from the group consisting of: Plasmodiumfalciparum; 15 Circumsporozoa; Leishmania donovani; Toxoplasma gondii; Schistosoma mansoni; Schistosomajaponicum; Schistosoma hematobium; and Trypanosoma brucei. Particularly preferred CTL epitopes of P. falciparum are derived from an antigen selected from the group consisting of: circumsporozoite protein (CSP), sporozoite surface protein 2 (PfSSP2), liver stage antigen I (LSA 1), merozoite surface protein 1 20 (MSP 1), serine repeat antigen (SERA), and AMA-I antigen (Amante, et al. J. Immunol. 159, 5535-5544, 1997; .Chaba et al. Int. J. Immunopharm. 20, 259-273, 1998; Shi et al., Proc. Natl Acad. Sci (USA) 96, 1615-1620, 1999; Wang et al. Science 282, 476-479, 1998; and Zevering et al. Immunol. 94, 445-454, 1998). Particularly preferred CTL epitopes of L. donovani are derived from the Repetitive Peptide (Liew et 25 al., J. Exp. Med. 172, 1359 (1990)). Particularly preferred CTL epitopes of T gondii are derived from the P30 surface protein (Darcy et al., J. Immunol. 149, 3636 (1992)).

3295672 I.DOC 62 Particularly preferred CTL epitopes of S. mansoni are derived from the Sm-28GST antigen (Wolowxzuk et al., J. Immunol 146:1987 (1991)). Preferred virus-specific CTL epitopes are derived from Rotaviruses, Herpes viruses, Corona viruses, Picornaviruses (eg. Apthovirus), Respiratory Synctial virus, Influenza 5 Virus, Parainfluenza virus, Adenovirus, Pox viruses, Bovine herpes virus Type I, Bovine viral diarrhea virus, Bovine rotaviruses, Canine Distemper Virus (CDV), Foot and Mouth Disease Virus (FMDV), Measles Virus (MV), Human Immunodeficiency Viruses (HIV), Feline Immunodeficiency Viruses (FIV), Epstein-Barr virus (EBV), Human Cytomegalovirus (HCMV), or hepatitis viruses, and the like. 10 Particularly preferred CTL epitopes of HIV-1 are derived from the env, gag, or pol proteins. Particularly preferred CTL epitopes of influenza virus are derived from the nucleoprotein (Taylor et al., Immunogenetics 26, 267 (1989); Townsend et al., Nature 348, 674(1983)), matrix protein (Bednarek et al., J. Immunol. 147, 4047 (1991)) or polymerase protein (Jameson et al., J. Virol. 72, 8682-8689, 1998; and Gianfrani et al., 15 Human Immunol. 61, 438-452, 2000). Particularly preferred CTL epitopes of Lymphocytic choriomeningitis virus (LCMV) are derived from glycoprotein-1 antigen (Zinkernagel et al. Nature 248, 701-702, 1974). Particularly preferred CTL epitopes of cytomegalovirus are derived from an antigen selected from the group consisting of: of pp28, pp50, pp65, pp71, pp150, gB, gH, IE-1, IE-2, US2, US3, US6, US 11, and UL18 20 (eg. Diamond, USSN 6,074,645, June 13, 2000; Longmate et al., Immunogenet. 52, 165-173, 2000; Wills et al., J. Virol. 70, 7569-7579, 1996; Solache et al., J. Immunol. 163, 5512-5518, 1999; Diamond et al., Blood 90, 1751-1767, 1997; Kern et al., Nature Med. 4, 975-978, 1998; Weekes et al., J. Virol. 73, 2099-2108, 1999; Retiere et al., J. Virol. 74, 3948-3952, 2000; and Salquin et al., Eur. J. Immunol. 30, 2531-2539, 2000). 25 Particularly preferred CTL epitopes of Measles Virus are derived from the fusion glycoprotein (MV-F) and particularly from residues 438-446 thereof (Herberts et al. J. Gen Virol. 82, 2131-2142, 2001). Particularly preferred epitopes from Epstein-Barr virus (EBV) are derived from a latent nuclear antigen (EBNA) or latent membrane 3295672 I.DOC 63 protein (LMP) of EBV, such as, for example, EBNA 2A, EBNA 3A, EBNA 4A, or EBNA 14a from EBV type A; EBNA 2B, EBNA 3B, EBNA 4B, or EBNA 14b from EBV type B; LMP1; or LMP2 (International Patent Application No. PCT/AU95/00140 published Sep. 16, 1995; International Patent Application No. PCT/AU97/00328 5 published Nov. 24, 1997; and International Patent Application No. PCT/AU98/00531 published Jan. 10, 1998). Preferred bacteria-specific CTL epitopes are derived from Pasteurella, Actinobacillus, Haemophilus, Listeria monocytogenes, Mycobacterium tuberculosis, Staphylococcus, Neisseria gonorrhoeae, Helicobacter pylori, Streptococcus pneumoniae, Salmonella 10 enterica, E. coli, Shigella, and the like. Suitable bacterial CTL epitopes include, for example, those CTL epitopes derived from the Mycobacterium tuberculosis 65Kd protein (Lamb et al., EMBO J., 6, 1245 (1987)); M. tuberculosis ESAT-6 protein (Morten et al., Infect. Immun. 66, 717-723, 1998); Staphylococcus aureus nuclease protein (Finnegan et al., J. Exp. Med. 164, 897 15 (1986)); Escherichia coli heat stable enterotoxin (Cardenas et al., Infect. Immunity 61, 4629 (1993)); and Escherichia coli heat labile enterotoxin (Clements et al., Infect. Immunity 53, 685 (1986)). Preferred CTL epitopes from mammalian subjects are derived from and/or capable of generating T cell responses against a tumor CTL antigen. Tumor-specific CTL 20 epitopes are usually native or foreign CTL epitopes, the expression of which is correlated with the development, growth, presence or recurrence of a tumor. In as much as such CTL epitopes are useful in differentiating abnormal from normal tissue, they are useful as a target for therapeutic intervention. Such CTL epitopes are well known in the art. Indeed, several examples are well-characterized and are currently the 25 focus of great interest in the generation of tumor-specific therapies. Non-limiting examples of tumor CTL epitopes are derived from carcinoembryonic antigen (CEA), 3295672 I.DOC 64 prostate specific antigen (PSA), melanoma antigen (MAGE, BAGE, GAGE), and mucins, such as MUC-1. Preferred CTL epitopes for administering to a cancer patient are derived from a protein that induces cancer, such as, for example, an oncoprotein (e.g., p53, ras etc.). 5 In a particularly preferred embodiment, the CTL epitope will comprise or consist of an amino acid sequence selected from the group consisting of: (i) TYQRTRALV from the NP of PR8 virus (SEQ ID NO: 114); (ii) KPKDELDYENDIEKKICKMEKCS of P. falciparum CSP (SEQ ID NO: 126); (iii) DIEKKICKMEKCSSVFNVVNS from P. falciparum CSP (SEQ ID NO: 127); 10 (iv) KPIVQYDNF from P. falciparum LSAI (SEQ ID NO: 128); (v) GISYYEKVLAKYKDDLE from P. falciparum MSPI (SEQ ID NO: 129); (vi) EFTYMINFGRGQNYWEHPYQKS of P. falciparum AMA-I (SEQ ID NO: 130); (vii) DQPKQYEQHLTDYEKIKEG from P. falciparum AMA-I (SEQ ID NO: 131); 15 (viii) NMWQEVGKAM from HIV-1 env protein (SEQ ID NO: 132); (ix) APTKAKRRVV from HIV-1 env protein (SEQ ID NO: 133); (x) CTRPNNNTRK from HIV-1 env protein (SEQ ID NO: 134); (xi) TVYYGVPVWK from HIV-1 env protein (SEQ ID NO: 135); (xii) RPVVSTQLL from HIV-1 env protein (SEQ ID NO: 136); 20 (xiii) SLYNTVATLY from HIV-1 gag protein (SEQ ID NO: 137); 3295672 I.DOC 65 (xiv) ELRSLYNTVA from HIV-1 gag protein (SEQ ID NO: 138); (xv) KIRLRPGGKK from HIV-1 gag protein (SEQ ID NO: 139); (xvi) IRLRPGGKKK from HIV-1 gag protein (SEQ ID NO: 140); (xvii) RLRPGGKKK from HIV-1 gag protein (SEQ ID NO: 141); 5 (xviii) GPGHKARV LA from HIV-I gag protein (SEQ ID NO: 142); (xix) SPIETVPVKL from HIV-1 pol protein (SEQ ID NO: 143); (xx) ILKEPVHGVY from HIV-1 pol protein (SEQ ID NO: 144); (xxi) AIFQSSMTK from HIV-1 pol protein (SEQ ID NO: 145); (xxii) SPAIFQSSMT from HIV-1 pol protein (SEQ ID NO: 146); 10 (xxiii) QVRDQAEH LK from HIV-1 pol protein (SEQ ID NO: 147); (xxiv) GPKVKQWPLT from HIV-1 pol protein (SEQ ID NO: 148); (xxv) TYQRTRALV from influenza virus nucleoprotein (SEQ ID NO: 149); (xxvi) TYQRTRALVRTGMDP from influenza nucleoprotein (SEQ ID NO: 150); (xxvii) IASNENMDAMESSTL from influenza virus nucleoprotein (SEQ ID NO: 151); 15 (xxviii)KAVYNFATM from LCMV gpl (SEQ ID NO: 152); (xxix) QVKWRMTTL from EBV (SEQ ID NO: 153); (xxx) VFSDGRVAC from EBV (SEQ ID NO: 154); (xxxi) VPAPAGPIV from EBV (SEQ ID NO: 155); 3295672 I.DOC 66 (xxxii) TYSAGIVQI from EBV (SEQ ID NO: 156); (xxxiii)LLDFVRFMGV from EBV (SEQ ID NO: 157); (xxxiv)QNGALAINTF from EBV (SEQ ID NO: 158); (xxxv) VSSDGRVAC from EBV (SEQ ID NO: 159); 5 (xxxvi)VSSEGRVAC from EBV (SEQ ID NO: 160); (xxxvii) VSSDGRVPC from EBV (SEQ ID NO: 161); (xxxviii) VSSDGLVAC from EBV (SEQ ID NO: 162); (xxxix)VSSDGQVAC from EBV (SEQ ID NO: 163); (xl) VSSDGRVVC from EBV (SEQ ID NO: 164); 10 (xli) VPAPPVGPIV from EBV (SEQ ID NO: 165); (xlii) VEITPYEPTG from EBV (SEQ ID NO: 166); (xliii) VEITPYEPTW from EBV (SEQ ID NO: 167); (xliv) VELTPYKPTW from EBV (SEQ ID NO: 168); (xlv) RRIYDLIKL from EBV (SEQ ID NO: 169); 15 (xlvi) RKIYDLIEL from EBV (SEQ ID NO: 170); (xlvii) PY LFWLAGI. from EBV (SEQ ID NO: 171); (xlviii) TSLYNLRRGTALA from EBV (SEQ ID NO: 172); (xlix) DTPLIPLTIF from EBV (SEQ ID NO: 173); 3295672 I.DOC 67 (1) TVFYNIPPMPL from EBV (SEQ ID NO: 174); (li) VEITPYKPTW from EBV (SEQ ID NO: 175); (lii) VSFIEFVGW from EBV (SEQ ID NO: 176); (liii) FRKAQIQGL from EBV (SEQ ID NO: 177); 5 (liv) FLRGRAYGL from EBV (SEQ ID NO: 178); (lv) QAKWRLQTL from EBV (SEQ ID NO: 179); (lvi) SVRDRLARL from EBV (SEQ ID NO: 180); (lvii) YPLHEQHGM from EBV (SEQ ID NO: 181); (lviii) HLAAQGMAY from EBV (SEQ ID NO: 182); 10 (lix) RPPIFIRRL from EBV (SEQ ID NO: 183); (lx) RLRAEAGVK from EBV (SEQ ID NO: 184); (lxi) IVTDFSVIK from EBV (SEQ ID NO: 185); (lxii) AVFDRKSDAK from EBV (SEQ ID NO: 186); (lxiii) NPTQAPVIQLVHAVY from EBV (SEQ ID NO: 187); 15 (lxiv) LPGPQVTAVLLHEES from EBV (SEQ ID NO: 188); (lxv) DEPASTEPVHDQLL from EBV (SEQ ID NO: 189); (lxvi) RYSIFFDY from EBV (SEQ ID NO: 190); (lxvii) AV LLHEESM from EBV (SEQ ID NO: 191); 3295672 I.DOC 68 (lxviii) RRARSLSAERY from EBV (SEQ ID NO: 192); (lxix) EENLLDFVRF from EBV (SEQ ID NO: 193); (lxx) KEHVIQNAF from EBV (SEQ ID NO: 194); (lxxi) RRIYDLIEL from EBV (SEQ ID NO: 195); 5 (lxxii) QPRAP IRPI from EBV (SEQ ID NO: 196); (lxxiii) EGGVGWRHW from EBV (SEQ ID NO: 197); (lxxiv) CLGGLLTMV from EBV (SEQ ID NO: 198); (lxxv) RRRWRRLTV from EBV (SEQ ID NO: 199); (lxxvi) RAKFKQLL from EBV (SEQ ID NO: 200); 10 (lxxvii)RKCCRAKFKQLLQHYR. from EBV (SEQ ID NO: 201); (lxxviii) YLLEMLWRL from EBV (SEQ ID NO: 202); (lxxix) YFLEILWGL from EBV (SEQ ID NO: 203); (lxxx) YLLEILWRL from EBV (SEQ ID NO: 204); (lxxxi) YLQQNWWTL from EBV (SEQ ID NO: 205); 15 (lxxxii)LLLALLFWL from EBV (SEQ ID NO: 206); (lxxxiii) LLVDLLWLL from EBV (SEQ ID NO: 207); (lxxxiv) LLLIALWNL from EBV (SEQ ID NO: 208); (lxxxv)WLLLFLAIL from EBV (SEQ ID NO: 209); 3295672 I.DOC 69 (lxxxvi) TLLVDLLWL from EBV (SEQ ID NO: 210); (lxxxvii) LLWLLLFLA from EBV (SEQ ID NO: 211); (lxxxviii) ILLIIALYL from EBV (SEQ ID NO: 212); (lxxxix) VLFIFGCLL from EBV (SEQ ID NO: 213); 5 (xc) RLGATIWQL from EBV (SEQ ID NO: 214); (xci) ILYFIAFAL from EBV (SEQ ID NO: 215); (xcii) SLVIVTTFV from EBV (SEQ ID NO: 216); (xciii) LMIIPLINV from EBV (SEQ ID NO: 217); (xciv) TLFIGSHVV from EBV (SEQ ID NO: 218); 10 (xcv) LIPETVPYI from EBV (SEQ ID NO: 219); (xcvi) VLQWASLAV from EBV (SEQ ID NO: 220); (xcvii) QLTPHTKAV from EBV (SEQ ID NO: 221); (xcviii)SVLGPISGHVLK from HCMV pp65 (SEQ ID NO: 222); (xcix) FTSQYRIQGKL from HCMV pp65(SEQ ID NO: 223); 15 (c) FVFPTKDVALR from HCMV pp65 (SEQ ID NO: 224); (ci) FPTKDVAL from HCMV pp65 (SEQ ID NO: 225); (cii) NLVPMVATV from HCMV pp65 (SEQ ID NO: 226); (ciii) MLNIPSINV from HCMV pp65 (SEQ ID NO: 227); 3295672 I.DOC 70 (civ) RIFAELEGV from HCMV pp65 (SEQ ID NO: 228); (cv) TPRVTGGGGAM from HCMV pp65 (SEQ ID NO: 229); (cvi) RPHERNGFTVL from HCMV pp65 (SEQ ID NO: 230); (cvii) RLLQTGIHV from HCMV pp 6 5 (SEQ ID NO: 231); 5 (cviii) VIGDQYVKV from HCMV pp65 (SEQ ID NO: 232); (cix) ALFFFDIDL from HCMV pp65 (SEQ ID NO: 233); (cx) YSEHPTFTSQY from HCMV pp65 (SEQ ID NO: 234); (cxi) VLCPKNMII from HCMV pp65 (SEQ ID NO: 235); (cxii) DIYRIFAEL from HCMV pp65 (SEQ ID NO: 236); 10 (cxiii) ILARNLVPMV from HCMV pp65 (SEQ ID NO: 237); (cxiv) EFFWDANDIY from HCMV pp65 (SEQ ID NO: 238); (cxv) IPSINVHHY) from HCMV pp65 (SEQ ID NO: 239); (cxvi) YILEETSVM from HCMV IE-1 (SEQ ID NO: 240); (cxvii) CVETMCNEY from HCMV IE-1 (SEQ ID NO: 241); 15 (cxviii)RRIEEICMK from HCMV IE-1 (SEQ ID NO: 242); (cxix) TTVYPPSSTAK from HCMV ppl50 (SEQ ID NO: 243); (cxx) RRYPDAVYL from Measles Virus Fusion glycoprotein (SEQ ID NO: 244); (cxxi) GYKDGNEYI from Listeria monocytogenes (SEQ ID NO: 245); 3295672 I.DOC 71 (cxxii) SIINFEKL from ovalbumin (SEQ ID NO: 246); and (cxxiii)DLMGYIPLV from the core protein of hepatitis C virus (SEQ ID NO: 249). It will be apparent from the preceding description that the polypeptide moiety of the subject lipopeptide is synthesized conveniently as a single amino acid chain, thereby 5 requiring no post-synthesis modification to incorporate both epitopes. As exemplified herein, a polypeptide moiety comprising an amino acid sequence selected from the group consisting of the following is preferred: (i) ALNNRFQIKGVELKSTYQRTRALV (SEQ ID NO: 115); (ii) ALNNRFQIKGVELKSKTYQRTRALV (SEQ ID NO: 116); 10 (iii) KLIPNASLIENCTKAELKTYQRTRALV (SEQ ID NO: 117); (iv) KLIPNASLIENCTKAELKNLVPMVATV (SEQ ID NO: 118); (v) AELGEYEKLLNSVLEPIKNLVPMVATV (SEQ ID NO: 119); (vi) TAAQITAGIALHQSNLNKNLVPMVATV (SEQ ID NO: 120); (vii) PRYIATNGYLISNFDESKNLVPMVATV (SEQ ID NO: 121); 15 (viii) KLIPNASLIENCTKAELKYLLEMLWRL (SEQ ID NO: 122); (ix) AELGEYEKLLNSVLEPIKYLLEMLWRL (SEQ ID NO: 123); (x) TAAQITAGIALHQSNLNKYLLEMLWRL (SEQ ID NO: 124); (xi) PRYIATNGYLISNFDESKYLLEMLWRL (SEQ ID NO: 125); (xii) KLIPNASLIENCTKAELKSIINFEKL (SEQ ID NO: 247); 20 (xiii) KLIPNASLIENCTKAELKGYKDGNEYI (SEQ ID NO: 248) and 3295672 I.DOC 72 (xiv) KLIPNASLIENCTKAELKDLMGYIPLV (SEQ ID NO: 250). For the purposes of nomenclature, SEQ ID Nos: 115-116 relate to synthetic peptides comprising a T-helper epitope from the light chain of influenza virus hemagglutinin (i.e. SEQ ID NO: 18) and an immunodominant H-2d-restricted CTL epitope from the 5 nucleoprotein of influenza virus strain PR8 (i.e. SEQ ID NO: 114) wherein the internal lysine residue that provides a lipid attachment site at its epsilon-amino group is indicated in bold type. In SEQ ID No: 116, an additional internal lysine residue has been engineered between the T-helper and CTL epitope (K16 in SEQ ID NO: 116). SEQ ID No: 117 relates to a synthetic peptide comprising a T-helper epitope from 10 canine distemper virus (CDV-F; SEQ ID NO: 24) that is active in dogs, mice, and humans and an immunodominant H-2d-restricted CTL epitope from the nucleoprotein of influenza virus strain PR8 (i.e. SEQ ID NO: 114) wherein the internal lysine residue that provides a lipid attachment site at its epsilon-amino group is indicated in bold type. In this peptide, an additional internal lysine residue has been engineered between the T 15 helper and CTL epitope (K 18 in SEQ ID NO: 117). SEQ ID No: 118 relates to a synthetic peptide comprising a T-helper epitope from canine distemper virus (CDV-F; SEQ ID NO: 24) that is active in dogs, mice, and humans and an immunodominant HLA A2-restricted CTL epitope from the immunodominant pp65 antigen of the cytomegalovirus of humans (i.e. HCMV pp65 20 antigen) (i.e. SEQ ID NO: 226) wherein the internal lysine residue that provides a lipid attachment site at its epsilon-amino group is indicated in bold type. In this peptide, an additional internal lysine residue has been engineered between the T-helper and CTL epitope (K18 in SEQ ID NO: 118). SEQ ID No: 119 relates to a synthetic peptide comprising a T-helper epitope from 25 canine distemper virus (CDV-F; SEQ ID NO: 26) that is active in dogs, mice, and humans and an immunodominant HLA A2-restricted CTL epitope from HCMV pp65 3295672 I.DOC 73 antigen (i.e. SEQ ID NO: 226) wherein the internal lysine residue that provides a lipid attachment site at its epsilon-amino group is indicated in bold type. In this peptide, an additional internal lysine residue has been engineered between the T-helper and CTL epitope (K18 in SEQ ID NO: 119). 5 SEQ ID No: 120 relates to a synthetic peptide comprising a T-helper epitope from canine distemper virus (CDV-F; SEQ ID NO: 20) that is active in dogs, mice, and humans and an immunodominant HLA A2-restricted CTL epitope from HCMV pp65 antigen (i.e. SEQ ID NO: 226) wherein the internal lysine residue that provides a lipid attachment site at its epsilon-amino group is indicated in bold type. In this peptide, an 10 additional internal lysine residue has been engineered between the T-helper and CTL epitope (K18 in SEQ ID NO: 120). SEQ ID No: 121 relates to a synthetic peptide comprising a T-helper epitope from canine distemper virus (CDV-F; SEQ ID NO: 44) that is active in dogs, mice, and humans and an immunodominant HLA A2-restricted CTL epitope from HCMV pp65 15 antigen (i.e. SEQ ID NO: 226) wherein the internal lysine residue that provides a lipid attachment site at its epsilon-amino group is indicated in bold type. In this peptide, an additional internal lysine residue has been engineered between the T-helper and CTL epitope (K18 in SEQ ID NO: 121). SEQ ID No: 122 relates to a synthetic peptide comprising a T-helper epitope from 20 canine distemper virus (CDV-F; SEQ ID NO: 24) that is active in dogs, mice, and humans and an immunodominant HLA A2-restricted CTL epitope from Epstein-Barr virus LMPI antigen (i.e. EBV LMPI; SEQ ID NO: 202) wherein the internal lysine residue that provides a lipid attachment site at its epsilon-amino group is indicated in bold type. In this peptide, an additional internal lysine residue has been engineered 25 between the T-helper and CTL epitope (K18 in SEQ ID NO: 122).

3295672_.DOC 74 SEQ ID No: 123 relates to a synthetic peptide comprising a T-helper epitope from canine distemper virus (CDV-F; SEQ ID NO: 26) that is active in dogs, mice, and humans and an immunodominant HLA A2-restricted CTL epitope from EBV LMPI (SEQ ID NO: 202) wherein the internal lysine residue that provides a lipid attachment 5 site at its epsilon-amino group is indicated in bold type. In this peptide, an additional internal lysine residue has been engineered between the T-helper and CTL epitope (K18 in SEQ ID NO: 123). SEQ ID No: 124 relates to a synthetic peptide comprising a T-helper epitope from canine distemper virus (CDV-F; SEQ ID NO: 20) that is active in dogs, mice, and 10 humans and an immunodominant HLA A2-restricted CTL epitope from EBV LMPI (SEQ ID NO: 202) wherein the internal lysine residue that provides a lipid attachment site at its epsilon-amino group is indicated in bold type. In this peptide, an additional internal lysine residue has been engineered between the T-helper and CTL epitope (K18 in SEQ ID NO: 124). 15 SEQ ID No: 125 relates to a synthetic peptide comprising a T-helper epitope from canine distemper virus (CDV-F; SEQ ID NO: 44) that is active in dogs, mice, and humans and an immunodominant HLA A2-restricted CTL epitope from EBV LMPI (SEQ ID NO: 202) wherein the internal lysine residue that provides a lipid attachment site at its epsilon-amino group is indicated in bold type. In this peptide, an additional 20 internal lysine residue has been engineered between the T-helper and CTL epitope (K18 in SEQ ID NO: 125). SEQ ID No: 247 relates to a synthetic peptide comprising a T-helper epitope from canine distemper virus (CDV-F; SEQ ID NO: 24) that is active in dogs, mice, and humans and an immunodominant CTL epitope from ovalbumin(i.e. SEQ ID NO: 246) 25 wherein the internal lysine residues that provide possible lipid attachment sites at its epsilon-amino group are indicated in bold type. Preferably, the lipid is attached via 3295672 I.DOC 75 K18 in SEQ ID NO: 247, which is an additional internal lysine residue that has been engineered between the T-helper and CTL epitope. SEQ ID No: 248 relates to a synthetic peptide comprising a T-helper epitope from canine distemper virus (CDV-F; SEQ ID NO: 24) that is active in dogs, mice, and 5 humans and an immunodominant CTL epitope from a Listeria monocytogenes antigen (i.e. SEQ ID NO: 245) wherein the internal lysine residues that provide possible lipid attachment sites at its epsilon-amino group are indicated in bold type. Preferably, the lipid is attached via KI 8 in SEQ ID NO: 248 which is an additional internal lysine residue that has been engineered between the T-helper and CTL epitope. 10 SEQ ID No: 250 relates to a synthetic peptide comprising a T-helper epitope from canine distemper virus (CDV-F; SEQ ID NO: 24) that is active in dogs, mice, and humans and an immunodominant CTL epitope from the core protein of hepatitis C virus (SEQ ID NO: 249) wherein the internal lysine residues that provide possible lipid attachment sites at its epsilon-amino group are indicated in bold type. Preferably, the 15 lipid is attached via KI 8 in SEQ ID NO: 250. The skilled artisan will readily be able to synthesize additional polypeptide moieties to those exemplified herein for use in the subject lipopeptides, by substituting the T-helper epitope and/or the CTL epitope of anyone of SEQ ID Nos: 115-125, 247, 248 or 250 with another T-helper epitope or CTL epitope, such as, for example a T-helper epitope 20 set forth in any one of SEQ ID Nos: 1 and 19-56, or a CTL epitope set forth in any one of SEQ ID Nos: 126-246 or 249. Moreover, the selection of appropriate T-helper epitope and CTL combinations will be apparent to the skilled artisan from the disclosure provided herein, according to the target species and the CTL epitope against which an immune response is sought. 25 The amino acid sequences of the polypeptide moities described herein, including those exemplified polypeptides set forth in SEQ ID Nos: 115-125, 247, 248 and 250 may be 3295672 I.DOC 76 modified for particular purposes according to methods well known to those of skill in the art without adversely affecting their immune function. For example, particular peptide residues may be derivatized or chemically modified in order to enhance the immune response or to permit coupling of the peptide to other agents, particularly 5 lipids. It also is possible to change particular amino acids within the peptides without disturbing the overall structure or CTL immunogenicity of the peptide. Such changes are therefore termed "conservative" changes and tend to rely on the hydrophilicity or polarity of the residue. The size and/or charge of the side chains also are relevant factors in determining which substitutions are conservative. 10 It is well understood by the skilled artisan that, inherent in the definition of a biologically functional equivalent protein or peptide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalent peptides are thus defined herein as those peptides in 15 which specific amino acids may be substituted. Particular embodiments encompass variants that have one, two, three, four, five or more variations in the amino acid sequence of the peptide. Of course, a plurality of distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention. Those skilled in the art are well aware that the following substitutions are permissible 20 conservative substitutions (i) substitutions involving arginine, lysine and histidine; (ii) substitutions involving alanine, glycine and serine; and (iii) substitutions involving phenylalanine, tryptophan and tyrosine. Peptides incorporating such conservative substitutions are defined herein as biologically functional equivalents. The importance of the hydropathic amino acid index in conferring interactive biological 25 function on a protein is generally understood in the art (Kyte & Doolittle, J. Mol. Biol. 157, 105-132, 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar 3295672 I.DOC 77 biological activity. The hydropathic index of amino acids also may be considered in determining a conservative substitution that produces a functionally equivalent molecule. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, as follows: isoleucine (+4.5); valine (+4.2); 5 leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate ( 3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices 10 are within .+/- 0.2 is preferred. More preferably, the substitution will involve amino acids having hydropathic indices within +/- 0.1, and more preferably within about +/ 0.05. It is also understood in the art that the substitution of like amino acids is made effectively on the basis of hydrophilicity, particularly where the biological functional 15 equivalent protein or peptide thereby created is intended for use in immunological embodiments, as in the present case (e.g. US Patent No. 4,554,101). As detailed in US Patent No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +/- 0.1); glutamate (+3.0 +/- 0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine ( 20 0.4); proline (-0.5 +/- 0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine ( 1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine ( 2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, it is preferred to substitute amino acids having hydrophilicity values within about +/- 0.2 of each other, more preferably within about +/- 0.1, and even more preferably within 25 about +/- 0.05. Having identified peptides suitable for use as immunogens, it also is contemplated that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure. Such compounds, which may be termed peptidomimetics, may be 3295672_.DOC 78 used in the same manner as the peptides of the invention and hence are also functional equivalents. The generation of a structural functional equivalent may be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the 5 present invention. Another method for determining the "equivalence" of modified peptides involves a functional approach. For example, a given peptide is used to generate monoclonal or polyclonal antibodies. These antibodies can then, in turn, be used to screen libraries of degenerate peptides that include thousands or hundreds of thousands of other peptides, 10 thereby identifying structures that are, at least to a certain extent, immunologically equivalent. Of course, these structures may bear some primary sequence homology to the peptide used to generate the antibodies, but they also may be quite different. In another example, a suitable variant peptide will comprise an amino acid sequence that interacts at a significant level with a MHC Class I allele as determined using a 15 predictive algorithm for determining MHC Class I-binding epitopes, such as, for example, the SYFPEITHI algorithm of the University of Tuebingen, Germany, or the algorithm of the HLA Peptide Binding Predictions program of the BioInformatics and Molecular Analysis Section (BIMAS) of the National Institutes of Health of the Government of the United States of America. Such variant sequences will also bind to 20 and/or stabilize an MHC Class I molecule on the surface of an APC (eg in the PBMC fraction or buffy coat fraction of serum) and/or will induce a memory CTL response or elicit IFN-y production and/or will stimulate CTL activity in a standard cytotoxicity assay. The determination of such functionalities is readily achievable by those skilled in the art. 25 The polypeptide moiety of the lipopeptide is readily synthesized using standard techniques, such as the Merrifield method of synthesis (Merrifield, J Am Chem Soc, 85,:2149-2154, 1963) and the myriad of available improvements on that technology (see e.g., Synthetic Peptides: A User's Guide, Grant, ed. (1992) W.H. Freeman & Co., 3295672_.DOC 79 New York, pp. 382; Jones (1994) The Chemical Synthesis of Peptides, Clarendon Press, Oxford, pp. 230.); Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York; WUnsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Methoden der 5 Organischen Chemie (Mler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474). 10 The lipid moiety may comprise any C 2 to C 3 0 saturated, monounsaturated, or polyunsaturated linear or branched fatty acyl group, and preferably a fatty acid group selected from the group consisting of: palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl and decanoyl. Lipoamino acids are particularly preferred lipid moieties within the present context. As used herein, the term "lipoamino acid" refers to a molecule 15 comprising one or two or three or more lipids covalently attached to an amino acid residue, such as, for example, cysteine or serine, lysine or an analog thereof. In a particularly preferred embodiment, the lipoamino acid comprises cysteine and optionally, one or two or more serine residues. The structure of the lipid moiety is not essential to activity of the resulting lipopeptide 20 and, as exemplified herein, palmitic acid and/or cholesterol and/or PamCys and/or Pam 2 Cys and/or Pam 3 Cys can be used. The present invention clearly contemplates a range of other lipid moieties for use in the lipopeptides, such as, for example, lauric acid, stearic acid or octanoic acid, without loss of immunogenicity. Accordingly, the present invention is not to be limited by the structure of the lipid moiety, unless 25 specified otherwise, or the context requires otherwise. Similarly, the present invention is not to be limited by a requirement for a single lipid moiety unless specified otherwise or the context requires otherwise. The additon of 3295672 I.DOC 80 multiple lipid moieties to the peptide moiety, such as, for example, to a position within the T-helper epitope, and to a position between the T-helper epitope and the B-cell or CTL epitope, is clearly contemplated. The lipid moiety is preferably a compound having a structure of General Formula 5 (VII): R1 N - C! COOH

(CH

2 )m X

(CH

2 )n

R

2 -CH

R

3

-CH

2 wherein: (i) X is selected from the group consisting of sulfur, oxygen, disulfide (-S-S-), and methylene (-CH 2 -), and amino (-NH-); 10 (ii) m is an integer being 1 or 2; (iii) n is an integer from 0 to 5; (iv) R, is selected from the group consisting of hydrogen, carbonyl (-CO-), and R' CO- wherein R' is selected from the group consisting of alkyl having 7 to 25 carbon atoms, alkenyl having 7 to 25 carbon atoms, and alkynyl having 7 to 25 carbon atoms, 15 wherein said alkyl, alkenyl or alkynyl group is optionally substituted by a hydroxyl, amino, oxo, acyl, or cycloalkyl group; 3295672_.DOC 81 (v) R 2 is selected from the group consisting of R'-CO-O-, R'-O-, R'-O-CO-, R'-NH-CO-, and R'-CO-NH-, wherein R' is selected from the group consisting of alkyl having 7 to 25 carbon atoms, alkenyl having 7 to 25 carbon atoms, and alkynyl having 7 to 25 carbon atoms, wherein said alkyl, alkenyl or alkynyl group is optionally 5 substituted by a hydroxyl, amino, oxo, acyl, or cycloalkyl group; and (vi) R 3 is selected from the group consisting of R'-CO-O-, R'-O-, R'-O-CO-, R'-NH-CO-, and R'-CO-NH-, wherein R' is selected from the group consisting of alkyl having 7 to 25 carbon atoms, alkenyl having 7 to 25 carbon atoms, and alkynyl having 7 to 25 carbon atoms, wherein said alkyl, alkenyl or alkynyl group is optionally 10 substituted by a hydroxyl, amino, oxo, acyl, or cycloalkyl group and wherein each of RI, R 2 and R 3 are the same or different. Depending upon the substituent, the lipid moiety of General Formula VII may be a chiral molecule, wherein the carbon atoms directly or indirectly covalently bound to integers Ri and R 2 are asymmetric dextrorotatory or levorotatory (i.e. an R or S) 15 configuration. Preferably, X is sulfur; m and n are both 1; R, is selected from the group consisting of hydrogen, and R'-CO-, wherein R' is an alkyl group having 7 to 25 carbon atoms; and

R

2 and R 3 are selected from the group consisting of R'-CO-O-, R'-O-, R'-O-CO-, R' NH-CO-, and R'-CO-NH-, wherein R' is an alkyl group having 7 to 25 carbon atoms. 20 Preferably, R' is selected from the group consisting of: palmitoyl, myristoyl, stearoyl and decanoyl. More preferably, R' is selected from the group consisting of: palmitoyl, stearoyl, lauroyl, and octanoyl, and decanoyl. Most preferably, R' is palmitoyl. Each integer R' in said lipid moiety may be the same or different.

3295672_.DOC 82 In a particularly preferred embodiment, X is sulfur; m and n are both 1; R, is hydrogen or R'-CO- wherein R' is palmitoyl; and R 2 and R 3 are each R'-CO-O-wherein R' is palmitoyl. These particularly preferred compounds are shown by Formula (I) and Formula (II) supra. 5 The lipid moiety can also have the following General Formula (VIII): H R4-HN-C-COOH

R

5 wherein: (i) R 4 is selected from the group consisting of: (i) an alpha-acyl-fatty acid residue consisting of between about 7 and about 25 carbon atoms; (ii) an alpha-alkyl-beta 10 hydroxy-fatty acid residue; (iii) a beta-hydroxy ester of an alpha-alkyl-beta-hydroxy fatty acid residue wherein the ester group is preferably a straight chain or branched chain comprising more than 8 carbon atoms; and (iv) a lipoamino acid residue; and (ii) R 5 is hydrogen or the side chain of an amino acid residue. Preferably, R 4 consists of between about 10 and about 20 carbon atoms, and more 15 preferably between about 14 and about 18 carbon atoms. Optionally, wherein R 4 is a lipoamino acid residue, the side-chain of the integers R 4 and R 5 can form a covalent linkage. For example, wherein R 4 comprises an amino acid selected from the group consisting of lysine, ornithine, glutamic acid, aspartic acid, a derivative of lysine, a derivative of ornithine, a derivative of glutamic acid, and a 20 derivative of aspartic acid, then the side chain of that amino acid or derivative is covalently attached, by virtue of an amide or ester linkage, to R 5

.

3295672_.DOC 83 Preferably, the structure set forth in General Formula VIII is a lipid moiety selected from the group consisting of: N,N'-diacyllysine; N,N'-diacylornithine; di(monoalkyl)amide or ester of glutamic acid; di(monoalkyl)amide or ester of aspartic acid; a N,O-diacyl derivative of serine, homoserine, or threonine; and a N,S-diacyl 5 derivative of cysteine or homocysteine. Amphipathic molecules, particularly those having a hydrophobicity not exceeding the hydrophobicity of Pam3Cys (Formula (I)) are also preferred. The lipid moieties of Formula (I), Formula (II), Formula (VI) or Formula (VIII) are further modified during synthesis or post-synthetically, by the addition of one or more 10 spacer molecules, preferably a spacer that comprises carbon, and more preferably one or more amino acid residues. These are conveniently added to the lipid structure via the terminal carboxy group in a conventional condensation, addition, substitution, or oxidation reaction. The effect of such spacer molecules is to separate the lipid moiety from the polypeptide moiety and increase immunogenicity of the lipopeptide product. 15 Arginine or serine dimers, trimers, tetramers, etc, or alternatively, 6-aminohexanoic acid, are particularly preferred for this purpose. Preferably, such spacers include a terminal protected amino acid residue to facilitate the later conjugation of the modified lipoamino acid to the polypeptide. Exemplary modified lipoamino acids produced according to this embodiment are 20 presented as Formulae (III) and (IV), which are readily derived from Formulae (I) and (II), respectively by the addition of a serine homodimer. As exemplified herein, Pam3Cys of Formula (I), or Pam 2 Cys of Formula (II) is conveniently synthesized as the lipoamino acids Pam 3 Cys-Ser-Ser of Formula (III), or Pam 2 Cys-Ser-Ser of Formula (IV) for this purpose.

3295672_.DOC 84 Formula (III): __H H H H

H

3 C - (CH 2

)

1 4 -CO-NH- C-CO-NH-C-CO-N-C---COOH

CH

2 CH 2

CH

2 S OH OH CH2

H

3

C-(CH

2

)

1 4 -CO-- CH

H

3

C-(CH

2

)

1 4

-OC-O-CH

2 5 Formula (IV): H H H H H-NH-C-CO-NH-C---CO-N-C-COOH

CH

2

CH

2

CH

2 S OH OH

CH

2

H

3

C-(CH

2

)

1 4 -CO-O-CH

H

3

C-(CH

2

)

1 4

-OC-O-CH

2 3295672 I.DOC 85 As an alternative to the addition of a spacer to the lipid moiety, the spacer may be added to the epsilon amino group of the internal lysine residue or to the terminal side chain group of a lysine analog in the polypeptide moiety, either as a short peptide, such as, for example an arginine or serine homodimer, homotrimer, homotetramer, etc, or 5 alternatively, by the sequential addition of amino acid residues, thereby producing a branched polypeptide chain. This approach takes advantage of the modified nature of the terminal side-chain group on the internal lysine or lysine analog to achieve specificity in the addition of the spacer. Naturally, to avoid sequential spacer addition, the terminal amino acid residue of the spacer should preferably be protected, such that 10 de-protection can facilitate conjugation.of the lipid moiety to the branched polypeptide. Alternatively, the spacer may be added to a non-modified epsilon amino group of the polypeptide by conventional nucleophilic substitution reaction. However, it is preferred to follow this approach if the polypeptide has an amino acid sequence comprising a single internal lysine residue and a blocked N-terminus. 15 The lipid moiety is prepared by conventional synthetic means, such as, for example, the methods described in US Patent Nos. 5,700,910 and 6,024,964, or alternatively, the method described by Wiesmuller et al., Hoppe Seylers Zur Physiol. Chem. 364, 593 (1983), Zeng et al., J. Pept. Sci 2, 66 (1996), Jones et al., Xenobiotica 5, 155 (1975), or Metzger et al., Int. J. Pept.Protein Res. 38, 545 (1991). Those skilled in the art will be 20 readily able to modify such methods to achieve the synthesis of a desired lipid for use conjugation to a polypeptide. Combinations of different lipids are also contemplated for use in the lipopeptides of the invention. For example, one or two myristoyl-containing lipids or lipoamino acids are attached via internal lysine or lysine analog residues to the polypeptide moiety, 25 optionally separated from the polypeptide by a spacer, with one or two palmitoyl containing lipid or lipoamino acid molecules attached to carboxy terminal lysine amino acid residues. Other combinations are not excluded.

3295672 I.DOC 86 The lipopeptides of the invention are readily modified for diagnostic purposes. For example, it is modified by addition of a natural or synthetic hapten, an antibiotic, hormone, steroid, nucleoside, nucleotide, nucleic acid, an enzyme, enzyme substrate, an enzyme inhibitor, biotin, avidin, polyethylene glycol, a peptidic polypeptide moiety 5 (e.g. tuftsin, polylysine), a fluorescence marker (e.g. FITC, RITC, dansyl, luminol or coumarin), a bioluminescence marker, a spin label, an alkaloid, biogenic amine, vitamin, toxin (e.g. digoxin, phalloidin, amanitin, tetrodotoxin), or a complex-forming agent. As exemplified herein, highly immunogenic and soluble lipopeptides are provided 10 comprising Pam 3 Cys of Formula (I), or Pam 2 Cys of Formula (II) or Ste 2 Cys or Lau 2 Cys or Oct 2 Cys conjugated via the epsilon amino group of an internal lysine residue of a polypeptide that comprises: (i) the amino acid sequence of a CD4+ T helper epitope derived from the light chain of influenza virus hemagglutinin (Jackson et al. Virol. 198, 613-623, 1994; i.e. amino acid sequence GALNNRFQIKGVELKS; SEQ 15 ID NO: 1) or a peptide derived from the CDV-F protein (SEQ ID NO: 24); (ii) a B-cell epitope-containing peptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence of luteinising hormone-releasing hormone (LHRH; Fraser et al., J. Endocrinol. 63, 399 (1974); Fraser and Baker, J. Endocrinol. 77, 85 (1978); i.e. "LHRH 1-10", amino acid sequence EHWSYGLRPG; SEQ ID NO: 20 2; "LHRH 2-10", amino acid sequence HWSYGLRPG; SEQ ID NO: 3; or "LHRH 6 10", amino acid sequence GLRPG; SEQ ID NO: 4), Group A Streptococcus (GAS) M protein (i.e., SEQ ID NO: 101), and pentagastrin (i.e., SEQ ID NO: 102) or a CTL epitope (i.e., SEQ ID NO: 126-246 and 250); (iii) a lysine residue positioned between said CD4+ T-helper epitope and said B-cell epitope or CTL epitope; and optionally (iv) 25 a lysine residue positioned within said CD4+ T-helper epitope.

3295672_.DOC 87 Preparation of Lipopeptides The invention provides a method of producing a lipopeptide comprising: (i) producing a polypeptide comprising an amino acid sequence that comprises: (a) the amino acid sequence of a T helper cell (Th) epitope and the amino acid 5 sequence of a B cell epitope or a CTL epitope, wherein said amino acid sequences are different; and (b) one or more internal lysine residues or internal lysine analog residues; and (iii) covalently attaching each of said one or more lipid moieties directly or indirectly to an epsilon-amino group of said one or more internal lysine residues or to 10 the terminal side-chain group of said one or more internal lysine analog residues so as to produce a lipopeptide having the lipid moiety attached to the epsilon amino group of said internal lysine residue or having the lipid moiety attached to the terminal side chain group of said internal lysine analog residue. Preferably, the method further comprises production of the lipid moiety. 15 Conventional chemical syntheses referred to herein are the preferred means for producing the polypeptide moiety and the lipid moiety. Preferably, the internal lysine or lysine analog is modified by selective removal of a blocking group (eg: Mtt) from the terminal side-chain group so as to permit the addition of an amino acid residue, a spacer or lipid moiety, including a lipoamino acid, 20 at that position. For attachment of the lipid to the polypeptide, it is convenient for the functional groups of the polypeptide to be protected in a manner known in the art of peptide synthesis, to 3295672 I.DOC 88 ensure that no undesirable reactions at those groups takes place at a significant reaction rate. By known coupling processes, the polypeptide is synthesized on a solid or soluble carrier, such as a polymer (for example Merrifield resin) and made available for 5 conjugation to a spacer, amino acid, or lipid. For example, the terminal side chain group of the lysine or lysine analog (eg. epsilon amino group of the internal lysine) is protected by one of a number of protecting groups. Blocking groups (also called protecting groups or masking groups) are used to protect the amino group of the amino acid having an activated carboxyl group that is involved in the coupling reaction, or to 10 protect the carboxyl group of the amino acid having an acylated amino group that is involved in the coupling reaction. For coupling to occur, a blocking group must be removed without disrupting a peptide bond, or any protecting group attached to another part of the peptide. For solid phase peptide synthesis, blocking groups that are stable to the repeated 15 treatments necessary for removal of the amino blocking group of the growing peptide chain and for repeated amino acid couplings, are used for protecting the amino acid side-chains. Additionally, the peptide-resin anchorage that protects the C-terminus of the peptide must be protected throughout the synthetic process until cleavage from the resin is required. Accordingly, by the judicious selection of orthogonally protected 20 alpha-amino acids, lipids and/or amino acids are added at desired locations to a growing peptide whilst it is still attached to the resin. Preferred amino blocking groups are easily removable but sufficiently stable to survive conditions for the coupling reaction and other manipulations, such as, for example, modifications to the side-chain groups. Preferred amino blocking groups are selected 25 from the group consisting of: (i) a benzyloxycarbonyl group (Z or carbobenzoxy) that is removed easily by catalytic hydrogenation at room temperature and ordinary pressure, or using sodium in liquid ammonia and hydrobromic acid in acetic acid; (ii) a t- 3295672 I.DOC 89 Butoxycarbonyl group (Boc) that is introduced using t-butoxycarbonyl azide or di-tert butyldicarbonate and removed using mild acid such as, for example, trifluoroacetic acid (50% TFA in dichloromethane), or HCI in acetic acid/dioxane/ethylacetate; (iii) a 9 fluorenylmethyloxycarbonyl group (Fmoc) that is cleaved under mildly basic, non 5 hydrolytic conditions, such as, for example, using a primary or secondary amine (eg. 20% piperidine in dimethyl formamide); (iv) a 2-(4-biphenylyl) propyl(2)oxycarbonyl group (Bpoc); (v) a 2-nitro-phenylsulfenyl group (Nps); and (vi) a dithia-succionyl group (Dts). Side chain-protecting groups will vary for the functional side chains of the amino acids 10 forming the peptide being synthesized. Side-chain protecting groups are generally based on the Bzl group or the tBu group. Amino acids having alcohols or carboxylic acids in the side-chain are protected as Bzl ethers, Bzl esters, cHex esters, tBu ethers, or tBu esters. Side-chain protection of Fmoc amino acids requires blocking groups that are ideally base stable and weak acid (TFA) labile. For example, the epsilon-amino 15 group of Lysine is protected using Mtt (eg. Fmoc-lysine(Mtt)-OH). Alternatively, a halogenated benzyl derivative such as CIZ is used to protect the lysine side chain should enhanced acid stability be required. The thiol group of Cystine, the imidazole of Histidine, or guanidino group of Arginine, generally require specialised protection. Many different protecting groups for peptide synthesis have been described (see The 20 Peptides, Gross et al. eds., Vol. 3, Academic Press, New York, 1981). The two most widely used protection strategies are the Boc/Bzl- and the Fmoc/tBu strategies. In Boc/Bzl, Boc is used for amino protection and the side-chains of the various amino acids are protected using Bzl- or cHex-based protecting groups. A Boc group is stable under catalytic hydrogenation conditions and is used orthogonally along 25 with a Z group for protection of many side chain groups. In Fmoc/tBu, Fmoc is used for amino protection and the side-chains are protected with tBu-based protecting groups.

3295672_.DOC 90 Peptides are lipidated by methods well known in the art. Standard condensation, addition, substitution or oxidation (e.g. disulfide bridge formation or amide bond formation between a terminal amino group on the internal lysine or lysine analog with the carboxy terminal group of an incoming amino acid or peptide or lipoamino acid) 5 reactions result in the addition of lipid to the polypeptide. In an alternative embodiment, a peptide of the present invention for use as an immunogen is produced by chemoselective ligation or chemical conjugation or oxime chemistry. Such methods are well-known in the art, and allow for the individual peptide components to be produced by chemical or recombinant means, followed by 10 their chemoselective ligation in an appropriate configuration or conformation or order (eg. Nardin et al., Vaccine 16, 590 (1998); Nardin et al., J Immunol. 166, 481 (2001); Rose et al., Mol. Immunol. 32, 1031 (1995); Rose et al., Bioconjug. Chem 7, 552 (1996); and Zeng et al., Vaccine 18, 1031 (2000), which are incorporated herein by reference). 15 Lipopeptide formulations The lipopeptide is conveniently formulated in a pharmaceutically acceptable excipient or diluent, such as, for example, an aqueous solvent, non-aqueous solvent, non-toxic excipient, such as a salt, preservative, buffer and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic 20 esters such as ethyloleate. Aqueous solvents include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to routine skills in the art. 25 The addition of an extrinsic adjuvant to the lipopeptide formulation, although generally not required, is also encompassed by the invention. Such extrinsic adjuvants include all 3295672_.DOC 91 acceptable immunostimulatory compounds such as, for example, a cytokine, toxin, or synthetic composition. Exemplary adjuvants include IL-1, IL-2, BCG, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thur-MDP), N-acetyl-nor muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N 5 acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-2'-dipalmitoyl-sn-glycero-3 hydroxyphosphoryloxy)-ethylamine (CGP) 1983A, referred to as MTP-PE), lipid A, MPL and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. 10 It may be desirable to co-administer biologic response modifiers (BRM) with the lipopeptide, to down regulate suppressor T cell activity. Exemplary BRM's include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA, USA); Indomethacin (IND; 150 mg/d) (Lederle, NJ, USA); or low-dose Cyclophosphamide (CYP; 75, 150 or 300 mg/m.sup.2) (Johnson/Mead, NJ, USA). 15 Use of lipopeptides comprising CTL epitope in immunization The lipopeptide of the present invention also enhances CTL memory responses against the CTL epitope moiety when administered to an animal subject, without any requirement for an adjuvant to achieve a similar level of CTL activation. In addition, enhanced maturation of dendritic cells and other biological effects which include 20 induction of IFN-y producing CD8+ cells as well as viral, bacterial and tumour cell clearance have been observed following administration of vaccine. Accordingly, the invention also contemplates a method of enhancing cell mediated immunity against the organism from which the CTL epitope is derived in a subject comprising administering the lipopeptide of the invention or a derivative or a 25 functionally equivalent variant of said lipopeptide or a vaccine composition comprising 3295672_.DOC 92 said lipopeptide or variant or derivative for a time and under conditions sufficient to activate a CTL and/or a CTL precursor of said subject. By "CTL precursor" is meant a naive T cell (ie. a T cell that expresses one or more T cell receptors on its surface and is capable of proliferating and differentiating into a 5 memory T cell or effector T cell). Preferably, the lipopeptide or vaccine is administered prophylactically to a subject not harboring a latent or active infection by a parasite, bacterium or virus or suffering from a cancer or administered therapeutically to a subject harboring a latent or active infection by a parasite, bacteria or virus or suffering from a cancer. 10 In the present context, the term "activate" means that the ability of a T cell to recognize and lyse a cell harboring an antigen from which the CTL epitope is derived is enhanced, or that the ability of a T cell to recognize a T cell epitope of said antigen is enhanced, either transiently or in a sustained manner. The term "activate" shall also be taken to include a reactivation of a T cell population following activation of a latent 15 infection by a parasite or bacteria or virus, or following re-infection with a parasite or bacteria or virus, or following immunization of a previously-infected subject with a lipopeptide or composition of the invention. Those skilled in the art are aware that optimum T cell activation requires cognate recognition of antigen/MHC by the T cell receptor (TcR), and a co-stimulation 20 involving the ligation of a variety of cell surface molecules on the T cell with those on an antigen presenting cell (APC). The costimulatory interactions CD28/B7, CD40L/CD40 and OX40/OX40L are preferred, but not essential for T cell activation. Other costimulation pathways may operate. For determining the activation of a CTL or precursor CTL or the level of epitope 25 specific activity, standard methods for assaying the number of CD8+ T cells in a specimen can be used. Preferred assay formats include a cytotoxicity assay, such as for 3295672 I.DOC 93 example the standard chromium release assay, the assay for IFN-y production, such as, for example, the ELISPOT assay. These assay formats are described in detail in the accompanying examples. MHC class I Tetramer assays can also be utilized, particularly for CTL epitope-specific 5 quantitation of CD8+ T cells (Altman et al., Science 274, 94-96, 1996; Ogg et al., Curr Opin Immunol. 10, 393-396, 1998). To produce tetramers, the carboxyl terminus of an MHC molecule, such as, for example, the HLA A2 heavy chain, is associated with a specific peptide epitope or polyepitope, and treated so as to form a tetramer complex having bound thereto a suitable reporter molecule, preferably a fluorochrome such as, 10 for example, fluoroscein isothiocyanate (FITC), phycoerythrin, phycocyanin or allophycocyanin. Tetramer formation is achieved, for example, by producing the MHC-peptide fusion protein as a biotinylated molecule and then mixing the biotinylated MHC-peptide with deglycosylated avidin that has been labeled with a fluorophore, at a molar ratio of 4:1. The Tetramers produced bind to a distinct set of 15 CD8+ T cell receptors (TcRs) on a subset of CD8+ T cells derived from the subject (eg in whole blood or a PBMC sample), to which the peptide is HLA restricted. There is no requirement for in vitro T cell activation or expansion. Following binding, and washing of the T cells to remove unbound or non-specifically bound Tetramer, the number of CD8+ cells binding specifically to the HLA-peptide Tetramer is readily 20 quantified by standard flow cytometry methods, such as, for example, using a FACSCalibur Flow cytometer (Becton Dickinson). The Tetramers can also be attached to paramagnetic particles or magnetic beads to facilitate removal of non-specifically bound reporter and cell sorting. Such particles are readily available from commercial sources (eg. Beckman Coulter, Inc., San Diego, CA, USA). Tetramer staining does not 25 kill the labeled cells; therefore cell integrity is maintained for further analysis. MHC Tetramers enable the accurate quantitative analyses of specific cellular immune responses, even for extremely rare events that occur at less than 1% of CD8+T cells 3295672_.DOC 94 (Bodinier et al., Nature Med. 6, 707-710, 2000; Ogg et al., Curr Opin Immunol. 10, 393-396, 1998). The total number of CD8+ cells in a sample can also be determined readily, such as, for example, by incubating the sample with a monoclonal antibody against CD8 5 conjugated to a different reporter molecule to that used for detecting the Tetramer. Such antibodies are readily available (eg. Becton Dickinson). The relative intensities of the signals from the two reporter molecules used allows quantification of both the total number of CD8+ cells and Tetramer-bound T cells and a determination of the proportion of total T cells bound to the Tetramer. 10 Because CD4+ T-helper cells function in CMI as producers of cytokines, such as, for example IL-2, to facilitate the expansion of CD8+ T cells or to interact with the APC thereby rendering it more competent to activate CD8+ T cells, cytokine production is an indirect measure of T cell activation. Accordingly, cytokine assays can also be used to determine the activation of a CTL or precursor CTL or the level of cell mediated 15 immunity in a human subject. In such assays, a cytokine such as, for example, IL-2, is detected or production of a cytokine is determined as an indicator of the level of epitope-specific reactive T cells. Preferably, the cytokine assay format used for determining the level of a cytokine or cytokine production is essentially as described by Petrovsky and Harrison, J. Immunol. 20 Methods 186, 37-46, 1995, which assay reference is incorporated herein. Preferably, the cytokine assay is performed on whole blood or PBMC or buffy coat. Preferably, the lipopeptide or derivative or variant or vaccine composition is administered for a time and under conditions sufficient to elicit or enhance the expansion of CD8+ T cells.

3295672 I.DOC 95 Still more preferably, the lipopeptide or derivative or variant or vaccine composition is administered for a time and under conditions sufficient for cell mediated immunity (CMI) to be enhanced in the subject. By "CMI" is meant that the activated and clonally expanded CTLs are MHC-restricted 5 and specific for a CTL epitope. CTLs are classified based on antigen specificity and MHC restriction, (ie., non-specific CTLs and antigen-specific, MHC-restricted CTLs). Non-specific CTLs are composed of various cell types, including NK cells and can function very early in the immune response to decrease pathogen load, while antigen specific responses are still being established. In contrast, MHC-restricted CTLs achieve 10 optimal activity later than non-specific CTL, generally before antibody production. Antigen-specific CTLs inhibit or reduce the spread of a pathogen and preferably terminate infection. CTL activation, clonal expansion, or CMI can be induced systemically or compartmentally localized. In the case of compartmentally localized effects, it is 15 preferred to utilize a vaccine composition suitably formulated for administration to that compartment. On the other hand, there are no such stringent requirements for inducing CTL activation, expansion or CMI systemically in the subject. The effective amount of lipopeptide to be administered, either solus or in a vaccine composition to elicit CTL activation, clonal expansion or CMI will vary, depending 20 upon the nature of the immunogenic epitope, the route of administration, the weight, age, sex, or general health of the subject immunized, and the nature of the CTL response sought. All such variables are empirically determined by art-recognized means. The lipopeptide, optionally formulated with any suitable or desired carrier, adjuvant, 25 BRM, or pharmaceutically acceptable excipient, is conveniently administered in the form of an injectable composition. Injection may be intranasal, intramuscular, sub- 3295672 I.DOC 96 cutaneous, intravenous, intradermal, intraperitoneal, or by other known route. For intravenous injection, it is desirable to include one or more fluid and nutrient replenishers. The optimum dose to be administered and the preferred route for administration are 5 established using animal models, such as, for example, by injecting a mouse, rat, rabbit, guinea pig, dog, horse, cow, goat or pig, with a formulation comprising the lipopeptide, and then monitoring the CTL immune response using any conventional assay. The use of HLA A2/Kb transgenic mice carrying a chimeric human-mouse Class I major histocompatibility complex (MHC) locus composed of the al and a2 domains of 10 the human HLA A*0201 allele and the c 3 domain of the mouse H-2Kb Class I molecules (Vitiello et al., J. Exp. Med. 173, 1007, 1991) is particularly preferred for testing responses in vivo to a lipopeptide of the invention that comprises a HLA A2 restricted CTL epitope or a vaccine composition comprising same. Without being bound by any theory or mode of action, we believe that the biological 15 effects of the lipopeptides are exerted through their ability to stimulate and mature dendritic cells. It is the dendritic cells which then activate CD4+ and CD8+ T cells in the draining lymph nodes. For this reason, we would not nor would it be possible to activate T cells directly as envisaged. The following section has therefore been modified accordingly to accommodate the notion of dendritic cell activation. 20 In a related embodiment, the invention provides a method of enhancing the cell mediated immunity of a subject, said method comprising contacting ex vivo cells, preferably dendritic cells, obtained from a subject with an immunologically active lipopeptide of the invention or a derivative or variant thereof or a vaccine composition comprising said lipopeptide or derivative or variant for a time and under conditions 25 sufficient to mature said dendritic cells. Said dendritic cells are then capable of conferring epitope specific activation of T cells.

3295672 I.DOC 97 In a preferred embodiment, the invention provides a method of enhancing the cell mediated immunity of a subject, said method comprising: (i) contacting ex vivo dendritic cells obtained from a subject with an immunologically active lipopeptide of the invention or a derivative or variant thereof or 5 a vaccine composition comprising said lipopeptide or derivative or variant for a time and under conditions sufficient to mature said dendritic cells; and (ii) introducing the activated dendritic cells autologously to the subject or syngeneically to another subject in order that T cell activation occurs. The T cell may be a CTL or CTL precursor cell. 10 The subject from whom the dendritic cells are obtained may be the same subject or a different subject to the subject being treated. The subject being treated can be any subject carrying a latent or active infection by a pathogen, such as, for example, a parasite, bacterium or virus or a subject who is otherwise in need of obtaining vaccination against such a pathogen or desirous of obtaining such vaccination. The 15 subject being treated may also be treated for a tumour that they are carrying. By "epitope specific activity" is meant that the T cell is rendered capable of being activated as defined herein above (ie. the T cell will recognize and lyze a cell harboring a pathogen from which the CTL epitope is derived, or is able to recognize a T cell epitope of an antigen of a pathogen either transiently or in a sustained manner). 20 Accordingly, it is particularly preferred for the T cell to be a CTL precursor which by the process of the invention is rendered able to recognize and lyze a cell harboring the pathogen or able to recognize a T cell epitope of an antigen of the pathogen either transiently or in a sustained manner.

3295672 I.DOC 98 For such an ex vivo application, the dendritic cells are preferably contained in a biological sample obtained from a subject, such as, for example, blood, PBMC or a buffy coat fraction derived therefrom. Another aspect of the invention provides a method of providing or enhancing immunity 5 against a pathogen in an uninfected subject comprising administering to said subject an immunologically active lipopeptide of the invention or a derivative or variant thereof or a vaccine composition comprising said lipopeptide or derivative or variant for a time and under conditions sufficient to provide immunological memory against a future infection by the pathogen. As with the other embodiments described herein, the 10 pathogen may be a parasite, virus or bacterium, and is preferably a parasite, virus or bacterium referred to herein above from which a CTL epitope has been identified. In a related embodiment, the invention provides a method of enhancing or conferring immunity against a pathogen in an uninfected subject comprising contacting ex vivo dendritic cells obtained from said subject with an immunologically active lipopeptide 15 of the invention or a derivative or variant thereof or a vaccine composition comprising said lipopeptide or derivative or variant for a time and under conditions sufficient to confer epitope specific activity on T cells. Accordingly, this aspect of the invention provides for the administration of a prophylactic vaccine to the subject, wherein the active substituent of said vaccine (i.e. 20 the lipopeptide of the invention) induces immunological memory via memory T cells in an uninfected individual. The preferred embodiments of vaccination protocols described herein for enhancing the cell mediated immunity of a subject apply mutatis mutandis to the induction of immunological memory against the pathogen in a subject. The present invention is further described with reference to the following non-limiting 25 examples and the drawings. The examples provided herein in mice are accepted models for equivalent diseases in humans and the skilled person will readily be capable 3295672_.DOC 99 of extending the findings presented herein for such models to a human disease context without undue experimentation. Use of the lipopeptides comprising B cell epitopes in immunization The novel lipopeptides of the invention differ in essential aspects from known 5 lipopeptide conjugates of CTL epitopes in having the lipid moiety conjugated exclusively through the terminal side-chain group of an internal lysine or lysine analog residue, thereby enhancing T cell responses without the administration of additional adjuvant. Accordingly, a particular utility of the lipopeptides of the present invention is in the fields of eliciting a T cell response either in vivo or ex vivo, synthetic vaccine 10 preparation, diagnostic methods employing T cells, and immunotherapy for veterinary and human medicine. More particularly, the lipopeptide of the present invention comprising a B cell epitope induces the specific production of a high titer antibody against the B cell epitope moiety when administered to an animal subject, without any requirement for an 15 adjuvant to achieve a similar antibody titer. This utility is supported by the enhanced maturation of dendritic cells following administration of the subject lipopeptides (i.e. enhanced antigen presentation compared to lipopeptides having N-terminally coupled lipid). Accordingly, the invention provides a method of eliciting the production of antibody 20 against an antigenic B cell epitope comprising administering an isolated lipopeptide comprising a polypeptide conjugated to one or more lipid moieties to said subject for a time and under conditions sufficient to elicit the production of antibodies against said antigenic B cell epitope, wherein: (i) said polypeptide comprises: 3295672_.DOC 100 (a) the amino acid sequence of a T helper cell (Th) epitope and the amino acid sequence of a B cell epitope, wherein said amino acid sequences are different; and (b) one or more internal lysine residues or internal lysine analog residues for covalent attachment of each of said lipid moieties via an epsilon-amino group of said 5 internal lysine or via a terminal side-chain group of said internal lysine analog; and (ii) each of said one or more lipid moieties is covalently attached directly or indirectly to an epsilon-amino group of said one or more internal lysine residues or to a terminal side-chain group of said one or more internal lysine analog residues. The effective amount of lipopeptide used in the production of antibodies varies upon 10 the nature of the immunogenic B cell epitope, the route of administration, the animal used for immunization, and the nature of the antibody sought. All such variables are empirically determined by art-recognized means. Reference herein to antibody or antibodies includes whole polyclonal and monoclonal antibodies, and parts thereof, either alone or conjugated with other moieties. Antibody 15 parts include Fab and F(ab)2 fragments and single chain antibodies. The antibodies may be made in vivo in suitable laboratory animals, or, in the case of engineered antibodies (Single Chain Antibodies or SCABS, etc) using recombinant DNA techniques in vitro. In accordance with this aspect of the invention, the antibodies may be produced for the 20 purposes of immunizing the subject, in which case high titer or neutralizing antibodies that bind to the B cell epitope will be especially preferred. Suitable subjects for immunization will, of course, depend upon the immunizing antigenic B cell epitope. It is contemplated that the present invention will be broadly applicable to the immunization of a wide range of animals, such as, for example, farm animals (e.g. 25 horses, cattle, sheep, pigs, goats, chickens, ducks, turkeys, and the like), laboratory animals (e.g. rats, mice, guinea pigs, rabbits), domestic animals (cats, dogs, birds and 3295672_.DOC 101 the like), feral or wild exotic animals (e.g. possums, cats, pigs, buffalo, wild dogs and the like) and humans. Alternatively, the antibodies may be for commercial or diagnostic purposes, in which case the subject to whom the lipopeptide is administered will most likely be a 5 laboratory or farm animal. A wide range of animal species are used for the production of antisera. Typically the animal used for production of antisera is a rabbit, a mouse, rat, hamster, guinea pig, goat, sheep, pig, dog, horse, or chicken. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies. However, as will be known to those skilled in the art, larger 10 amounts of immunogen are required to obtain high antibodies from large animals as opposed to smaller animals such as mice. In such cases, it will be desirable to isolate the antibody from the immunized animal. Preferably, the antibody is a high titer antibody. By "high titer" means a sufficiently high titer to be suitable for use in diagnostic or therapeutic applications. As will be 15 known in the art, there is some variation in what might be considered "high titer". For most applications a titer of at least about 103_104 is preferred. More preferably, the antibody titer will be in the range from about 104 to about 105 , even more preferably in the range from about 105 to about 106. More preferably, in the case of B cell epioptes from pathogens, viruses or bacteria, the 20 antibody is a neutralizing antibody (i.e. it is capable of neutralizing the infectivity of the organism fro which the B cell epitope is derived). To generate antibodies, the lipopeptide, optionally formulated with any suitable or desired carrier, adjuvant, BRM, or pharmaceutically acceptable excipient, is conveniently administered in the form of an injectable composition. Injection may be 25 intranasal, intramuscular, sub-cutaeous, intravenous, intradermal, intraperitoneal, or by other known route. The lipopeptides of the present invention have demonstrated 3295672 I.DOC 102 efficacy when administered intranasally. For intravenous injection, it is desirable to include one or more fluid and nutrient replenishers. Means for preparing and characterizing antibodies are well known in the art. (See, e.g., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, 1988, incorporated 5 herein by reference). The efficacy of the lipopeptide in producing an antibody is established by immunizing an animal, for example, a mouse, rat, rabbit, guinea pig, dog, horse, cow, goat or pig, with a formulation comprising the lipopeptide, and then monitoring the immune response to the B cell epitope, as described in the Examples. Both primary and 10 secondary immune responses are monitored. The antibody titer is determined using any conventional immunoassay, such as, for example, ELISA, or radio immunoassay. The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, may be given, if required to achieve a desired antibody titer. The process of 15 boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal is bled and the serum isolated and stored, and/or the animal is used to generate monoclonal antibodies (Mabs). For the production of monoclonal antibodies (Mabs) any one of a number of well known techniques may be used, such as, for example, the procedure exemplified in US 20 Patent No. 4,196,265, incorporated herein by reference. For example, a suitable animal will be immunized with an effective amount of the lipopeptide of the invention and under conditions sufficient to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep, or frog cells is also possible. The use of rats may provide certain 25 advantages, but mice are preferred, with the BALB/c mouse being most preferred as the 3295672 I.DOC 103 most routinely used animal and one that generally gives a higher percentage of stable fusions. Following immunization, somatic cells capable of producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells 5 may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody 10 titer removed. Spleen lymphocytes are obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 107 to 2 x 108 lymphocytes. The B cells from the immunized animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal that was 15 immunized with the lipopeptide formulation. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells, or hybridomas. Any one of a number of myeloma cells may be used and these are known 20 to those of skill in the art (e.g. murine P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 4 1, Sp2lO-Agl4, FO, NSO/U, MPC-1 1, MPCI 1-X45-GTG 1.7 and S194/5XX0; or rat R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GMI500-GRG2, LICR LON-HMy2 and UC729-6). A preferred murine myeloma cell is the NS-I myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS 25 Human Genetic Mutant Cell Repository under Accession No. GM3573. Alternatively, a murine myeloma SP2/0 non-producer cell line which is 8-azaguanine-resistant is used.

3295672 I.DOC 104 To generate hybrids of antibody-producing spleen or lymph node cells and myeloma cells, somatic cells are mixed with myeloma cells in a proportion between about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have 5 been described by Kohler and Milstein, Nature 256, 495-497, 1975; and Kohler and Milstein, Eur. J. Immunol. 6, 511-519, 1976. Methods using polyethylene glycol (PEG), such as 37% (v/v) PEG, are described in detail by Gefter et al., Somatic Cell Genet. 3, 231-236, 1977. The use of electrically induced fusion methods is also appropriate. 10 Hybrids are amplified by culture in a selective medium comprising an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the 15 media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. The preferred selection medium is HAT, because only those hybridomas capable of operating nucleotide salvage pathways are able to survive in HAT medium, whereas 20 myeloma cells are defective in key enzymes of the salvage pathway, (e.g., hypoxanthine phosphoribosyl transferase or HPRT), and they cannot survive. B cells can operate this salvage pathway, but they have a limited life span in culture and generally die within about two weeks. Accordingly, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells. 25 The amplified hybridomas are subjected to a functional selection for antibody specificity and/or titer, such as, for example, by immunoassay (e.g. radioimmunoassay, 3295672 I.DOC 105 enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunobinding assay, and the like). The selected hybridomas are serially diluted and cloned into individual antibody producing cell lines, which clones can then be propagated indefinitely to provide 5 MAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma is injected, usually in the peritoneal cavity, into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, 10 such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they are readily obtained in high concentrations. MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic 15 methods such as HPLC or affinity chromatography. Monoclonal antibodies of the present invention also include anti-idiotypic antibodies produced by methods well-known in the art. Monoclonal antibodies according to the present invention also may be monoclonal heteroconjugates, (i.e., hybrids of two or more antibody molecules). In another embodiment, monoclonal antibodies according 20 to the invention are chimeric monoclonal antibodies. In one approach, the chimeric monoclonal antibody is engineered by cloning recombinant DNA containing the promoter, leader, and variable-region sequences from a mouse anti-PSA producing cell and the constant-region exons from a human antibody gene. The antibody encoded by such a recombinant gene is a mouse-human chimera. Its antibody specificity is 25 determined by the variable region derived from mouse sequences. Its isotype, which is determined by the constant region, is derived from human DNA.

3295672 I.DOC 106 In another embodiment, monoclonal antibodies according to the present invention is a "humanized" monoclonal antibody, produced by techniques well-known in the art. That is, mouse complementary determining regions ("CDRs") are transferred from heavy and light V-chains of the mouse Ig into a human V-domain, followed by the 5 replacement of some human residues in the framework regions of their murine counterparts. "Humanized" monoclonal antibodies in accordance with this invention are especially suitable for use in in vivo diagnostic and therapeutic methods. As stated above, the monoclonal antibodies and fragments thereof according to this invention are multiplied according to in vitro and in vivo methods well-known in the 10 art. Multiplication in vitro is carried out in suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements, e.g., feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like. In vitro production provides relatively 15 pure antibody preparations and allows scale-up to give large amounts of the desired antibodies. Techniques for large scale hybridoma cultivation under tissue culture conditions are known in the art and include homogenous suspension culture, (e.g., in an airlift reactor or in a continuous stirrer reactor or immobilized or entrapped cell culture). 20 Large amounts of the monoclonal antibody of the present invention also may be obtained by multiplying hybridoma cells in vivo. Cell clones are injected into mammals which are histocompatible with the parent cells, (e.g., syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as Pristane (tetramethylpentadecane) prior to 25 injection. In accordance with the present invention, fragments of the monoclonal antibody of the invention are obtained from monoclonal antibodies produced as described above, by 3295672 I.DOC 107 methods which include digestion with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction. The monoclonal conjugates of the present invention are prepared by methods known in the art, e.g., by reacting a monoclonal antibody prepared as described above with, for 5 instance, an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents, or by reaction with an isothiocyanate. Conjugates with metal chelates are similarly produced. Other moieties to which antibodies may be conjugated include radionuclides such as, for example, 3H, 1251, .32P, .35S, 14C, 51Cr, 36C, 57Co, 10 58Co, 59Fe, 75Se, and 152Eu. Radioactively labeled monoclonal antibodies of the present invention are produced according to well-known methods in the art. For instance, monoclonal antibodies are iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the 15 invention may be labeled with technetium99 by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labeling techniques, (e.g., by incubating pertechnate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the antibody). 20 Any immunoassay may be used to monitor antibody production by the lipopeptide formulations. Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. 25 However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used. Most preferably, the assay will be capable of generating quantitative results.

3295672 .DOC 108 For example, antibodies are tested in simple competition assays. A known antibody preparation that binds to the B cell epitope and the test antibody are incubated with an antigen composition comprising the B cell epitope, preferably in the context of the native antigen. "Antigen composition" as used herein means any composition that 5 contains some version of the B cell epitope in an accessible form. Antigen-coated wells of an ELISA plate are particularly preferred. In one embodiment, one would pre-mix the known antibodies with varying amounts of the test antibodies (e.g., 1:1, 1:10 and 1:100) for a period of time prior to applying to the antigen composition. If one of the known antibodies is labeled, direct detection of the label bound to the antigen is 10 possible; comparison to an unmixed sample assay will determine competition by the test antibody and, hence, cross-reactivity. Alternatively, using secondary antibodies specific for either the known or test antibody, one will be able to determine competition. An antibody that binds to the antigen composition will be able to effectively compete 15 for binding of the known antibody and thus will significantly reduce binding of the latter. The reactivity of the known antibodies in the absence of any test antibody is the control. A significant reduction in reactivity in the presence of a test antibody is indicative of a test antibody that binds to the B cell epitope (i.e., it cross-reacts with the known antibody). 20 In one exemplary ELISA, the antibodies against the B cell epitope are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a composition containing the B cell epitope is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound epitope may be detected. Detection is generally achieved by the 25 addition of a second antibody that is known to bind to the B cell epitope and is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of said second antibody, followed by the addition 3295672 I.DOC 109 of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label. Induction of sterility An appropriately configured lipopeptide of the present invention comprising an 5 antigenic B cell epitope of a reproductive hormone or a hormone receptor is capable of inducing infertility in a subject. Accordingly, the invention further provides a method of inducing infertility in a subject comprising administering to said subject an isolated lipopeptide comprising a polypeptide conjugated to one or more lipid moieties, wherein: 10 (i) said polypeptide comprises: (a) the amino acid sequence of a T helper cell (Th) epitope and the amino acid sequence of a B cell epitope of a reproductive hormone or hormone receptor, and wherein said amino acid sequences are different; and (b) one or more internal lysine residues or internal lysine analog residues for 15 covalent attachment of each of said lipid moieties via an epsilon-amino group of said internal lysine or via a terminal side-chain group of said internal lysine analog; and (ii) each of said one or more lipid moieties is covalently attached directly or indirectly to an epsilon-amino group of said one or more internal lysine residues or to a terminal side-chain group of said one or more internal lysine analog residues; and 20 (iii) said lipopeptide is administered for a time and under conditions sufficient to elicit a humoral immune response against said antigenic B cell epitope. The lipopeptides may be administered in the form of any convenient lipopeptide formulation as described herein.

3295672 I.DOC 110 By "humoral immune response" means that a secondary immune response is generated against the B cell epitope sufficient to prevent oogenesis, spermatogenesis, fertilization, implantation, or embryo development.. Preferably, the humoral immunity generated includes a sustained level of antibodies 5 against the B cell epitope in the subject. By a "sustained level of antibodies" is meant a sufficient level of circulating antibodies against the B cell epitope to prevent oogenesis, spermatogenesis, fertilization, implantation, or embryo development. Preferably, antibodies levels are sustained for at least a single reproductive cycle of an immunized female subject, and more preferably for at least about six months or 9 10 months or 12 months or 2 years. Preferably, the B cell epitope is derived from the amino acid sequence of luteinising hormone-releasing hormone (LHRH), follicle stimulating hormone (FSH), luteinising hormone (LH), human chorionic gonadotropin (hCG), a zona pellucida protein such as ZP3, or a FSH receptor ZP3a of humans or other mammals, such as pigs. 15 Particularly preferred B cell epitopes within this category include the C-terminal portion (CTP) of B-hCG; amino acid residues 323-341 of human ZP3; amino acid residues 8-18 or residues 272-283 or residues 319-330 of porcine ZP3a. Even more preferably, the B cell epitope comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID 20 NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84. The T-helper epitope preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 44, however any one of SEQ ID Nos: 1 or 18-56 can be used.

3295672 I.DOC 111 In a particularly preferred embodiment of the invention, the T-helper epitope comprises an amino acid sequence as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 44, and the B-cell epitope comprises an amino acid sequence of LHRH as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ 5 ID NO: 4. In accordance with such a preferred embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 5-16, 103 or 104. Also in accordance with this preferred embodiment, it is preferred (albeit not essential) that the lipid moiety comprise a lipoamino acid selected from the group consisting of: (i) Pam 2 Cys; (ii) Ste 2 Cys; (iii) Lau 2 Cys; and (iv) Oct 2 Cys. 10 The sustained production of antibodies against LHRH achieved by the lipopeptides of the invention demonstrates the general utility of the subject lipopeptides as an active agent in a vaccine preparation for inducing sterility, or as a contraceptive agent. Accordingly, the invention further contemplates a contraceptive agent comprising a pharmaceutically acceptable diluent and a lipopeptide comprising an isolated 15 polypeptide conjugated to one or more lipid moieties wherein: (i) said polypeptide comprises: (a) the amino acid sequence of a T helper cell (Th) epitope and the amino acid sequence of a B cell epitope of a reproductive hormone or hormone receptor, wherein said amino acid sequences are different; and 20 (b) one or more internal lysine residues or internal lysine analog residues for covalent attachment of each of said lipid moieties via an epsilon-amino group of said internal lysine or via a terminal side-chain group of said internal lysine analog; and (ii) each of said one or more lipid moieties is covalently attached directly or indirectly to an epsilon-amino group of said one or more internal lysine residues or to a 25 terminal side-chain group of said one or more internal lysine analog residues.

3295672_.DOC 112 The vaccine/contraceptive agent of the invention may comprise one or more carriers or excipients or other agents as described herein above under "lipopeptide formulations". Similarly, administration of the subject vaccine/contraceptive agent is achieved by means described herein above. Preferably, the subject is a human, or an animal subject 5 such as, for example, a farm animal, laboratory animal, domestic animal, feral animal or wild exotic animal. Immunization against Group A Streptococcus Group A streptococcus (GAS) is the bacterial agent of relatively mild illnesses such as, for example, "strep throat," and impetigo, as well as rarer severe and even life 10 threatening diseases such as, for example, necrotizing faciitis and streptococcal toxic shock syndrome. Severe, sometimes life-threatening, GAS disease may occur when bacteria get into parts of the body where bacteria usually are not found, such as the blood, muscle, or the lungs, an infection termed "invasive GAS disease". Two of the most severe forms of invasive GAS disease are necrotizing fasciitis and Streptococcal 15 Toxic Shock Syndrome (STSS). Necrotizing fasciitis destroys muscles, fat, and skin tissue. STSS causes blood pressure to drop rapidly and organs (e.g., kidney, liver, lungs) to fail. About 20% of patients with necrotizing fasciitis and more than half with STSS die. About 10%-15% of patients with other forms of invasive group A streptococcal disease die. There were about 9,400 cases of invasive GAS disease in the 20 United States alone in 1999. Invasive GAS infections generally occur when the bacteria get past the defenses of the person who is infected, such as, for example, when a person has sores or other breaks in the skin that allow the bacteria to get into the tissue, or when the person's ability to fight off the infection is decreased because of chronic illness or an illness that affects 25 the immune system, incuding HIV/AIDS. Also, some virulent strains of GAS are more likely to cause severe disease than others. People suffering from chronic illnesses like 3295672_.DOC 113 cancer, diabetes, and kidney dialysis, and those who use medications such as steroids have a higher risk. As exemplified herein, an appropriately configured lipopeptide of the present invention comprising an antigenic B cell epitope of a Group A streptococcus antigen, preferably 5 protein M, is capable of immunizing an animal host against GAS, and more particularly inducing serum IgG, saliva IgA and fecal IgA against the M protein of GAS, and also providing a protective immune response against a subsequent challenge by GAS thereby reducing GAS-induced mortality. Accordingly the invention further provides a method of inducing an immune response 10 against a Group A streptococcus antigen in a subject comprising administering to said subject an isolated lipopeptide comprising a polypeptide conjugated to one or more lipid moieties, wherein: (iv) said polypeptide comprises: (b) the amino acid sequence of a T helper cell (Th) epitope and the amino acid 15 sequence of a B cell epitope of a Group A streptococcus antigen, wherein said amino acid sequences are different; and (c) one or more internal lysine residues or internal lysine analog residues for covalent attachment of each of said lipid moieties via an epsilon-amino group of said internal lysine or via a terminal side-chain group of said internal lysine analog; and 20 (v) each of said one or more lipid moieties is covalently attached directly or indirectly to an epsilon-amino group of said one or more internal lysine residues or to a terminal side-chain group of said one or more internal lysine analog residues; and (vi) said lipopeptide is administered for a time and under conditions sufficient to elicit a humoral immune response against said antigenic B cell epitope.

3295672_.DOC 114 The lipopeptides may be administered in the form of any convenient lipopeptide formulation as described herein. By "humoral immune response" means that a secondary immune response is generated against the B cell epitope sufficient to induce serum IgG, saliva IgA or fecal IgA 5 against a peptide comprising the B-cell epitope, or alternatively or in addition, providing a protective immunity against a subsequent challenge with Group A streptococcus. Preferably, the humoral immunity generated includes a sustained level of antibodies against the B cell epitope in the subject. By a "sustained level of antibodies" is meant a 10 sufficient level of circulating antibodies against the B cell epitope to prevent the spread of infection by a Group A streptococcus following a subsequently challenge, and/or reduce morbidity or mortality in a subject that is subsequently challenged with a Group A streptococcus. Preferably, antibodies levels are sustained for at least about 6 months or 9 months or 12 15 months or 2 years. Preferably, the B cell epitope is derived from the amino acid sequence of the M protein of Group A streptococcus. Particularly preferred B cell epitopes within this category include a peptide that comprises the amino acid sequence set forth in SEQ ID NO: 101. 20 The T-helper epitope preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 44, however any one of SEQ ID Nos: I or 18-56 can be used. In a particularly preferred embodiment of the invention, the T-helper epitope comprises an amino acid sequence as set forth in SEQ ID NO: 24 and the B-cell epitope 25 comprises an amino acid sequence set forth in SEQ ID NO: 101. In accordance with 3295672 I.DOC 115 such a preferred embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 105-108. Also in accordance with this preferred embodiment, it is preferred (albeit not essential) that the lipid moiety comprise a lipoamino acid of Formula (I) or (II), however any lipid as described herein 5 will be useful. The sustained production of antibodies against the J14 peptide achieved by the lipopeptides of the invention demonstrates the general utility of the subject lipopeptides as an active agent in a vaccine preparation for providing protective immunity against Group A streptococcus. 10 Accordingly, the invention further provides a vaccine against Group A streptococcus comprising a pharmaceutically acceptable diluent and a lipopeptide comprising an isolated polypeptide conjugated to one or more lipid moieties wherein: (iii) said polypeptide comprises: (b) the amino acid sequence of a T helper cell (Th) epitope and the amino acid 15 sequence of a B cell epitope of a Group A streptococcus antigen, wherein said amino acid sequences are different; and (c) one or more internal lysine residues or internal lysine analog residues for covalent attachment of each of said lipid moieties via an epsilon-amino group of said internal lysine or via a terminal side-chain group of said internal lysine analog; and 20 (iv) each of said one or more lipid moieties is covalently attached directly or indirectly to an epsilon-amino group of said one or more internal lysine residues or to a terminal side-chain group of said one or more internal lysine analog residues. The vaccine of the invention may comprise one or more carriers or excipients or other agents as described herein above under "lipopeptide formulations".

3295672 I.DOC 116 Similarly, administration of the subject vaccine is achieved by means described herein above, preferably by an intranasal route. Preferably, the subject is a human, or an animal subject such as, for example, a farm animal, laboratory animal, domestic animal, feral animal or wild exotic animal. 5 Inhibition or prevention of excessive and unregulated gastric acid secretion Gastrin is known to stimulate gastric acid secretion by parietal cells, an activity mediated by binding of gastrin to gastrin receptors or cholecystekinin receptors. The terminal four-to-five amino acid residues of gastrin provide the same receptor specificity and activity as the full-length protein. The terminal five amino acid residues 10 of gastrin are termed "pentagastrin". Unregulated gastrin expression or secretion causes hypergastrinemia, which can lead to Zollinger-Ellison syndrome, the formation of gastric and duodenal ulcers, or gastrinoma in the pancreas or duodenum, as a consequence of excessive and unregulated gastric acid secretion. Immunoneutralization of gastrin using antibodies against gastrin is also known to block 1 5 secretion of gastric acid in response to intragastric secretion of gastrin peptides. As exemplified herein, an appropriately configured lipopeptide of the present invention comprising an antigenic B cell epitope of a gastrin peptide is capable of immunizing an animal host against gastrin or an effect of excessive gastrin production in a mouse model of other mammals in which inhibition of gastric acid secretion is indicated. The 20 data provided herein demonstrate the general utility of the subject lipopeptides in inducing humoral immunity against gastrin and immunoneutralization of gastrin, to thereby block secretion of gastric acid, in an animal suffering from hypergastrinemia, Zollinger-Ellison syndrome, gastric ulceration or duodenal ulceration due to excessive and unregulated secretion of gastric acid, or to reduce or prevent the formation of 25 gastrin-secreting tumors in the pancreas or duodenum (i.e. the prophylaxis and/or therapy of gastrinoma).

3295672 I.DOC 117 Accordingly, the invention further provides a method of inducing an immune response against a gastrin peptide in a subject comprising administering to said subject an isolated lipopeptide comprising a polypeptide conjugated to one or more lipid moieties, wherein: 5 (vii) said polypeptide comprises: (c) the amino acid sequence of a T helper cell (Th) epitope and the amino acid sequence of a B cell epitope of a gastrin peptide antigen, wherein said amino acid sequences are different; and (d) one or more internal lysine residues or internal lysine analog residues for 10 covalent attachment of each of said lipid moieties via an epsilon-amino group of said internal lysine or via a terminal side-chain group of said internal lysine analog; and (viii) each of said one or more lipid moieties is covalently attached directly or indirectly to an epsilon-amino group of said one or more internal lysine residues or to a terminal side-chain group of said one or more internal lysine analog residues; and 15 (ix) said lipopeptide is administered for a time and under conditions sufficient to elicit a humoral immune response against said antigenic B cell epitope. The lipopeptides may be administered in the form of any convenient lipopeptide formulation as described herein. By "humoral immune response" means that a secondary immune response is generated 20 against the B cell epitope sufficient to induce serum IgG against a gastrin peptide comprising the B-cell epitope. Preferably, the humoral immunity generated includes a sustained level of antibodies against the B cell epitope in the subject. By a "sustained level of antibodies" is meant a 3295672 I.DOC 118 sufficient level of circulating antibodies against the B cell epitope to prevent excessive or unregulated gastric acid secretion in response to gastrin. Preferably, antibodies levels are sustained for at least about 6 months or 9 months or 12 months or 2 years. 5 Preferably, the B cell epitope is contained within a pentagastrin peptide. Particularly preferred B cell epitopes within this category include a peptide that comprises the amino acid sequence set forth in SEQ ID NO: 102, however the full length gastrin protein or any immunogenic fragment thereof comprising a B-cell epitope may also be used. 10 The T-helper epitope preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 44, however any one of SEQ ID Nos: 1 or 18-56 can be used. In a particularly preferred embodiment of the invention, the T-helper epitope comprises an amino acid sequence as set forth in SEQ ID NO: 24 and the B-cell epitope 15 comprises an amino acid sequence set forth in SEQ ID NO: 102. In accordance with such a preferred embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos: 109-112. Also in accordance with this preferred embodiment, it is preferred (albeit not essential) that the lipid moiety comprise a lipoamino acid of Formula (I) or (II), however any lipid as described herein 20 will be useful. The sustained production of antibodies against pentagastrin or gastrin that is achieved by the lipopeptides of the invention demonstrates the general utility of the subject lipopeptides as an active agent in a vaccine preparation for reducing an adverse effect of gastrin in a subject in need thereof.

3295672 I.DOC 119 Accordingly, the invention further provides a vaccine against a disease or condition induced by excessive gastrin secretion in a subject comprising a pharmaceutically acceptable diluent and a lipopeptide comprising an isolated polypeptide conjugated to one or more lipid moieties wherein: 5 (v) said polypeptide comprises: (c) the amino acid sequence of a T helper cell (Th) epitope and the amino acid sequence of a B cell epitope of a gastrin peptide antigen, wherein said amino acid sequences are different; and (d) one or more internal lysine residues or internal lysine analog residues for 10 covalent attachment of each of said lipid moieties via an epsilon-amino group of said internal lysine or via a terminal side-chain group of said internal lysine analog; and (vi) each of said one or more lipid moieties is covalently attached directly or indirectly to an epsilon-amino group of said one or more internal lysine residues or to a terminal side-chain group of said one or more internal lysine analog residues. 15 The vaccine of the invention may comprise one or more carriers or excipients or other agents as described herein above under "lipopeptide formulations". Similarly, administration of the subject vaccine is achieved by means described herein above. Preferably, the subject is a human. The present invention is further described with reference to the following non-limiting 20 examples and the drawings. In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non limiting examples.

3295672_.DOC 120 EXAMPLE 1: Materials and Methods Chemicals Unless otherwise stated chemicals were of analytical grade or its equivalent. N,N' dimethylformamide (DMF), piperidine, trifluoroacetic acid (TFA), O'benzotriazole 5 N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), I hydroxybenzotriazole (HOBt) and diisopropylethylamine (DIPEA) and diisopropylcarbodiimide (DIPCDI) were obtained from Auspep Pty. Ltd., Melbourne, Australia and Sigma-Aldrich Pty. Ltd., Castle Hill, Australia. O'benzotriazole N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) was obtained from Bachem, (Bachem AG, Switzerland). 10 Dichloromethane (DCM) and diethylether were from Merck Pty Ltd. (Kilsyth, Australia). Phenol and triisopropylsilane (TIPS) were from Aldrich (Milwaulke, WI) and trinitrobenzylsulphonic acid (TNBSA) and diaminopyridine (DMAP) from Fluka; 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was obtained from Sigma and palmitic acid was from Fluka. 15 Viruses The type A influenza viruses used in this study were an H3Nl subtype virus referred to as Mem 71, which was derived by genetic reassortment of A/Memphis/l/71 (H3N2) X A/Bellamy/42 (H[N 1). Virus was grown for 2 days in the allantoic cavity of 10-day embryonated hen's eggs. Allantoic fluid containing virus was stored in aliquots at 20 70*C. Infectious virus titers were obtained by assay of plaque formation in monolayers of Madin-Darby canine kidney (MDCK) cells (Tannock et al, Infect. Immun. 43, 457 462, 1984) and are expressed as PFU/milliliter. Bacteria Listeria monocytogenes EGD was cultured overnight at 37 0 C on Horse Blood Agar 25 (HBA) plates. The bacteria were washed off the plates using sterile PBS and the 3295672 I.DOC 121 concentration adjusted to 5 x 10 3 Listeria cells/ml. Balb/c mice were infected intravenously with 1 x 10 3 Listeria cells. The dose was checked retrospectively by plating serial 10-fold dilutions on HBA plates. Peptide Syntheses 5 The general procedure used for the peptide synthesis has been described by Jackson et al., Vaccine 18, 355 (1999). To enable lipid attachment between the CD4+ T cell epitope and B-cell epitope, Fmoc-lysine(Mtt)-OH was inserted at a point between the two epitopes in the approximate centre of the resin-bound peptide. If lipid was to be added to another position within the peptide, such as, for example, the Lys- 14 residue 10 of SEQ ID NO: 24, then the Fmoc-lysine(Mtt)-OH was also inserted at that position. Following completion of peptide synthesis the Mtt group was removed by continual flow washing with 1% TFA in dichloromethane over a period of 30-45 mins to expose the epsilon amino group of the lysine residue. Two serine residues were coupled to this epsilon amino group in the case where two serine residues were used as spacer. 15 Alternatively, two arginine residues were coupled to this epsilon amino group in the case where two arginine residues were used as spacer. Alternatively, 6-aminohexanoic acid was coupled to this epsilon amino group. The subsequent coupling of the lipid moiety, such as, for example, Pam 3 Cys, Pam 2 Cys, Ste 2 Cys, Oct 2 Cys, or Lau 2 Cys is described below. 20 All resin-bound peptide constructs were cleaved from the solid phase support with reagent B (88% TFA, 5% phenol, 2% TIPS, 5% water) for 2 hr, and purified by reversed phase chromatography as described by Zeng et al., Vaccine 18, 1031 (2000). Analytical reversed phase high pressure liquid chromatography (RP-HPLC) was carried out using a Vydac C4 column (4.6 x 250 mm) installed in a Waters HPLC system and 25 developed at a flow rate of Iml/min using 0.1% TFA in H 2 0 and 0.1% TFA in CH 3 CN as the limit solvent. All products presented as a single major peak on analytical RP- 3295672 I.DOC 122 HPLC and had the expected mass when analysed by MALDI-TOF mass spectrometry on a Bruker BIFLEX instrument equipped with delayed ion extraction. The final quantitation of the immunogens was done by measuring the absorption at 280 nm exploiting the presence of a tryptophan and a tyrosine residue in the peptide constructs 5 (molar extinction coefficient of 6.6 x 103). Synthetic immunogens were assembled by conventional solid-phase methodology using Fmoc chemistry. The general procedure used for the peptide synthesis has been described by Jackson et al., Vaccine 18, 355 (1999). To enable lipid attachment between the CD4+ T helper epitope and the CTL epitope, Fmoc-lysine(Mtt)-OH was 10 inserted at a point between the two epitopes in the approximate centre of the resin bound peptide. Following completion of peptide synthesis the Mtt group was removed by continual flow washing with 1% TFA in dichloromethane over a period of 30-45 mins. To investigate the effect of serine by incorporating two residues between the peptide 15 and lipid moieties of the Pam 3 Cys-containing peptides and Pam 2 Cys-containing peptides, two serine residues were added sequentially to the peptide prior to covalent attachment of the lipid moiety (the structures of which are shown in Figure 1). Summaries of their characteristics, carried out by analytical RP-HPLC and mass spectrometry, are presented in Tables 1 and 2. 20 3295672 I.DOC 123 Table 1 HPLC elution and mass characteristics of peptide vaccines based upon influenza virus hemagglutinin T-helper epitope (SEQ ID NO: 1) and LHRH B-cell epitope (SEQ ID NO: 2) 'Peptide construct 'Retention time Expected mass Experimentally (min) (Da) determined mass (Da) [Thl-[B1 26.3 2957.1 2957.3 [Thl-Lvs-[B] 26.0 3085.5 3084.7 Pam Cys-Ser-Ser-[Thl-[B1 51.5 4022.4 4020.8 Pam2Cys-Ser-Ser-[Thl-[B1 41.8 3785.1 3785.5 Pam7Cys-[Thl-[B] 40.7 3609.3 3605.7 [Thl-Lvs(PanmCys)-[B1 50.4 3977.4 3969.5 rThl-Lvs(Pam,Cvs)-rBl 40.7 3739.5 3739.6 [Th]-Lys(Pam 2 Cys-Ser-Ser-)-[B] 40.3 3913.5 3912.1 5 'Reversed phase chromatography was carried out on Vydac C4 column (4.6 x 250 mm) installed in a Waters HPLC system and developed at a flow rate of I ml/min using 0.1% TFA in H20 and 0.1% TFA in CH3CN as the limit solvent. 2Mass spectrometry was carried out using a Bruker Biflex MALDI-TOF instrument equipped with delayed ion extraction. Analysis was carried out in the linear mode.

3295672 I.DOC 124 Table 2 HPLC elution and mass characteristics of peptide vaccines based upon CDV-F P25 T helper epitope (SEQ ID NO: 24) and pentagastrin B-cell epitope (SEQ ID NO: 102) Retention time Expected mass 2 Experimentally 'Peptide construct (min) (Da) determined mass (Da) [Thl-Lvs-fB1 31.4 2621.5 2620.7 Pam2Cys-Ser-Ser-[Thl-[B1 54.9 3449.7 3450.3 [Th]-Lys(Pam 2 Cys-Ser-Ser-)-[B] 53.9 3505.7 3506.7 Reversed phase chromatography was carried out on Vydac C4 column (4.6 x 300 mm) installed in a 5 Waters HPLC system and developed at a flow rate of I ml/min using 0.1% TFA in H20 and 0.1% TFA in CH3CN as the limit solvent. 2Mass spectrometry was carried out using an Agilent I 10 LC/MSD ion trap mass spectrometer. Peptides comprising influenza virus CTL epitopes 10 A panel of immunogens was synthesized that incorporated peptides representing a minimal determinant for CD8+ T cells and/or a determinant for CD4+ T cells, both from influenza virus. The peptide NP (147-155) with the sequence TYQRTRALV (a CTL determinant present in the NP of PR8 virus; SEQ ID NO: 114) is the dominant CD8+ T-cell determinant recognized by BALB/c mice and is common to all type A 15 influenza virus strains (Bodmer et al, Cell 52, 253-258, 1988; and Sherman et al, J. Exp. Med. 175, 1221-1226, 1992). The peptide HA2 (166-180), with the sequence ALNNRFQIKGVELKS (SEQ ID NO: 18), is a CD4+ T-helper determinant present within the HA2 chain of Mem 71 influenza virus hemmagglutinin elicits CD4+ T cells 3295672_.DOC 125 that are crossreactive with all viruses of the H3 subtype (Jackson et al, Virology 198, 153-170, 1994). Peptides comprising L. monocytogenes CTL epitopes An immunogenic peptide was synthesized that incorporated a minimal CTL epitope 5 with amino acid sequence GYKDGNEYI (residues 91-99 of the protein literialysin) from L. monocytogenes (ie. SEQ ID NO: 245) and a T-helper epitope from CDV-F (SEQ ID NO: 24). Peptides comprising a CTL epitope expressed by B16-OVA tumour cell line An immunogenic peptide was synthesized that incorporated a CTL epitope with amino 10 acid sequence SIINFEKL (SEQ ID NO: 246) and a T-helper epitope from CDV-F (SEQ ID NO: 24). Peptides comprising a CTL epitope from the core protein of hepatitis C virus An immunogenic peptide was synthesized that incorporated a CTL epitope with amino acid sequence DLMGYIPLV (SEQ ID NO: 249) and a T-helper epitope from CDV-F 15 (SEQ ID NO: 24). Synthetic immunogens were assembled by conventional solid-phase methodology using Fmoc chemistry. The general procedure used for the peptide synthesis has been described by Jackson et al., Vaccine 18, 355 (1999). To enable lipid attachment between the CD4+ T helper epitope and the CTL epitope, Fmoc-lysine(Mtt)-OH was 20 inserted at a point between the two epitopes in the approximate centre of the resin bound peptide. Following completion of peptide synthesis the Mtt group was removed by continual flow washing with 1% TFA in dichloromethane over a period of 30-45 mins.

3295672_.DOC 126 Synthesis of lipid moieties of Formulae (I) Pam 3 Cys was prepared according to the method described by Weismuller et al., Hoppe Seylers Z Physiol Chem 364, 593 (1983), as modified according to the method described by Zeng et al., J Pept Sci 2, :66 (1996). The lipoamino acid Pam 3 Cys is 5 coupled to the exposed epsilon-amino group of lysine according to the procedure described by Zeng et al. (supra). Briefly, a 2-fold excess of Pam 3 Cys, TBTU and HOBt was dissolved in DCM and a 3-fold excess of DIPEA added. This solution was then added to the resin-bound peptide to generate the lipopeptide. Synthesis of lipid moieties of Formulae (I) 10 The synthesis of Pam 2 Cys was adapted from previously described methods as described by Jones et al., Xenobiotica 5, 155 (1975) and Metzger et al., Int J Pept Protein Res 38, 545 (1991), with the exception that 3-bromo-propan-1,2-diol was used instead of 3 chloro-propan-1,2-diol, and centrifugation and not filtration was used to recover the product. 15 Synthesis of lipopeptides Lipopeptides produced in this study had the general structures shown in Figures 1 and 18. Amino acid sequences of the peptide moieties comprising B cell epitopes included in the various lipopeptides are shown in Figure 2. Pam 2 Cys was coupled to peptides according to the methods described by Jones et al., Xenobiotica 5, 155 (1975) and 20 Metzger et al., Int J Pept Protein Res 38, 545 (1991), with the following modifications: Synthesis of S-(2,3-Dihydroxypropyl)cysteine Triethylamine (6 g, 8.2 ml, 58 mmoles) was added to L-cysteine hydrochloride (3 g, 19 mmole) and 3-bromo-propan-1,2-diol (4.2 g, 2.36 ml, 27 mmole) in water and the homogeneous solution kept at room temperature for 3 days. The solution was reduced 25 in vacuo at 40*C to a white residue which was boiled with methanol (100ml), 3295672_.DOC 127 centrifuged and the residue dissolved in water (5ml). This aqueous solution was added to acetone (300ml) and the precipitate isolated by centrifugation. The precipitate was purified by several precipitations from water with acetone to give S-(2,3 dihydroxypropyl)cysteine as a white amorphous powder (2.4 g, 12.3 mmol, 64.7%). 5 Synthesis of N-Fluorenylmethoxycarbonyl-S-(2,3-dihydroxypropyl)-cysteine (Fmoc Dhc-OH) S-(2,3-dihydroxypropyl)cysteine (2.45 g, 12.6 mmole) was dissolved in 9% sodium carbonate (20 ml). A solution of fluorenylmethoxycarbonyl-N-hydroxysuccinimide (3.45 g, 10.5 mmole) in acetonitrile (20 ml) was added and the mixture stirred for 2 h, 10 then diluted with water (240 ml), and extracted with diethyl ether (25 ml x 3). The aqueous phase was acidified to pH 2 with concentrated hydrochloric acid and was then extracted with ethyl acetate (70 ml x 3). The extract was washed with water (50 ml x 2) and saturated sodium chloride solution (50 ml x 2), dried over sodium sulfate and evaporated to dryness. Recrystalisation from ether and ethyl acetate at -20*C yielded a 15 colourless powder (2.8 g, 6.7 mmole, 63.8%). Coupling of Fmoc-Dhc-OH to resin-bound peptide Fmoc-Dhc-OH (100mg, 0.24 mmole) was activated in DCM and DMF (1:1, v/v, 3 ml) with HOBt (36 mg, 0.24 mmole) and DICI (37 ul, 0.24 mmol) at 0 'C for 5 min. The mixture was then added to a vessel containing the resin-bound peptide (0.04 mmole, 20 0.25g amino-peptide resin). After shaking for 2 h the solution was removed by filtration and the resin was washed with DCM and DMF (3 x 30 ml each). The reaction was monitored for completion using the TNBSA test. If necessary a double coupling was performed. 25 3295672 I.DOC 128 Palmitoylation of the two hydroxy groups of the Fmoc-Dhc-peptide resin Palmitic acid (204 mg, 0.8 mmole), DICI (154 ul, 1 mmole) and DMAP (9.76 mg, 0.08 mmole) were dissolved in 2 ml of DCM and I ml of DMF. The resin-bound Fmoc Dhc-peptide resin (0.04 mmole, 0.25 g) was suspended in this solution and shaken for 5 16 h at room temperature. The solution was removed by filtration and the resin was then washed with DCM and DMF thoroughly to remove any residue of urea. The removal of the Fmoc group was accomplished with 2.5% DBU (2 x 5mins). Stearoylation of the two hydroxy groups of the Fmoc-Dhc-peptide resin Stearic acid (about 0.8 mmole), DICI (154 ul, 1 mmole) and DMAP (9.76 mg, 0.08 10 mmole) were dissolved in 2 ml of DCM and 1 ml of DMF. The resin-bound Fmoc Dhc-peptide resin (0.04 mmole, 0.25 g) was suspended in this solution and shaken for 16 h at room temperature. The solution was removed by filtration and the resin was then washed with DCM and DMF thoroughly to remove any residue of urea. The removal of the Fmoc group was accomplished with 2.5% DBU (2 x 5mins). 15 Lauroylation of the two hydroxy groups of the Fmoc-Dhc-peptide resin Lauric acid (about 0.8 mmole), DICI (154 ul, 1 mmole) and DMAP (9.76 mg, 0.08 mmole) were dissolved in 2 ml of DCM and 1 ml of DMF. The resin-bound Fmoc Dhc-peptide resin (0.04 mmole, 0.25 g) was suspended in this solution and shaken for 16 h at room temperature. The solution was removed by filtration and the resin was 20 then washed with DCM and DMF thoroughly to remove any residue of urea. The removal of the Fmoc group was accomplished with 2.5% DBU (2 x 5mins). Octanoylation of the two hydroxy groups of the Fmoc-Dhc-peptide resin Octanoic acid (about 0.8 mmole), DICI (154 ul, 1 mmole) and DMAP (9.76 mg, 0.08 mmole) were dissolved in 2 ml of DCM and I ml of DMF. The resin-bound Fmoc 25 Dhc-peptide resin (0.04 mmole, 0.25 g) was suspended in this solution and shaken for 3295672 I.DOC 129 16 h at room temperature. The solution was removed by filtration and the resin was then washed with DCM and DMF thoroughly to remove any residue of urea. The removal of the Fmoc group was accomplished with 2.5% DBU (2 x 5mins). Enzyme-linked immunosorbent assays (ELISA) 5 ELISA assays were carried out on serum samples as described essentially by Ghosh et al., Int Immun. 11, 1103, (1999), using the immunizing antigen (eg., LHRH, J14 or pentagastrin) as the coating antigen. The titres of antibody are expressed as the reciprocal of the highest dilution of serum to achieve an OD of 0.2, which represents approximately 5 times the background binding in the absence of antibody. The isotype 10 of antibodies specific for LHRH or J14 was determined using rabbit antisera directed against mouse 1gM, IgGI, IgG2a, IgG2b, IgG3 or IgA (ICN Pharmaceuticals Inc., Costa Mesa, CA) as previously described by Ghosh et al., Int Immun. 11, 1103, (1999). Fertility studies After being inoculated with peptide immunogen and following exposure to male mice, 15 female mice were tested for their ability to drop litters. A group of female mice immunized with saline in CFA was used as a control. A male mouse was introduced into a cage in which two or three female mice were kept and male mice rotated between each cage to expose each group of female mice to every male. Males and females were kept together for a total of 3 weeks at the end of which time the males were removed 20 and the females kept under observation. Immunization protocols Peptides comprising influenza virus CTL epitopes Groups of female BALB/c mice, 6 to 8 weeks old, were inoculated at day 0 and again on day 28. Alternatively, female outbred Quackenbush mice, 4-6 weeks old, were 25 immunized intranasally and provided with boosts as per the primary immunization at 3295672 I.DOC 130 21-day intervals. For subcutaneous (s.c.) inoculations 9nmoles of lipopeptide constructs were prepared in 100 [l volume of saline per dose and non-lipidated peptides formulated as an emulsion in an equal volume of complete Freund's adjuvant (CFA) for the primary injection or incomplete Freund's adjuvant for the secondary 5 inoculation. For intranasal (i.n.) inoculations, 9nmoles of peptide in 50 Pl of saline were applied to the nares of mice anaesthetised with penthrane for inhalation. Sera were prepared from blood taken at 4 weeks after the primary inoculation and two weeks after the secondary inoculation, or altenatively, from tail bleeds seven days following the final immunization. 10 Peptides comprising a CTL epitope of L. monocytogenes 5 BALB/c mice were inoculated with 9nmoles of non-lipidated peptide ([P25]-Lys [LLO91-99]), or lipidated peptide ([P25]-Lys(Pam2Cys-Ser-Ser)-[LLO91-99]) in which lipid was attached between the two epitopes at the approximate centre of the molecule, or with 1000 bacteria. In the case of peptide vaccine, inoculation was 15 subcutaneous and in the case of bacteria inoculation was intravenous. The number of interferon-y producing cells present in spleen was measured on day 28 following in vitro stimulation with the CTL epitope or no antigen. The vertical axis shows the number of interferon- y producing cells per 1,000,000 splenocytes. Peptides comprising a CTL epitope of ovalbumin 20 Each of 9 C57BL/6 mice (8-10 wks) were immunised subcutaneously with 20 nmoles of lipidated [P25]-Lys(Pam 2 Cys-Ser-Ser)-[SIINFEKL] or non-lipidated [P25]-Lys [SIINFEKL] peptide in 100 pl volume of saline. In the case of lipidated peptide, lipid was attached between the two epitopes at the approximate centre of the molecule. 25 3295672 I.DOC 131 Peptides comprising a CTL epitope of hepatitis C virus core protein Human monocyte-derived dendritic cells were incubated with lipopeptide [P25] Lys(Pam 2 Cys-Ser-Ser)-[HCV) (5pg/mL) for 48 hours before staining with FITC conjugated antibodies for HLA-DR, CD83 and CD86 before analysis by flow 5 cytometry. Challenge of immunized mice with influenza virus Penthrane anesthetized mice previously immunized with peptides comprising CTL epitopes of influenza virus were challenged intranasally (i.n.) with 104.5 PFU of infectious Mem 71 influenza virus. Each mouse received 50 pl of virus in the form of 10 allantoic fluid diluted in PBS. At 5 days after challenge, the mice were killed by cervical dislocation, and the lungs were removed and transferred aseptically to bottles containing 1.5 ml of Hank's balanced salt solution supplemented with 100 U of penicillin, 100 pg of streptomycin, and 30 pg of gentamicin per ml. Lung homogenates were prepared by using a tissue homogenizer, and the cell material was pelleted by 15 centrifugation at 300 X g for 5 min. The supernatants were removed, divided into aliquots and stored at -70'C until required. Titers of infectious virus in the lung supernantants were determined by plaque assay on monolayers of MDCK cells (Tannock et al, Infect. Immun. 431, 457-462, 1984 ). Challenge of immunized mice with L. monocytogenes 20 Mice immunized s.c. with 9nmol peptide immunogen or PBS, or i.v. with 1000 bacteria, were challenged by i.v. injection with bacteria 28 days after priming and the number of colony forming units of bacteria present in the liver determined 28 days after challenge.

3295672 I.DOC 132 Challenge of immunised mice with tumour cells Melanoma Challenge 14 days after inoculation with non-lipidated [P25]-Lys-[SI[NFEKL] or lipidated peptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[SIINFEKL], 6 mice from each group were 5 challenged with 2x10 5 melanoma cells expressing ovalbumin [B 16-OVA] and therefore expressed the CTL epitope SIINFEKL (Bellone, et al, J. Immunol. 165:2651-2656). Hair around the injection site was removed with an electric shaver prior to injection to facilitate measurement of the emerging tumors. Growing tumors were monitored, and the animals were sacrificed when tumor size reached 15 by 15 mm. Mean tumor area 10 was calculated for each treatment group at the indicated number of days after the tumor challenge. Lewis Lung Carcinoma Challenge Mice were injected with 3x10 4 Lewis Lung tumour cells that had been transfected with ovalbumin and therefore expressed the CTL epitope (Nelson et al., J Immunol. 166: 15 5557-5566, 2001). Four days after receiving tumour cells, animals were inoculated with 20 nmoles of lipidated peptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[SIINFEKL], non lipidated peptide [P25]-Lys-[SIINFEKL] or with PBS subcutaneously in the base of the tail. A second dose of immunogen was administered eleven days after receiving the tumour cells. Animals were monitored for tumour incidence and survival; animals 20 were euthanased when tumour area exceeded 100 mm 2 . Tetramer staining ofpeptide-specific CD8+ T cells CD8+ T cells specific for an immunodominant H-2Kd-restricted CTL epitope consisting of amino acid residues 147-155 of the nucleoprotein of influenza virus strain A/Puerto Rico/8/34 (PR8;HlNI) in the lipopeptide immunogen, as set forth in SEQ ID 25 NO: 1, were identified using tetrameric complexes of the H-2Kd glycoprotein with bound CTL peptide (TYQRTRALV; SEQ ID NO: 2) (Bodmer et al, Cell 52: 253-258, 3295672_.DOC 133 1988; Sherman et al, J. Exp. Med. 175: 1221-1226, 1992). The monomer was a gift from Professor Peter Doherty, Department of Microbiology and Immunology, University of Melbourne and was made at St. Jude Children's Research Hospital, Memphis TN, USA. Tetramer was made by incubating the monomer with 5 Streptavidin-phycoerythrin (Molecular Probes, Eugene, OR, USA) at a 4:1 molar ratio. Lymphocytes from the lung were first treated with 20 pL of normal mouse serum (NMS) for 5 mins at room temperature and then stained for 60 min with the tetrameric complexes at a 1:25 dilution. This was followed by staining with anti-CD8a (53-6.7) conjugated with Allophycocyanin for 30 mins on ice and washed twice and analysed by 10 a fluorescence-activated cell sorter (FACSort, Becton Dickinson, San Jose's, USA). The data were analysed by FlowJo (Tree Star, Inc, CA, USA). T-Cell culture medium T-cell culture medium consisted of RPM1 1640 (CSL Ltd.) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 2 mM L-glutamine, 2 mM sodium pyruvate, 15 30 pg of gentamicin/ml, 100 Og of streptomycin/ml, 100 IU of penicillin/ml, and 104 M 2-mercaptoethanol. Cytotoxic T-cell assays Secondary effector cells were generated either from inguinal and popliteal lymph nodes of mice that had been immunized s.c. 7 days previously with lipopeptide immunogens 20 or from spleen cells of mice primed at least 28 days previously with the lipopeptide immunogens. Briefly, 4 x 107 lymph node cells or spleen cells, depleted of erythrocytes by treatment with Tris-buffered ammonium chloride (0.15 M NH 4 CI in 17 mM Tris HCl at pH 7.2), were cultured with 107 irradiated (2,200 rads, 60Co source) virus infected or lipopeptide-pulsed syngeneic spleen cells in 25-cm2 tissue culture flasks 25 (Falcon) containing 15 ml of T-cell culture medium. The virus-infected spleen cells had been preincubated at 37'C for 30 min with 3,000 hemagglutinating units of either 3295672 I.DOC 134 infectious Mem 71 or PR8 virus in 1 ml of serum-free RPMI and washed once prior to addition to the flask. The lipopeptide-pulsed spleen cells had been preincubated at 37*C for 60 min with 100 pg of the CTL lipopeptide/ml and also washed once prior to addition to the flask. After 5 days of culture at 37'C in a humidified atmosphere 5 containing 5% C0 2 , the cells were washed three times and used in "Cr-release assays. The 5 1 Cr-release assays were performed in triplicate as described previously (Harling McNabb et al, Int. Immunol. 11, 1431-1439, 1999) by using P815 mastocytoma cells (H-2d, DBA/2) as targets. In vivo Cytotoxic T-cell assays 10 The ability of various peptide-based immunogens to induce epitope-specific CTL was determined in vivo. Groups of three mice were inoculated intranasally with various lipopeptides in 501l PBS and challenged with Mem7l on day 28. In order to analyze CTL determinant specific cytotoxicity in vivo, syngeneic spleen cells were pulsed with the CTL determinant and labelled with high intensity CFSE (2.5IM). Antigen-specific 15 lysis was controlled by co-injecting syngeneic spleen cells labelled with low intensity CFSE (0.25pM). A mixture of 15 x 106 cells of each target cell population was injected intraveniously on day 4 post-infection. The mice were killed 16 hr later and spleens were analysed for the presence of CFSE-high and CFSE-low cell populations by flow cytometry. A total of 1 x 106 lymphocytes were analysed for each sample. 20 Individual mice are represented by the closed squares and the bars represent the geometric mean titre. ELISPOT assay for IFN-y-secreting cells CTL-specific IFN-y-secreting cells were enumerated by an ELISPOT assay modified from that of Murali-Krishna et al, Immunity 8, 177-187, 1998. Flat-bottom polyvinyl 25 chloride microtiter plates (96-well: Dynatech) were coated overnight with 50 Pl of rat anti-(mouse IFN-y) antibody (clone R4-14a2) at 5 pg/ml in PBS. Unoccupied sites on 3295672 I.DOC 135 the wells were then blocked by incubation for 1 h with 10 mg of bovine serum albumin/ml in PBS, and the plates were washed three times with PBS containing 0.05% Tween 20 (PBST). Twofold dilutions of spleen or lymph node cells in T-cell medium were then added to the wells, together with 5 x 105 irradiated (2,200 rads, 60 Co source) 5 syngeneic spleen cells from unimmunized mice and 10 U of recombinant human interleukin-2 (Pharmingen, San Diego, Calif.)/well. Cells were incubated at 37'C in 5%

CO

2 for 18 h in the presence or absence of the CTL peptide at a concentration of 1 pg of peptide/ml. Cells were then lysed and removed by rinsing the plates, initially with distilled water and then PBST. Then, 50 pl of a 1/500 dilution of biotinylated anti 10 (mouse IFN-y) antibody (clone XMG 1.2; Pharmingen) was added, and the plates were incubated at room temperature for 2 h. Plates were again washed, and 50 p1 of streptavidin-alkaline phosphatase (Pharmingen; 1/400 dilution in 5 mg of bovine serum albumin/ml of PBST) was added to each well; the mixtures were then incubated for a further 2 h. The plates were washed. and 100 pl of ELISPOT substrate (Sedgwick et 15 al, J. Immunol. Methods 57, 301-309, 1983) containing 1 mg of BCIP (5-bromo-4 chloro-3-indolyphosphate) per ml of 2-amino-2-methyl-I-propynol buffer (Sigma) was added to each well. When blue-green spots had developed, the plates were washed with water and dried, and the spots were counted with the aid of an inverted microscope. 20 DI Dendritic cell cultures Dendritic cells (DC) were cultured in medium based on complete IDDM. This consisted of Iscove's Modified Dulbecco's Medium (IMDM) containing 25 mM HEPES and without alpha-thioglycerol or L-glutamine (JRH Bioscience, Lenexa, USA), supplemented with 10% (v/v) heat inactivated (56'C, 30 min) foetal calf serum 25 (CSL Ltd., Parkville, Victoria, Australia), gentamicin (24 pg/mL), glutamine (2 mM), sodium pyruvate (2 mM), penicillin (100 IU/mL), streptomycin (180 pg/mL) and 2 mercaptoethanol (0.1 mM). For DC generation complete IMDM was further supplemented with 30% supernatant from cultured NIH/3T3 cells and 5% GM-CSF in 3295672_.DOC 136 the form of a supernatant from Ag8653 cells transfected with the GM-CSF gene (DC medium). The culture method for immature dendritic cells was adapted from Winzler et al., J. Exp Med. 185, 317 (1997). Spleen cells from a BALB/c mouse were seeded at 1.5 x 5 106 cells per 55 mm dish (Techno-Plas, S.A., Australia) in 3 ml DC medium and incubated at 37*C with 5% CO 2 . All the equipment used for culturing was pyrogen free. The medium was changed every 4 days and all cells returned to the dish. On day 12, both suspended and weakly adherent cells were collected by forcefully pipetting and then aspirating the medium. The procedure was repeated with 2 ml of PBS. The 10 remaining strongly adherent cells were discarded. The collected cells were pelleted by centrifugation and reseeded into a new dish. Cells were subsequently maintained on a 4 day alternating cycle of media change and passage. After 1 month of continuous culturing, the floating and semi-adherent cells took on the appearance and staining characteristics of immature DC and are referred to as Dl cells. Under these passage 15 conditions the majority of cultured Dl cells maintain an immature phenotype characterized by an intermediate expression level of cell surface MHC class II molecules. Flow cytometric analysis of DI cells DI cells (I x 105 cells per sample) were seeded in a new Petri dish with 1 mL of DC 20 media and incubated with 0.0045 nmole of lipopeptide, dissolved in complete IMDM medium. Lipopolysaccharide (LPS) purified from E. coli serotype 0111 :B4 (Difco, Detroit, Michigan, USA, was used at 5 pg/mL as a positive control for DC maturation. After overnight incubation, the cells were harvested and washed once with PBS with 1% FCS. To prevent non-specific binding to FCORII/III, the cells were pre-incubated 25 with 20 pL of normal mouse serum for 5 mins at room temperature. The cells were then exposed to FITC-conjugated monoclonal antibody 14-4-4S (IgG2a , anti-I-Ek,d; Ozato et al., J. Immunol.,124, 533 (1980)) for 30 min on ice. Monoclonal antibody 3295672 I.DOC 137 36/1 (Brown et al., Arch Virol 114: 1, 1990), which is specific for the antigen of influenza virus from which the T-helper epitope is derived, was used as an isotype control. All antibodies were used at 2.5 pig/mL. The samples were washed once with PBS containing 1% FCS and fixed with PBS containing 4% paraformaldehyde on ice 5 for 15 minutes. Flow cytometry analysis was performed using a FACSort (Becton Dickinson, San Jose, USA) and the data were analysed using FlowJo software (Tree Star, Inc., San Carlos,CA, USA). Human Dendritic cell cultures Generation of monocyte-derived dendritic cells 10 Peripheral blood mononuclear cells (PMBCs) were prepared from buffy coat preparations obtained from blood donors (Red Cross Blood Bank, Melbourne, Australia) by Ficoll Paque (Amersham Pharmacia, Sweden) gradient separation. The cells were washed three times in PBS and incubated with optimal amounts of murine anti-CD14 hybridoma supernatant (3C10, American Type Culture Collection) for 45 15 minutes on ice. After two washes, cells were further incubated with goat anti-murine IgG microbeads (Miltenyi Biotech, Germany) according to the manufacturer's protocol. CD14+ monocytes were then positively selected by affinity purification using a magnet-activated cell sorting (MACS) column. Immature DC were generated by culturing the monocytes in GM-CSF and IL-4 (40ng/ml and 20ng/ml, respectively 20 [Schering Plough, USA]) supplemented RPMI-1640 (Gibco, USA) containing 10% FCS (CSL, Australia), 2mmol/L glutamine, 2mmol/L sodium pyruvate, 100 U/ml penicillin, 100pg/ml streptomycin, 30pg/ml gentamicin and 0.1mmol/L 2 mercaptoethanol. Cells were cultured for 5 days before use with half volume changes of media every 2 days. 25 3295672_.DOC 138 Measurement of DC maturation The ability of peptide and lipopeptide-based immunogens to up-regulate the expression of MHC class II, CD83 and CD86 on human monocyte-derived dendritic cells was determined by incubating 5 x 105 cells per ml for 2 days in medium supplemented with 5 GM-CSF and IL-4 and either LPS (5pg/mL), non-lipidated peptide [Th]-Lys-[CTL] (5pg/mL) or lipopeptide [Th]-Lys(Pam 2 Cys-Ser-Ser)-[CTL] (5pg/mL) for 48 hours. Phenotypic analysis of surface markers was performed by staining with fluorochrome conjugated monoclonal antibodies to HLA-DR (G46-6 [L243]), CD83 (HB15e), CD86 (Cat. No. 2331 [FUN-i]) and appropriate isotype matched antibodies (MOPC-21 and 10 G155-178) from Becton Dickinson (USA), according to the manufacturer's protocols. Cells were then washed, fixed in 1% formaldehyde and analysed on a flow cytometer. The histograms are representative of large granular cells gated on the forward and side scatter dot plot. The shaded regions of the histograms and the associated numerical values identify the percentage of cell populations expressing high levels of CD83, 15 CD86 or H LA-DR. EXAMPLE 2: Studies on lipopeptides comprising LHRH B cell epitopes Solubility properties of lipopeptides comprising LHRH Visual inspection of the different lipopeptide preparations comprising LHRH showed 20 that they differed markedly in their solubilities (Figure 2). Enhanced solubility was most evident in those cases where lipid was attached between the two epitopes at the approximate centre of the molecule. The lipopeptides designated [Th]-Lys(Pam 2 Cys) [B] and [Th]-Lys(Pam 3 Cys)-[B] were soluble in saline at concentrations of at least 8 mg/mi (no higher concentrations were examined), whereas constructs in which lipid 25 was attached to the N-terminus of the sequence formed opalescent solutions at concentrations as low as 0.25 mg/ml.

3295672 I.DOC 139 Efforts to further enhance the solubility of peptides with N-terminally linked lipid by the incorporation of two hydrophilic serine residues between the lipid and peptide moieties (i.e. Pam 2 Cys-Ser-Ser-[Th]-[B] and Pam 3 Cys-Ser-Ser-[Th]-[B]), proved unsuccessful. In fact the lipopeptide Pam 3 Cys-Ser-Ser-[Th]-[B] was so insoluble that it 5 could not be purified by RP-HPLC under conditions used for the other lipopeptides. We considered that the insoluble nature of this construct would prevent it from being considered as a viable proposition for manufacture as a vaccine. Immunogenicity of lipopeptides comprising LHRH B cell epilopes The three lipopeptides designated Pam 2 Cys-Ser-Ser-[Th]-[B], [Th]-Lys(Pam 2 Cys)-[B] 10 and [Th]-Lys(Pam 3 Cys)-[B], when administered s.c. in saline induced high levels of anti-LHRH antibody. In fact, antibody titres induced after two doses of these lipopeptides were similar to those obtained with [Th]-[B] or [Th]-Lys-[B] when administered in CFA (Figure 3). The titres of anti-LHRH antibodies in sera of mice that had received Pam 3 Cys-Ser-Ser-[Th]-[B] or Pam 2 Cys-[Th]-[B] were slightly lower. 15 The two soluble lipopeptides [Th]-Lys(Pam 2 Cys)-[B], [Th]-Lys(Pam 3 Cys)-[B] induced 10 to 100-fold higher levels of anti-LHRH antibody following the primary inoculation than did the other less soluble lipopeptide constructs. Two groups of five mice receiving [Th]-[B] admixed with Pam 3 Cys-Ser-(Lys) 4 in the ratio 1:1 or 1:5 did not elicit significant levels of anti-LHRH antibody, a finding that contrasts with other 20 results reported using Pam 3 Cys-Ser-(Lys) 4 as an adjuvant (Jung, G., and W. G. Bessler. (1995) In: "Immunological recognition ofpeptides in medicine and biology ", N. D. Zegers, W. J. A. Boersma, and E. Claassen, eds.. CRC Press, Boca, New York, London, Tokyo, p. 159). The results of the fertility study carried out two weeks after the second inoculation with 25 the various lipopeptides are shown in Table 3.

3295672_.DOC 140 None of the mice that received either of the two soluble lipopeptide constructs, [Th] Lys(Pam 2 Cys)-[B] or [Th]-Lys(Pam3Cys)-[B], administered in saline or the two non lipidated constructs [Th]-[B] or [Th)-Lys-[B] administered in CFA, became pregnant. One mouse from the group that received Pam 2 Cys-Ser-Ser-[Th]-[B], and two animals 5 from the groups that received Pam 3 Cys-Ser-Ser-[Th]-[B] or Pam 2 Cys-[Th]-[B] dropped litters. All members of control groups of mice that received saline in CFA or the peptide [Th]-[B] co-admixed with Pam 3 Cys-S-(Lys) 4 dropped litters. Antibody levels were followed up to 7 months after the second dose of peptide vaccine. The titres of anti-LHRH antibody present in lipopeptide-primed mice and in mice 10 primed with non lipidated peptide administered in CFA decrease between 4 and 20 fold during a 26 week period. Three months following the secondary inoculation a fertility study carried out on all mice yielded similar results to the 2 week post-immunization trial. Mice that had received the soluble lipopeptides, [Th]-Lys(Pam 2 Cys)-[B] or [Th] Lys(Pam 3 Cys)-[B], in saline or the non-lipidated [Th]-[B] and [Th]-Lys-[B] in CFA 15 were still infertile.

V) 0 ~O Co N Co oD oD oD o e - - - o o o o. M. cc CD CD CD in oo -o 0 tf 0f0 LrN tr) 00~L~ IJ Lr -H - - -H -H -H -H -n coo C) 0 0 0c E v - 5 U . NJ - N 0 0 0 0 a 00 0 -H -H -eH - H o0 r- C o m -o e -H -HJ -H -H - -H1 -H "C r.) 6 t 6 0 0 0 0 0 \ - 4 .N oo N o \ + - - - -H -H E C- t 00 It N 000 C.). 3 '-3 - oN -- (J (J oo 6 6 6 6 006 - C - a, -- cc a a 0t Co C) CD C -H -H -H -H +H 00 004 \C 0Z \Z 1 0) 00 0 rV N 0r0 A 6 6..606 6 -H -H -H -H -H -H- -H W 0000~ 00 00 (NJ C> Z Z _ C14 C1 S CS S -o 0 00 ~0 ~J - ~0 t CSE +ZH + -H + + 6u6 0 ~~~rc '0 0 0 0 N) - (NJ CS N ~ 1 ' O~4 r4 V o _______ M4 OL 142 Pam 2 Cys is a more potent adjuvant than Pam 3 Cys The results presented in Figure 3 and Table 2 indicate that the two branched lipopeptides [Th]-Lys(Pam 2 Cys)-[B] and [Th]-Lys(Pam 3 Cys)-[B] were not only more soluble but also elicited higher antibody titres, particularly in the primary antibody 5 response, than did the immunogens Pam 2 Cys-[Th]-[B], Pam 2 Cys-Ser-Ser-[Th]-[B] and Pam 3 Cys-Ser-Ser-[Th]-[B]. To examine this further, we investigated the effect of decreasing the dose on the immunogenicity of [Th]-Lys(Pam 2 Cys)-[B] and [Th]-Lys(Pam 3 Cys)-[B]. At doses of 10 nmole and 1 nmole, [Th]-Lys(Pam 2 Cys)-[B] induced higher antibody titres than did 10 [Th]-Lys(Pam 3 Cys)-[B] (Table 4). A more striking difference was observed in the mating trial; I of 5 and 0 of 5 mice receiving 10 and I nmole [Th]-Lys(Pam 2 Cys)-[B], respectively, dropped litters whereas 3 of 5 and 5 of 5 mice receiving [Th] Lys(Pam3Cys)-[B] at these doses dropped litters (Table 4). These results indicate that Pam 2 Cys-containing peptides are better immunogens than Pam 3 Cys- containing 15 peptides. The effect of including two additional serine residues into the Pam 2 Cys-containing immunogens had little or no effect on the fertility status of animals although there was an improvement in the antibody titres that were generated following the second dose (Table 4).

143 Table 4 Anti-LHRH antibody titres and fertility status of mice inoculated with different doses of peptide vaccines. Mean anti-LHRH 2Pregnancy status Inoculum antibody titre logoi) 2 (No. of animals per weeks following second group that dropped dose litters) [Th]-Lys(Pam3Cys)-[B] 3.76±0.36 3/5 [Th]-Lys(Pam3Cys)-[B] 3.22±0.51 5/5 [Th]-Lys(Pam 2 Cys)-[B] 4.22±0.33 1/5 [Th]-Lys(Pam 2 Cys)-[B] 3.61±1.18 0/5 [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] 4.64±0.23 0/5 [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] 3.92±0.65 1/5 [Th]-[B] in CFA 4.72±0.21 1/5 [Th]-[B] in CFA 3.56±0.22 3/5 Saline in CFA <2 5/5 5 'Lipopeptides were administered in saline and the non-lipidated peptide [Th]-[B] and saline controls were inoculated in CFA for the primary inoculation and incomplete Freunds adjuvant for the secondary inoculation. All vaccines were administered by the subcutaneous route. 2 Fertility experiments were initiated two weeks after the second dose of vaccine.

144 Systemic antibody responses following intranasal (i.n.) immunization We inoculated [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] and Pam 2 Cys-Ser-Ser-[Th]-[B] in saline by the intranasal route. The same vaccines were also inoculated by the subcutaneous route and the systemic anti-LHRH antibody responses were measured. 5 The solution used for inoculation of [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] was clear and the one for Pam 2 Cys-Ser-Ser-[Th]-[B] was opalescent indicating solubility differences between the two preparations. Following two intranasal inoculations, each of the vaccines induced similar titres of serum anti-LHRH antibodies which were slightly lower than those induced following 10 subcutaneous inoculation (Table 5). The more soluble [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] induced significantly higher levels of anti-LHRH antibody 4 weeks after a single dose than did the less soluble Pam 2 Cys-Ser-Ser-[Th]-[B] (p = 0.00007); in fact this was similar to the result obtained following subcutaneous inoculation. The fertility trial showed that two intranasal inoculations of [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] prevented 15 all mice from becoming pregnant in contrast to those animals receiving Pam 2 Cys-Ser Ser-[Th]-[B] intranasally in which 3 of 5 mice became pregnant. A comparison of the longevity of the responses induced by the two constructs when administered by the two different routes is also shown in Table 5. Twenty six weeks following the second dose of vaccine the levels of antibody in all mice had dropped 20 below those observed 2 weeks after receiving the second dose. The decrease in anti LHRH antibody in the group that received [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] subcutaneously, however, was much less apparent again indicating the superiority of a configuration in this context wherein Pam 2 Cys-Ser-Ser is attached at the approximate centre of the molecule. 25 -o 0. 0. .e .145 co o u -C 0 EE -oo .. -4 N N N -- m os m + - 00n i No -H -H -H -H -H L-.CC MU o 0 nC 0 -, -C) -- - eo -H -H -H -H -H w 00 - 0 o~ ~~~C V) o --'o - -H O 0- -H -H -H-> CA -H -H o t Ln (A 'A u0 -- .o N - .o 0 --- c c o-- E u U - .rn 146 We also determined the titres of individual antibody isotypes that were directed towards LHRH and obtained from animals following two subcutaneous or intranasal doses of the soluble lipopeptide [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] (Figure 4). Intranasal inoculation appeared to induce higher levels of IgG3, IgG2b and possibly IgM than did subcutaneous 5 inoculation even though the amount of total Ig induced by intranasal inoculation was less. Exposure of DC to peptides and lipopeptides induce different levels of cell surface MHC class II molecules The priming of naYve CD4+ T cells in secondary lymphoid organs by dendritic cells is preceded by maturation of DC upon exposure to antigen. This maturation is characterised 10 by up-regulation of MHC products and co-stimulatory molecules on the DC surface. We therefore determined whether the various peptides and lipopeptides could differentially activate dendritic cells in an attempt to explain the different immunogenic properties of these vaccine candidates. The results of experiments in which a line of immature DC, Dl cells, were exposed to 15 peptides, stained for surface expression of MHC class II molecules then analysed by flow cytometry, demonstrated that [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] was the most effective and Pam 2 Cys-[Th]-[B] was the least effective in causing maturation of DC (Figure 5). The ability of [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] to up-regulate class II expression approached that of bacterial lipopolysaccharide (LPS) and Pam 2 Cys-Ser-Ser[Th]-[B] and [Th] 20 Lys(Pam 2 Cys)-[B] displayed intermediate levels of activation. The non-lipidated peptide was unable to induce maturation of Dl cells greater than the 26% which occurs spontaneously in culture. The ability of the lipopeptides to induce the maturation of DI cells was concentration-dependent (data not shown). The relative abilities of these lipopeptides to induce maturation of DI cells directly reflected their ability to induce 25 antibody, providing a possible mechanism for differences in immunogenicity. Antibody responses to the C-terminal pentapeptide of LHRH As shown in Figure 6, approximately equivlaent antibody responses are elicited by lipidated [Th]-Lys(Pam 2 Cys)-[B] in which [Th] consists of CD4' T cell epitope from the light chain of influenza hemagglutinin (SEQ ID NO: 1) and [B] is LHRH 1-10 (SEQ ID 30 NO: 2) or LHRH 6-10 (i.e., the last C-terminal 5 residues of LHRH; SEQ ID NO: 4), with 147 or without a serine spacer (Ser-Ser) positioned between the lipid and peptide moieties. These data support the proposition that the usefulness of the lipopeptides is not limited to any specific amino acid sequence being used as the immunizing antigen. Lipids other than Pam 2 Cys are useful in the lipopeptide constructs 5 Groups of BALB/c mice (6-8 weeks old) were inoculated subcutaneously with 20 nmoles of the peptide immunogens shown in Figure 7, comprising the lipid moieties Pam 2 Cys; Ste 2 Cys; Lau 2 Cys; or Oct 2 Cys conjugated to the amino acid sequence set forth in SEQ ID NO: 9 (i.e. a peptide comprising the CDV-F T-helper epitope of SEQ ID NO: 24 conjugated to LHRH 2-10 as set forth in SEQ ID NO: 3, with an internal lysine residue 10 positioned between these epitopes), for both primary and secondary vaccinations. Peptide structures are shown in Figure 7. All lipopeptides were administered in saline. The non lipidated peptides was administered in CFA as a control. Sera were obtained from blood taken at 4 weeks following the primary vaccination and 2 weeks following the secondary vaccination. 15 Data shown in Figure 8 indicate that strong primary and secondary antibody responses can be obtained when the Pam 2 Cys moiety is substituted for another lipid moiety in the lipopeptide constructs. Different spacers can be used to separate lipid from peptide in the lipopeptides Groups of BALB/c mice (6-8 weeks old) were inoculated subcutaneously with 20 nmoles 20 of the peptide immunogens shown in Figure 7, comprising the lipid moiety Pam 2 Cys conjugated to the amino acid sequence set forth in SEQ ID NO: 9 and separated therefrom using a spacer consisting of a serine homodimer, arginine homodimer or 6-aminohexanoic acid. Peptide structures are shown in Figure 7. All lipopeptides were administered in saline. The non lipidated peptides was administered in CFA as a control. Sera were 25 obtained from blood taken at 4 weeks following the primary vaccination and 2 weeks following the secondary vaccination. Data shown in Figure 9 indicate that strong primary and secondary antibody responses can be obtained when the Pam 2 Cys moiety is separated from the peptide moiety in the lipopeptide constructs using a variety of different spacers. 30 148 The lipid moiety can be attached to an internal lysine residue within the T-helper epitope To determine the stringency of a requirement for positioning of the internal lysine residue to which the lipid moiety is attached, we also studied the immunogenicity of a lipopeptide construct wherein the lipid was attached to an internal lysine residue within the T-helper 5 epitope. Groups of BALB/c mice (o weeks and 4 weeks old) were inoculated subcutaneously with 20 nmoles of the peptide immunogens comprising the lipid moiety Pam 2 Cys conjugated to the amino acid sequence set forth in SEQ ID NO: 9 between the T helper epitope and B-cell epitope, or alternatively, conjugated to the amino acid sequence set forth in SEQ ID NO: 103 at position Lys-]4 within the T-helper epitope. Peptide 10 structures are shown in Figures 7 and 10. All lipopeptides were administered in saline. The non lipidated peptide was administered in CFA as a control. Sera were obtained from blood taken at 4 weeks following the primary vaccination and 2 weeks following the secondary vaccination. Data shown in Figure 11 indicate that strong antibody responses are obtainable using 15 lipopeptides wherein the lipid moiety is attached to either position, suggesting that strict placement of the internal lysine and, as a consequence, the lipid moiety, is not essential to immunogenicity. Discussion In this study we describe the assembly of a variety of lipopeptide immunogens composed 20 of a CD4' T cell epitope, the self peptide LHRH which includes one or more B cell epitopes and Pam 3 Cys or Pam 2 Cys. Without placing any strict requirement on the need for approximate central positioning of the lipid, we found that the solubility of the resulting vaccine was greatly improved by placing lipids in the approximate centre of the peptide immunogen between the T cell 25 epitope and LHRH instead of at the more usual position at the N-terminus. A clear solution in saline at the concentration required for inoculation could easily be obtained with these branched structures. In contrast, the immunogens in which the lipid was coupled at the N-terminus were less soluble, giving a cloudy or opalescent solution in saline. Investigation of the antibody responses and subsequent fertility trials indicated that the 30 water-soluble lipopeptides induced higher antibody titres 4 weeks after the primary inoculation and were also more efficient in preventing pregnancy than were the less 149 soluble lipopeptides where lipid was attached to the N-terminus. A water-soluble self adjuvanting vaccine has clear advantages over partially soluble or insoluble material allowing for simplification of the manufacturing process and also more accurate metering of dose. 5 Investigations into thte stringency of a requirement for positioning the lipid moiety indicated that some fexibility is possible, since antibody responses were also observed in immunized animals when the lipid was positioned within the T-helper epitope, rather than between the T-helper epitope and the B-cell epitope. Investigations into the effects of varying the lipopeptide dose indicated that Pam 2 Cys 10 containing lipopeptides are better immunogens than are Pam 3 Cys-containing peptides. However, other lipidopeptides were also useful in generating strong antibody responses, such as, for example, Ste 2 Cys-containing lipopeptides, Lau 2 Cys-containing lipopeptides, and Oct 2 Cys-containing lipopeptides. We found in the present study that insertion of two serine residues or two arginine residues 15 between the lipid moiety and the peptide sequence increased the potency of the resulting Pam 2 Cys-containing immunogens. When lipid is attached to the N-terminus, the two serine residues could either be acting as an inert spacer between the lipid and the peptide sequence or as an extension of the T helper cell epitope and perhaps modulating immunological activity. In those cases where lipid is coupled to the epsilon-amino group 20 of a lysine residue at the centre of the molecule, the two serine residues or two arginine residues are acting as a spacer, because the inert spacer, 6-aminohexanoic acid achieved similar results. We also found that the immunogenicity of lipopeptide constructs was not dependent upon the specific amino acid sequence of the T-helper epitope or the B-cell epitope used, 25 indicating general utility of the approach taken to producing a wide range of lipopeptides against different antigenic B-cell epitopes and in a number of different animal hosts. It is understood that macrophages are stimulated by microbial products which bind to cell surface receptors; the signal resulting from this binding event is transmitted via Toll-like receptors and results in the production of pro-inflammatory cytokines and chemokines. 30 These receptors are also present in populations of DC, and, when engaged, transmit signals 150 for cellular maturation and migration as well as for the production of molecules required for efficient antigen presentation. The various synthetic lipopeptide vaccines used in this study were found to induce the up regulation of class II MHC molecules, a marker used to assess DC maturation, on the 5 surface of immature DC. In contrast, the non-lipidated peptide construct was unable to cause maturation of DC indicating that the lipid moiety is responsible for the effect. The hierarchy of lipopeptide-induced maturation of DC reflects the hierarchy of immunogenicity exhibited by the peptide constructs implies that the ability of the vaccine to interact with and induce maturation of DC leads to a better immune response, possibly 10 by increasing the efficiency of CD4* T cell priming by DC that have been signalled to mature and migrate to the draining lymph node. The lipopeptides can trigger an immune response in the absence of additional adjuvant and can therefore be delivered by non-parenteral routes. We therefore investigated the antibody response following intranasal inoculation of Pam 2 Cys-containing peptides. The 15 results obtained here showed that intranasal inoculation of [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] or Pam 2 Cys-Ser-Ser-[Th]-[B] induced lower titres of systemic anti-LHRH antibody than those induced by inoculation by the subcutaneous route and also that the isotype profiles of immunoglobulins were different. Intranasal inoculation of the soluble lipopeptide [Th] Lys(Pam 2 Cys-Ser-Ser)-[B] induced higher levels of IgG2b and IgG3, but lower levels of 20 IgGI and IgG2a compared to subcutaneous immunization. This may indicate that the two routes of immunization result in the induction of somewhat different subsets of T cells providing help for antibody production which may, in part, be due to the different populations of DCs encountered at different sites. It may also reflect a preference that dendritic cells have for molecules with unusual geometries. 25 Intranasal inoculation of the water-soluble peptide construct [Th]-Lys(Pam 2 Cys-Ser-Ser) [B] induced significantly higher anti-LHRH antibody titres 4 weeks after the first dose of vaccine than did insoluble Pam 2 Cys-Ser-Ser-[Th]-[B]. Fertility trials carried out with these mice demonstrated that only intranasal inoculation with [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B] was able to totally prevent reproduction. Although similar antibody titres were apparent in 30 both groups of mice following the second dose of antigen, high titres of antibody were only elicited during the primary response to [Th]-Lys(Pam 2 Cys-Ser-Ser)-[B]. It is 151 therefore possible that for an immunocontraceptive vaccine to be effective, the time for which high titres of antibody are present is an important determinant of efficacy. Taken together, the measurements of antibody titres and the results of the fertility trials demonstrate that placement of Pam 2 Cys between the B cell epitope and the T helper 5 epitope, at the approximate centre of a totally synthetic peptide vaccine increases the solubility and also the immunogenicity of the vaccine. This improved immunogenicity is further improved by the introduction of two serine residues between the lipid and the peptide sequence of these branched peptide vaccines. The finding that incorporation of lipid, self-adjuvanting moieties into different positions of peptide-based vaccines 10 profoundly alters physical, immunogenic and biological properties provides another strategy for successful vaccine design. EXAMPLE 3: Studies on lipopeptides comprising a B cell epitope from the M protein of Group A streptococcus 15 The effect of multiple lipids To test whether or not immunogenicicty of the lipopeptides was dependent upon the number of lipids conjugated to the peptides, and to demonstrate that effective lipopeptides could be formulated against different antigenic B-cell epitope-containing peptides, we produced lipopeptides comprising a peptide moiety that comprises the CDV-F P25 T 20 helper epitope and a Group A Streptococcus B cell epitope J14 (i.e. the peptide moiety has the amino acid sequence of SEQ ID NO: 105), and one or two lipid moieties. The lipoamino acid moiety Pam 2 Cys-Ser-Ser was added to an internal lysine positioned between the T-helper epitope and the B-cell epitope and, in one construct, an additional lipoamino acid moiety Pam 2 Cys-Ser-Ser was also added to an N-terminal lysine in the T 25 helper epitope. Female outbred Quackenbush mice 4-6 weeks old (15/group) were inoculated intranasally with 60pg of peptide-based vaccine in a total volume of 30pl PBS. Mice received three doses of vaccine at 21-day intervals. Fecal IgA was determined 6 days following the last dose of antigen. Seven days following the final dose mice were bled from the tail vein and 30 J14-specific serum IgG was determined. Indirect bacteriocidal assays were also performed 152 to determine the ability of sera from immunized mice to opsonise or "kill" the M 1 GAS strain in vitro. Eight days following the final dose saliva was collected from individual mice and the average J14-specific salivary IgA antibody titres were determined by standard ELISA. Two weeks after the last dose of antigen, mice were challenged 5 intranasally with MI GAS strain and survival determined at various time points afterwards. Data in Figure 12 indicate that significant (P<0.05) serum IgG titres were elicited using either lipopeptide compared to non-lipidated peptides or PBS, indicating that the lipopeptide constructs are not dependent upon the selecton of T-helper or B-cell epitope, and that lipopeptides comprising single or multiple lipid moieties can be used to elicit high 10 serum IgG levels following intranasal immunization. Data presented in Figure 13 also indicate that sera collected from mice immunized with JI4-containing lipopeptides having one or two lipid moieties were also capable of significant (P<0.05) killing of GAS compared to sera collected from animals immunized with control non-lipidated peptides or PBS. 15 Data presented in Figure 14 indicate that mice inoculated J14-containing lipopeptides having one or two lipid moieties had significantly (P<0.05) higher saliva IgA titres than the control groups that were immunized control non-lipidated peptides or PBS. However, the monolipidated peptide was far superior than the bi-lipidated peptide in inducing saliva IgA levels by intranasal administration. 20 Interestingly, only mice inoculated with mono-lipidated J14-containing peptide, wherein the lipid moiety was positioned between the T-helper epitope and the B-cell epitope (i.e., [Th]-Lys(Pam 2 Cys-Ser-Ser)-[J14]) had significant (P<0.05) faecal IgA titres at 6 days following final immunization, compared to PBS or non-lipidated peptide (Figure 15). This may be a consequence of timing, since fecal IgA was determined before saliva IgA or 25 serum IgG levels were determined. Alternatively, it may be a consequence of the intranasal administration route. Other explanations cannot be excluded at present. As shown in Figure 16, mice inoculated with with mono-lipidated J14-containing peptide, wherein the lipid moiety was positioned between the T-helper epitope and the B-cell epitope (i.e., [Th]-Lys(Pam 2 Cys-Ser-Ser)-[J 14]) also demonstrated the best survival 30 following intransal challenge with GAS, compared to the bi-lipidated peptide or non- 153 lipidated peptide. However, some protective immunity was conferred by both the bi lipidated peptide and non-lipidated peptide compared to the J14 peptide alone or PBS. In summary, the data presented in Examples 2 and 3 indicate that the lipopeptide formulations of the present invention are broadly applicable to inducing strong antibody 5 responses in animals, particularly murine models, with a variety of T-helper epitopes and B-cell epitopes. Additionally, the lipopeptide formulations are particularly suited to intranasal administration, since strong IgG and IgA responses are obtained by this route. However, our data indicate that, at least for J14 immunogens, mono-lipidated peptides may serve as better mucosal adjuvants than lipopeptides comprising multiple lipid moieties. 10 EXAMPLE 4: Studies on lipopeptides comprising a B cell epitope from gastrin The immunogenicity of lipopeptide immunogens based on gastrin was determined. Female BALB/c mice were inoculated subcutaneously in the base of the tail with 20nmoles of peptide or lipopeptide immunogens. All lipopeptides were administered in PBS and the 15 non-lipidated peptides were administered in CFA. Saline emulsified with CFA was used as a negative control. The peptides used were Gastrin- 17 (sequence EGPWLEEEEEAYGWMDF; SEQ ID NO: 113); [P25]-Lys-[PentaGastrin] (SEQ ID NO: 110) in which PentaGastrin is the C-terminal sequence GWMDF of gastrin-17 (i.e., SEQ ID NO: 102); and [P25]-Lys(Pam 2 Cys-Ser-Ser)-[PentaGastrin]. 4 weeks after 20 immunisation sera was obtained from the animals and at the same time they received a second similar dose of antigen. Mice were bled a second time a further 2 week after the second dose of antigen and antibodies capable of reacting with the peptide gastrin-17 sequence determined in ELISA. As shown in Figure 17, mice inoculated with Gastrin-17 in CFA contained levels of anti 25 Gastrin-17 antibodies equivalent to the negative control of Saline in CFA. While immunisation with the non-lipidated peptide [P25]-Lys-[PentaGastrin] elicited very low levels of anti-Gastrin-17 antibodies, mice challenged with the lipopeptide [P25] Lys(Pam 2 Cys-Ser-Ser)-[PentaGastrin] demonstrated high antibody titres similar to that elicited after immunisation with the peptide in CFA. These data again illustrate that the 30 lipopeptide formulations of the present invention are broadly applicable to inducing strong antibody responses in animals, with a variety of T-helper epitopes and B-cell epitopes.

154 EXAMPLE 5: Immunogenecity of lipopeptides comprising CTL epitopes from influenza virus Lipopeptides having a CTL epitope from influenza virus and in particular the lipopeptides [Th]-Lys(Pam 2 Cys-Ser-Ser)-[CTL] and [Th]-Lys(Pam 3 Cys-Ser-Ser)-[CTL], which 5 comprise the amino acid sequence set forth in SEQ ID NO: 116 were tested for their ability to induce enhanced CTL-mediated viral clearance and to enhance dendritic cell maturation. As a negative control, a non-lipidated peptide having the amino acid sequence of SEQ ID NO: 116 was used in all experiments. Viral Clearance 10 The lipopeptides elicited a higher level of viral clearance than non-lipidated peptides (Figures 20, 21 a). Viral load in the lungs of mice primed with the lipopeptides and challenged with infectious Mem 71 virus 9 days later was reduced by 95% ([Th] Lys(Pam3Cys-Ser-Ser)-[CTL]; Figure 20) or 99% ([Th]-Lys(Pam 2 Cys-Ser-Ser)-[CTL]; Figure 20) compared to samples from mice immunized with PBS alone. In contrast, non 15 lipidated peptide achieved only a 65% reduction in viral load ([Th]-Lys-[CTL]; Figure 3). Enhanced viral clearance was also observed in lipopeptide-inoculated animals that had been challenged with Mem 71 virus 28 days after the initial inoculation. In contrast, the ability to clear virus is significantly weaker at this time point in mice inoculated with the non-lipidated peptide. 20 As shown in Figure 21 b, there was also enhanced CD8+ T cell activation in immunized mice receiving the lipopeptides referred to in the legend to Figure 19, compared to mice receiving only non-lipidated peptide or PBS as seen by the number of CD8+ T calls found in the BAL fluids. Dendritic Cell maturation 25 The priming of naive CD4+ T cells and CD8+ T cells in secondary lymphoid organs by dendritic cells is preceded by maturation of DC upon exposure to antigen epitope. This maturation is characterised by up-regulation of MHC products and co-stimulatory molecules on the DC surface. We therefore determined whether the various peptides and lipopeptides could differentially activate dendritic cells in an attempt to explain the 30 different immunogenic properties of these vaccine candidates.

155 The results of experiments in which a line of immature DC, Dl cells, were exposed to peptides, stained for surface expression of MHC class II molecules then analysed by flow cytometry, demonstrated that there was enhanced maturation of dendritic cells following their exposure to the peptides [Th)-Lys(Pam 3 Cys-Ser-Ser)-[CTL] or [Th]-Lys(Pam 2 Cys 5 Ser-Ser)-[CTL] compared to [Th]-Lys-[CTL] peptide or medium alone (Figure 21c). [Th]-Lys(Pam 2 Cys-Ser-Ser)-[CTL] was the most effective and the non-lipidated peptide [Th]-Lys-[CTL) was the least effective in causing maturation of DC, with [Th] Lys(Pam 3 Cys-Ser-Ser)-[CTL] being nearly as effective as [Th]-Lys(Pam 2 Cys-Ser-Ser) [CTL] (Figure 21c). The ability of the lipidated peptide [Th]-Lys(Pam 2 Cys-Ser-Ser) 10 [CTL] to up-regulate class II expression was the same as for bacterial lipopolysaccharide (LPS). The non-lipidated peptide was unable to induce maturation of Dl cells greater than about 26%, a level that occurs spontaneously in culture. The relative abilities of these lipopeptides to induce maturation of Dl cells directly reflected their ability to induce CTL mediated viral clearing responses and CD8+ T cells in the BAL. 15 Effects of different lipids on cylotoxicity and T cell proliferation in vitro and in vivo The effects of conjugating different lipids, including PamiCys, Pam 2 Cys, Pam 3 Cys, palmitic acid and cholesterol, to the peptide immunogen were also determined. As shown in Figure 22, viral load in the lungs of mice primed with Pam 2 Cys-containing lipopeptides were lower than for mice primed with lipopeptides comprising the other lipids 20 tested, suggesting that Pam 2 Cys is preferred for conferring protection against virus. All lipids however, offered some protection against virus. This effect was also reflected in the IFN-gamma CD8+ T cell count (Figure 23). Collectively, these data suggest that it is important to attach the lipid to the cysteine glycerol residue, as in the [Th]-Lys(Pam 2 Cys Ser-Ser)-[CTL] epitope structure, for maximum cytotoxic effect. 25 In tetramer assays, the highest number of tetramer positive CD8+ T cells per lung were observed for lipopeptides wherein the lipid moiety was added to the epsilon amino group of an internal lysine residue (e.g., lipopeptides [Th]-Lys(PamiCys-Ser-Ser)-[CTL], [Th] Lys(Pam 2 Cys-Ser-Ser)-[CTL], [Th]-Lys(Pam 3 Cys-Ser-Ser)-[CTL], and [Th] Lys(Chol2Lys-Ser-Ser)-[CTL] in Figure 24) compared to non-lipidated peptide or 30 lipopeptide having lipid added to the N-teminus of the peptide (e.g., construct Pal 2 LysLys[Th]-[CTL] in Figure 24). These data also confirm that the positioning of the 156 lipid internal to the peptide, by attachment to the epsilon amino group of an internal lysine residue, enhances cytotoxic activity of the CTL epitope. To analyze CTL determinant specific cytotoxicity in vivo, mice were inoculated intranasally with 9nmoles of various lipopeptides in PBS and challenged with Mem7l 5 virus on day 28. CTL determinant-specific cytotoxicity in vivo was measured using syngeneic spleen cells pulsed with the CTL determinant and labelled with high intensity CFSE. Non-pulsed spleen cells labelled with low intensity CFSE were used as a control. A mixture of cells of each target cell population was injected intraveniously on day 4 post infection. The mice were killed 16 hr later and spleens were analysed for the presence of 10 CFSE-high and CFSE-low cell populations by flow cytometry. A total of 1 x 106 lymphocytes were analysed for each sample. Data in Figure 25 is a graphical representation showing cytotoxic T cell activity in naive mice. Figure 26 indicates that the lipopeptide [Th]-Lys(Pam 2 Cys-Ser-Ser)-[CTL] comprising the CD4+ T-helper epitope set forth in SEQ ID NO: 18 and the H-2d-restricted CTL epitope set forth in SEQ ID NO: 113, 15 induced significant cytotoxicity in vivo. As shown in Figure 27, lipopeptides have higher activity than non-lipidated peptide, with the lipopeptides designated [Th]-Lys(PamiCys-Ser-Ser)-[CTL], [Th]-Lys(Pam 2 Cys-Ser Ser)-[CTL] and [Th]-Lys(Pam 3 Cys-Ser-Ser)-[CTL] providing a marked enhancement of specific lysis in vivo compared to the non-lipidated peptide [Th]-Lys-[CTL] and other 20 lipopeptides tested. These data again confirm that positioning of the lipid internal to the peptide, by attachment to the epsilon amino group of an internal lysine residue, enhances cytotoxic activity of the CTL epitope in vivo. EXAMPLE 6: Immunogenicity of lipopeptides comprising a CTL epitope from L. 25 monocytogenes A lipopeptide having a CTL epitope from L. monocytogenes and in particular the lipopeptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[LLO91-99] comprising the amino acid sequence set forth in SEQ ID NO: 248 was tested for its ability to induce a CD8+ T cell response, and to protect against a challenge with L. monocytogenes. As a negative control, PBS or a 30 non-lipidated peptide having the amino acid sequence of SEQ ID NO: 248 was used in all experiments. Isolated bacteria were used as a positive control.

157 IFN-y production by splenocytes The lipopeptide tested in this study induced a specific CD8+ T cell response against the immunizing CTL epitope, as evidenced by the enhanced number of IFN- y producing splenocytes present in mice immunized with lipidated peptide relative to non-lipidated 5 peptide. Mice immunized with 9nmoles lipidated peptide vaccine [P25]-Lys(Pam 2 Cys Ser-Ser)-[LLO91-99] comprising the amino acid sequence set forth in SEQ ID NO: 248 produced about 15-fold more IFN- y producing cells per million splenocytes than mice receiving non-lipidated peptide or a PBS control, indicating an enhanced activation of IFN y producing CD8+ T cells in the mice receiving the lipidated peptide (Figure 28). 10 Protection against challenge with isolated bacteria Data in Figure 29 indicate that the lipidated [P25]-Lys(Pam 2 Cys-Ser-Ser)-[LLO91-99] peptide successfully provides protection against a subsequent challenge with whole bacteria. A significantly enhanced protection was also observed in mice immunized with the lipidated [P25]-Lys(Pam 2 Cys-Ser-Ser)-[LLO91-99] peptide relative to mice 15 immunized with non-lipidated [P25]-Lys-[LLO91-99] peptide or PBS (i.e. non-immunized mice). EXAMPLE 7: Protection against challenge with tumour cells Protection against challenge with Melanoma cells 20 The ability of the lipopeptide vaccine containing the ovalbumin CTL epitope (SI[NFEKL) to induce protection against melanoma cells expressing this CTL epitope (B 16-OVA cells) was assessed. IFN- y production was determined in mice inoculated with lipopeptide comprising a CDV-F T-helper epitope (P25) and a CTL epitope (SIINFEKL) of ovalbumin linked via the epsilon amino group of an internal lysine residue positioned between said 25 epitopes to Pam 2 Cys (i.e. the peptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[SIINFEKL] listed in Figure 19 and based upon SEQ ID NO: 247). C57BL/6 mice were vaccinated with 20 moles lipidated peptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[SIINFEKL], non-lipidated peptide [P25]-Lys-[SIINFEKL] or with PBS subcutaneously in the base of the tail. Mice were then challenged subcutaneously on the back 14 days later with B 16-OVA cells. 30 Splenocytes were obtained from the inoculated animals and stimulated in vitro with the 158 CTL epitope having the sequence SIINFEKL and the number of IFN- y producing cells per 1,000,000 splenocytes was measured. Data show enhanced numbers of IFN- y producing cells for mice inoculated with lipopeptide (Table 6), indicating an enhanced ability of the lipopeptides to activate T cells relative to non-lipidated peptide. 5 Importantly, control of tumour growth was elicited by immunisation with lipopeptide compared to mice immunized with the non-lipidated peptide [P25]-Lys-[SIINFEKL] or PBS alone (Figure 30). No tumour growth was observed over a 15 day period in mice immunised with [P25]-Lys(Pam 2 Cys-Ser-Ser)-[SIINFEKL]. Conversely, tumours of greater than 75mm2 in diameter were observed in mice immunised with [P25)-Lys 10 [SIINFEKL] or PBS alone. Together, these data confirm the protective ability of the lipopeptide compared to non-lipidated peptide in protection against tumours. Table 6 Numbers of IFN-y secreting splenocytes in representative melanoma samples receiving [P25]-Lys(Pam 2 Cys-Ser-Ser)-[SI[NFEKL] lipopeptide compared to non-lipidated [P25] 15 Lys-[SIINFEKL] peptide or PBS PEPTIDE/LIPOPEPTIDE IMMUNOGEN No. IFN-y secreting [P25]-Lys(Pam 2 Cys-Ser-Ser)- [P25]-Lys- PBS splenocytes per 106 [SIINFEKL [SIINFEKL] splenocytes 284 18 5 205 14 0 192 10 0 Average 227 14 3 Std. deviation 49 4 24 Protection against challenge with Lewis Lung tumour cells The ability of the lipopeptide to provide protection against Lewis Lung tumor development in animals in vivo was also tested. Mice were injected with 3x10 4 Lewis Lung tumour cells transfected with ovalbumin and therefore expressing the CTL epitope SIINFEKL 159 (Nelson et al., J Immunol. 166: 5557-5566, 2001). Four days after receiving tumour cells, animals were inoculated subcutaneously in the base of the tail with 20nmoles lipidated peptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[SIfNFEKL], or alternatively, non-lipidated peptide [P25]-Lys-[SI[NFEKL] or PBS. A second and similar dose of immunogen was 5 administered eleven days after receiving the tumour cells. Data in Figure 31 indicate that the percentage of animals with fewer lesions developing was significantly higher for animals receiving the lipopeptide compared to animals receiving the non-lipidated peptide or PBS. As shown in Figure 32, animals receiving the lipopeptide immunogen also survived for longer than those receiving the non-lipidated peptide or PBS. These data 10 further confirm the protective ability of the lipopeptide compared to non-lipidated peptide for protection against tumours. EXAMPLE 8: Enhanced expression of MHC class II, CD83 and CD86 on human dendritic cells following administration of a lipopeptide comprising a CDV-F T 15 helper epitope and a CTL epitope from hepatitis C virus The lipopeptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[HCV] described in the legend to Figure 19 was tested for its ability to up-regulate the expression of MHC class II, CD83 and CD86 on human dendritic cells. Human monocyte-derived dendritic cells were incubated with media alone, LPS (5pg/mL), non-lipidated peptide [P25]-Lys-[HCV] (5pg/mL) or 20 lipopeptide [P25]-Lys(Pam 2 Cys-Ser-Ser)-[HCV] (5pg/mL) for 48 hours before staining with FITC-conjugated antibodies for HLA-DR, CD83 and CD86 before analysis by flow cytometry. Data shown in Figure 33 demonstrate a higher percentage of dendritic cell populations that express HLA-DR, CD83 and CD86 antigens on their cell surface are present following treatment with lipidated peptide than following treatment with non 25 lipidated peptide or PBS alone. The ability of the lipopeptide to induce maturation of human dendritic cells directly reflected the immunogenic ability of the lipopeptide compared to the non-lipidated peptide, providing a possible mechanism for immunogenicity. 30 160 Discussion In this study we describe the assembly of a variety of lipopeptide immunogens composed of a CD4+ T cell epitope, a CD8+ CTL epitope and Pam 3 Cys or Pam 2 Cys linked thereto via the epsilon amino group of an internal lysine residue. 5 The precise nature of the lipid moiety in generating an immune response was not shown to be critical, because a range of lipids, including cholesterol, palmitic acid, PamiCys, Pam 2 Cys, and Pam 3 Cys were shown to successfully elicit T cell proliferation and cytotoxicity. However, significant differences were observed in terms of protection and IFN-y production, at least in the case of lipopeptides directed against influenza virus, 10 suggesting that lipid structure may be an important consideration in vivo. In particular, at least for vaccines incorporating the influenza virus CTL epitope, Pam 2 Cys linked to the epsilon amino group of an internal lysione residue in the peptide weremost effective in conferring protection, suggesting that a linkage to the cysteine glycerol is preferred. The lipopeptides of the invention are effective in enhancing the CD8+ T cell responses of 15 immunized animals against bacterial and viral pathogens and also against tumour cells. Given the success of the self-adjuvanting peptides exemplified herein to protect against viral and bacterial pathogens as well as tumour cells, it is reasonable to expect that this technology is generally applicable to a wide range of vaccination protocols. Insertion of serine residues between the lipid moiety and the peptide sequence does not 20 adversely affect the potency of the resulting Pam2Cys-containing immunogens. The lipopeptides can trigger an immune response in the absence of additional adjuvant and can be delivered by both parenteral and non-parenteral routes, particularly intranasally. Taken together, the data provided herein demonstrate that placement of a wide range of lipids, including but not limited to Pam 2 Cys and Pam 3 Cys, between the CTL epitope and 25 the T helper epitope, at the approximate centre of a totally synthetic peptide vaccine increases the immunogenicity of the vaccine.

Claims

1. A lipopeptide comprising a T helper cell (Th) epitope and a B cell epitope or a CTL epitope, wherein the amino acid sequences of the Th epitope is different from the amino acid sequence of the B cell or CTL epitope; one or more internal lysine residues or internal 5 lysine analog residues and one or more lipid moieties wherein said lipid moieties are covalently attached to said internal lysine residues or internal lysine analog residues.

2. A lipopeptide according to claim I wherein said one or more lipid moieties is covalently attached to an epsilon-amino group of said one or more internal lysine residues or to a terminal side-chain group of said one or more internal lysine analog residues. 10

3. The lipopeptide of claim I or 2 wherein the lipid is attached to the epsilon-amino group of a lysine residue.

4. The lipopeptide of any one of claims 1 to 3 wherein the internal lysine residue to which a lipid moiety is attached is positioned between the Th epitope and the CTL epitope.

5. The lipopeptide of any one of claims 1 to 3 wherein the internal lysine residue to 15 which a lipid moiety is attached is positioned within the Th epitope.

6. The lipopeptide according to any one of claims I to 5 wherein the lipid moiety has a structure of General Formula (VII): Formula (VII) R1-NH-CH-COOH 20 (CH 2 )m (CH 2 )n R 2 -CH R3-CH2 25 wherein: 162 X is selected from the group consisting of sulfur, oxygen, disulfide (-S-S-), and methylene (-CH 2 -), and amino (-NH-); m is an integer being I or 2; n is an integer from 0 to 5; 5 R, is selected from the group consisting of hydrogen, carbonyl (-CO-), and R'-CO wherein R' is selected from the group consisting of alkyl having 7 to 25 carbon atoms, alkenyl having 7 to 25 carbon atoms, and alkynyl having 7 to 25 carbon atoms, wherein said alkyl, alkenyl or alkynyl group is optionally substituted by a hydroxyl, amino, oxo, acyl, or cycloalkyl group; 10 R 2 is selected from the group consisting of R'-CO-O-, R'-O-, R'-O-CO-, R'-NH-CO-, and R'-CO-NH-, wherein R' is selected from the group consisting of alkyl having 7 to 25 carbon atoms, alkenyl having 7 to 25 carbon atoms, and alkynyl having 7 to 25 carbon atoms, wherein said alkyl, alkenyl or alkynyl group is optionally substituted by a hydroxyl, amino, oxo, acyl, or cycloalkyl group; and 15 R 3 is selected from the group consisting of R'-CO-O-, R'-O-, R'-O-CO-, R'-NH-CO-, and R'-CO-NH-, wherein R' is selected from the group consisting of alkyl having 7 to 25 carbon atoms, alkenyl having 7 to 25 carbon atoms, and alkynyl having 7 to 25 carbon atoms, wherein said alkyl, alkenyl or alkynyl group is optionally substituted by a hydroxyl, amino, oxo, acyl, or cycloalkyl group 20 and wherein each of RI, R 2 and R 3 are the same or different.

7. The lipopeptide of claim 6 wherein X is sulfur; m and n are both 1; R, is selected from the group consisting of hydrogen, and R'-CO-, wherein R' is an alkyl group having 7 to 25 carbon atoms; and R 2 and R 3 are selected from the group consisting of R'-CO-O-, R' 0-, R'-O-CO-, R'-NH-CO-, and R'-CO-NH-, wherein R' is an alkyl group having 7 to 25 25 carbon atoms.

8. The lipopeptide of claim 7 wherein R' is selected from the group consisting of: palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, decanoyl, and cholesterol. 163

9. The lipopeptide according to any one of claims 6 to 8 wherein the lipid is contained within a lipoamino acid moiety selected from the group consisting of: PamiCys, Pam 2 Cys, Pam 3 Cys, Chol 2 Lys, Ste 2 Cys, Lau 2 Cys, and Oct 2 Cys.

10. The lipopeptide according to claim 9 wherein the lipoamino acid moiety is 5 Pam 2 Cys.

11. The lipopeptide according to any one of claims I to 5 wherein the lipid moiety has the following General Formula (VIII): ___H R 4 -HN C-COOH R5 wherein: 10 R 4 is selected from the group consisting of: (i) an alpha-acyl-fatty acid residue consisting of between about 7 and about 25 carbon atoms; (ii) an alpha-alkyl-beta-hydroxy-fatty acid residue; (iii) a beta-hydroxy ester of an alpha-alkyl-beta-hydroxy-fatty acid residue; and (iv) a lipoamino acid residue; and R 5 is hydrogen or the side chain of an amino acid residue. 15

12. The lipopeptide according to any one of claims I to 1I wherein the lipid moiety is separated from the peptide moiety by a spacer.

13. The lipopeptide of claim 12 wherein the spacer comprises arginine, serine or 6 aminohexanoic acid.

14. The lipopeptide of claim 12 or 13 wherein the spacer consists of a serine 20 homodimer.

15. The lipopeptide according to any one of claims 1 to 14 wherein the internal lysine or internal lysine analog is nested within a synthetic amino acid sequence having low immunogenicity. 164

16. The lipopeptide according to any one of claims I to 15 wherein the T-helper epitope is a T-helper epitope of influenza virus haemagglutinin or a T-helper epitope of canine distemper virus F (CDV-F) protein.

17. The lipopeptide of claim 16 wherein the T-helper epitope of influenza virus 5 haemagglutinin comprises the amino acid sequence set forth in SEQ ID NO: 1.

18. The lipopeptide of claim 16 wherein the T-helper epitope of CDV-F protein comprises the amino acid sequence set forth in SEQ ID NO: 20.

19. A lipopeptide comprising a T helper cell (Th) epitope and a B cell epitope or a CTL epitope, wherein the amino acid sequences of the Th epitope is different from the amino 10 acid sequence of the B cell or CTL epitope; one or more internal lysine residues or internal lysine analog residues and one or more lipid moieties wherein said lipid moieties are covalently attached to said internal lysine residues or internal lysine analog residues and said lipopeptide is of general Formula (VI): H epotipe-A- HN-C-CO- A- epitope (CH2)n X 15 wherein: one of the epitopes is a T-helper epitope and the other is a B cell epitope or a CTL epitope; A is either present or absent and consists of an amino acid spacer of about I to about 6 amino acids in length; n is an integer having a value of 0, 1, 2, 3, or 4; 20 X is a terminal side-chain group selected from the group consisting of NH, 0 and S; Y is either present of absent and consists of an amino acid spacer of about I to about 6 amino acids in length; and 165 Z is a lipid moiety.

20. The lipopeptide of claim 19 wherein A is absent.

21. The lipopeptide of claim 19 or 20 wherein Y is present and consists of a serine homodimer. 5

22. The lipopeptide according to any one of claims 19 to 21 wherein Z is selected from the group consisting of: PamiCys, Pam 2 Cys, Pam 3 Cys, Chol 2 Lys, Ste 2 Cys, Lau 2 Cys, and Oct 2 Cys.

23. A composition comprising the lipopeptide according to any one of claims 1 to 22 and a pharmaceutically acceptable excipient or diluent. 10 Dated: 6 June 2006 The Council of the Queensland Institute of Medical Research Patent Attorneys for the Applicant: 15 BLAKE DAWSON WALDRON PATENT SERVICES