EP2121007A1

EP2121007A1 - Circular ccr5 peptide conjugates and uses thereof

Info

Publication number: EP2121007A1
Application number: EP07858126A
Authority: EP
Inventors: Adrian Huber; Martin Bachmann; Gary Jennings; Alain Tissot; Johannes Langedijk; Peter Timmerman; Jelle Slootstra; Ronald Boshuizen
Original assignee: Cytos Biotechnology AG; Pepscan Systems BV
Current assignee: Cytos Biotechnology AG; Pepscan Systems BV
Priority date: 2006-12-21
Filing date: 2007-12-21
Publication date: 2009-11-25
Also published as: WO2008074895A1; AU2007336132A1; CA2672826A1

Abstract

The present invention is in the fields of medicine, public health, immunology, molecular biologyand virology. The invention provides composition comprising a virus-like particle (VLP) linked to atleast one antigen, wherein said antigen is CCR5 PNt domain comprising one looped peptidic structure. The invention also provides a process for producing the composition. The compositions ofthis invention are useful in the production of vaccines, in particular, for the prevention and treatment of HIV infection. Moreover, the compositions of the invention induce efficient immune responses, in particular antibody responses.

Description

CIRCULAR CCR5 PEPTIDE CONJUGATES AND USES THEREOF

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The present invention is in the fields of medicine, public health, immunology, molecular biology and virology. The invention provides composition comprising a virus-like particle (VLP) linked to at least one antigen, wherein said antigen is CCR5 PNt domain comprising one looped peptidic structure.

[0002] The invention also provides a process for producing the composition. The compositions of this invention are useful in the production of vaccines, in particular, for the prevention and treatment of HIV infection. Moreover, the compositions of the invention induce efficient immune responses, in particular antibody responses.

Related Art

[0003] HIV R5 strains use the cell surface molecules CD4 and CCR5 for attachment and entry into macrophages and CD4+ T cells. CCR5 is a 7-transmembrane receptor with an N-terminal sequence and three loops exposed to the extracellular space, which are called subsequently PNt, ECL-I, ECL-2, and ECL-3, respectively. The natural CCR5 ligands, RANTES, MIP- lα, MIP- IB and analogs thereof are able to block the virus-coreceptor interaction and further cause the internalization of CCR5 (Lederman et al, 2004, Science 306, p485). CCR5 specific auto-antibodies have been found in 12.5% women that were repeatedly exposed to HIV but remained uninfected (Lopalco et al., 2000, J. Immunology 164, 3426). These antibodies were shown to bind the first extracellular loop (ECL-I) of CCR5 and could inhibit R5-tropic HIV infection of peripheral blood mononuclear cells (PBMC). AlIo immunisation in women led to CCR5 specific antibodies that were capable of inhibiting R5-HIV infection in vitro (Wang et al., 2002, Clin. Exp. Immunol. 129, 493). [0004] Monoclonal CC-CCR5 antibodies are able to prevent HIV infection in vitro

(Olson et al., 1999, J. Virol. 73, 4145; Wu and LaRosa et al., 1997, J. Exp. Med. 186, 1373). Antibodies produced by immunizing monkeys with linear CCR5 peptides (from the N- terminal, the ECL-I, or the ECL-2 sequence) have viral inhibitory effect in vitro (Lehner et al., 2001, J. Immunology 166, 7446). The CCR5 PNt domain was displayed on papillomavirus like particles and immunized pig-tailed macaques. This vaccine induced high- avidity anti-CCR5 IgG autoantibody responses, and all five immunized macaques generated IgG that could block infection of CCR5-tropic simian/human immunodeficiency virus SHIV SF 162P3 in vitro (Chackerian et al, 2004, J. Virol. 78, 4037).

SUMMARY OF THE INVENTION

[0005] We have, now, surprisingly found that the inventive compositions and vaccines, respectively, comprising at least one CCR5 PNt domain comprising one looped peptidic structure linked to a virus-like particle, are capable of inducing immune responses, in particular antibody responses, leading to high antibody titer against CCR5. Moreover, we have surprisingly found that inventive compositions and vaccines, respectively, are capable of inducing immune responses, in particular antibody responses, with protective and/or therapeutic effect against the HIV infection. This indicates that the immune responses, in particular the antibodies generated by the inventive compositions and vaccines, respectively, are, thus, capable of specifically recognizing HIV and/or HIV infected cells in vivo, and neutralizing and inhibiting the infection of the virus.

[0006] Thus, in the first aspect, the present invention provides a composition comprising: (a) a virus-like particle with at least one first attachment site; (b) at least one antigenic peptide with at least one second attachment site, wherein said antigenic peptide comprises: (i) CCR5 PNt domain comprising an amino acid sequence KINVK (SEQ ID NO:25); (ii) a first amino acid comprising a first reactive group and wherein said first amino acid is located at the N-terminal of KINVK; (iii) a second amino acid comprising a second reactive group and wherein said second amino acid is located at the C-terminal of KINVK, wherein said first and said second amino acid does not comprise said second attachment site linking said first attachment site; wherein said first reactive group binds to said second reactive group by at least one covalent bond so that the peptide starting from said first amino acid and ending with said second amino acid is looped; and wherein said virus-like particle and said at least one antigenic peptide are linked through said first and said second attachment site.

[0007] In one preferred embodiment of the invention, the virus-like particle is a VLP of an RNA-bacteriophage. In one preferred embodiment, the virus-like particle suitable for use in the present invention comprises recombinant protein, preferably recombinant coat protein, mutants or fragments thereof, of a virus, preferably of an RNA bacteriophage. [0008] In another aspect, the present invention provides a method of preventing and/or treating HIV infection, wherein the method comprises administering the inventive composition or the inventive vaccine composition, respectively, to a human.

BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1 The antigenic peptides coupled to Qβ shown by coomassie stained

SDS-PAGE. Lane 1 : marker bands, Lane 2 and 4: Qβ-SMPH; lane 3: Qβ-P36; and lane 5: Qβ-P37.

[0010] FIG. 2 CCR5+ cells in a FACS staining assay. Individual sera from mice immunized with Qβ-P36 (day 21) (FIG. 2A) or Qβ-P37 (day 65) (FIG. 2B) were tested for staining CCR5+ cells in a FACS staining assay. Analysed was the percentage of live (propidium negative) cells to which the sera bound. A serum was considered positive, if more than 10% cells were stained CCR5+ by the serum. Monoclonal antibodies 45531 and 2D7 were used as positive controls.

[0011] FIG. 3 HIV neutralisation assay. R5 tropic pseudotype HIV viruses were used to infect human CD8 depleted PBMC cells. The y-axis represents the concentration of antibodies needed for 50% (black bar), 70% (white bar) or 90% (stripe bar) neutralization. Mab PAl 4 was used as appositive control and Total IgG raised against Qb was used as a negative control.

[0012] FIG. 4 Sequence of P16 peptide indicating the reactive groups and the looped structures.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0014] Antigen: As used herein, the term "antigen" refers to a molecule capable of being bound by an antibody or a T cell receptor (TCR) if presented by MHC molecules. The term "antigen", as used herein, also encompasses T-cell epitopes. An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes. This may, however, require that, at least in certain cases, the antigen contains or is linked to a Th cell epitope and is given in adjuvant. An antigen can have one or more epitopes (B- and T- epitopes). The specific reaction referred to above is meant to indicate that the antigen will preferably react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be evoked by other antigens. Antigens as used herein may also be mixtures of several individual antigens.

[0015] Antigenic site: The term "antigenic site" and the term "antigenic epitope", which are used herein interchangeably, refer to continuous or discontinuous portions of a polypeptide, which can be bound immunospecifically by an antibody or by a T-cell receptor within the context of an MHC molecule. Immunospecifϊc binding excludes non-specific binding but does not necessarily exclude cross-reactivity. Antigenic site typically comprise 5- 10 amino acids in a spatial conformation which is unique to the antigenic site. [0016] Associated: The term "associated" (or its noun association) as used herein refers to all possible ways, preferably chemical interactions, by which two molecules are joined together. Chemical interactions include covalent and non-covalent interactions. Typical examples for non-covalent interactions are ionic interactions, hydrophobic interactions or hydrogen bonds, whereas covalent interactions are based, by way of example, on covalent bonds such as ester, ether, phosphoester, amide, peptide, carbon-phosphorus bonds, carbon- sulfur bonds such as thioether, or imide bonds.

[0017] Attachment Site, First: As used herein, the phrase "first attachment site" refers to an element which is naturally occurring with the VLP or which is artificially added to the VLP, and to which the second attachment site may be linked. The first attachment site may be a protein, a polypeptide, an amino acid, a peptide, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (bio tin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxy 1 group, a guanidinyl group, histidinyl group, or a combination thereof. A preferred embodiment of a chemically reactive group being the first attachment site is the amino group of an amino acid such as lysine. The first attachment site is located, typically on the surface, and preferably on the outer surface of the VLP. Multiple first attachment sites are present on the surface, preferably on the outer surface of virus-like particle, typically in a repetitive configuration. In a preferred embodiment the first attachment site is associated with the VLP, through at least one covalent bond, preferably through at least one peptide bond. In a further preferred embodiment the first attachment site is naturally occurring with the VLP. Alternatively, in another preferred embodiment the first attachment site is artificially added to the VLP.

[0018] Attachment Site, Second: As used herein, the phrase "second attachment site" refers to an element which is naturally occurring with or which is artificially added to the antigenic peptide of the invention and to which the first attachment site may be linked. The second attachment site may be a protein, a polypeptide, a peptide, an amino acid, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidinyl group, histidinyl group, or a combination thereof. A preferred embodiment of a chemically reactive group being the second attachment site is a sulfhydryl group, preferably a sulfhydryl group of cysteine. The term "antigenic peptide with at least one second attachment site", as used herein, refers, to a construct comprising the antigenic peptide and at least one second attachment site. In one preferred embodiment, the second attachment site is naturally occurring within the antigenic peptide. In another preferred embodiment, the second attachment site is artificially added to the antigenic peptide. In one preferred embodiment the second attachment site is associated with the antigenic peptide through at least one covalent bond, preferably through at least one peptide bond. In one preferred embodiment, the antigenic peptide with at least one second attachment site further comprises a linker, preferably said linker comprises at least one second attachment site, preferably said linker is fused to the antigenic peptide by a peptide bond.

[0019] Bound: As used herein, the term "bound" refers to binding or attachment that may be covalent, e.g., by chemically coupling, or non-covalent, e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc. Preferably the term "bound" refers to binding or attachment that is covalent. Covalent bonds can be, for example, ester, ether, phosphoester, amide, peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds, and the like. [0020] CCR5 PNt domain: The term "CCR5 PNt domain", as used herein, should encompass any polypeptide comprising, consisting essentially of, or alternatively or preferably consisting of human CCR5 PNt domain of SEQ ID NO:22. Moreover, the term "CCR5 PNt domain", as used herein, should also encompass any polypeptide comprising, consisting essentially of, or alternatively or preferably consisting of, any natural or genetically engineered variant having more than 70%, preferably more than 80%, preferably more than 85%, even more preferably more than 90%, even more preferably more than 93%, again more preferably more than 95%, and most preferably more than 97% amino acid sequence identity with the CCR5 PNt domain as defined above with the proviso that said polypeptide comprises the sequence KINVK (SEQ ID NO:25). The term "CCR5 PNt domain" as used herein should furthermore encompass post-translational modifications including but not limited to glycosylations, acetylations, phosphorylations of the CCR5 PNt domain as defined above. Preferably the CCR5 CCR5 PNt domain, as defined herein, consists of at most 50, even more preferably at most 40 amino acids in length. Typically and preferably, CCR5 PNt domain, preferably when linked to the virus-like particle of the invention, is capable of inducing in vivo the production of antibody specifically binding to CCR5.

[0021] Coat protein: The term "coat protein" and the interchangeably used term

"capsid protein" within this application, refers to a viral protein, preferably a subunit of a natural capsid of a virus, preferably of an RNA-bacteriophage, which is capable of being incorporated into a virus capsid or a VLP.

[0022] Linked: The term "linked" (or its noun: linkage) as used herein, refers to all possible ways, preferably chemical interactions, by which the at least one first attachment site and the at least one second attachment site are joined together. Chemical interactions include covalent and non-covalent interactions. Typical examples for non-covalent interactions are ionic interactions, hydrophobic interactions or hydrogen bonds, whereas covalent interactions are based, by way of example, on covalent bonds such as ester, ether, phosphoester, amide, peptide, carbon-phosphorus bonds, carbon-sulfur bonds such as thioether, or imide bonds. In certain preferred embodiments the first attachment site and the second attachment site are linked through at least one covalent bond, preferably through at least one non-peptide bond, and even more preferably through exclusively non-peptide bond(s). The term "linked" as used herein, however, shall not only encompass a direct linkage of the at least one first attachment site and the at least one second attachment site but also, alternatively and preferably, an indirect linkage of the at least one first attachment site and the at least one second attachment site through intermediate molecule(s), and hereby typically and preferably by using at least one, preferably one, heterobifunctional cross-linker.

[0023] Linker: A "linker", as used herein, either associates the second attachment site with the antigenic peptide or already comprises, essentially consists of, or consists of the second attachment site. Preferably, a "linker", as used herein, already comprises the second attachment site, typically and preferably - but not necessarily - as one amino acid residue, preferably as a cysteine residue. A "linker" as used herein is also termed "amino acid linker", in particular when a linker according to the invention contains at least one amino acid residue. Thus, the terms "linker" and "amino acid linker" are interchangeably used herein. However, this does not imply that such a linker consists exclusively of amino acid residues, even if a linker consisting of amino acid residues is a preferred embodiment of the present invention. The amino acid residues of the linker are, preferably, composed of naturally occurring amino acids or unnatural amino acids known in the art, all-L or all-D or mixtures thereof. Further preferred embodiments of a linker in accordance with this invention are molecules comprising a sulfhydryl group or a cysteine residue and such molecules are, therefore, also encompassed within this invention. Further linkers useful for the present invention are molecules comprising a C1-C6 alkyl-, a cycloalkyl such as a cyclopentyl or cyclohexyl, a cycloalkenyl, aryl or heteroaryl moiety. Moreover, linkers comprising preferably a C1-C6 alkyl-, cycloalkyl- (C5, C6), aryl- or heteroaryl- moiety and additional amino acid(s) can also be used as linkers for the present invention and shall be encompassed within the scope of the invention. Association of the linker with the antigenic peptide is preferably by way of at least one covalent bond, more preferably by way of at least one peptide bond. In case of a second attachment site not naturally occurring with the antigenic peptide, the linker is associated to the at least one second attachment site, for example, a cysteine, preferably, by way of at least one covalent bond, more preferably by way of at least one peptide bond. [0024] Ordered and repetitive antigen array: As used herein, the term "ordered and repetitive antigen array" generally refers to a repeating pattern of antigen or, characterized by a typically and preferably high order of uniformity in spacial arrangement of the antigens with respect to virus-like particle, respectively. In one embodiment of the invention, the repeating pattern may be a geometric pattern. Certain embodiments of the invention, such as VLP of RNA-bacteriophage s, are typical and preferred examples of suitable ordered and repetitive antigen arrays which, moreoever, possess strictly repetitive paracrystalline orders of antigens, preferably with spacings of 1 to 30 nanometers, preferably 2 to 15 nanometers, even more preferably 2 to 10 nanometers, even again more preferably 2 to 8 nanometers, and further more preferably 1.6 to 7 nanometers.

[0025] Polypeptide: The term "polypeptide" as used herein refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). It indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides and proteins are included within the definition of polypeptide. Post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like are also encompassed. [0026] Recombinant VLP: The term "recombinant VLP", as used herein, refers to a VLP that is obtained by a process which comprises at least one step of recombinant DNA technology. The term "VLP recombinantly produced", as used herein, refers to a VLP that is obtained by a process which comprises at least one step of recombinant DNA technology. Thus, the terms "recombinant VLP" and "VLP recombinantly produced" are interchangeably used herein and should have the identical meaning.

[0027] Reactive group first/second: the term "the first/second reactive group" refers to part of the side chain of the first/second amino acid of the antigenic peptide of the invention, which associates to "the second/first reactive group" of the second/first amino acid of the antigenic peptide of the invention, either directly or preferably through at least one, preferably only one intermediate molecule. Part of the side chain that may serve as the first or second reactive group include thiol (C), amine (K), amido (QQ, arginine (R), carboxylic acid (DE), alcohol (ST), thioether (M), imidazol (K), phenyl (F), phenol (Y), indole (W), and aliphatic (AVILP).

[0028] Virus particle: The term "virus particle" as used herein refers to the morphological form of a virus. In some virus types it comprises a genome surrounded by a protein capsid; others have additional structures (e.g., envelopes, tails, etc.). [0029] Virus-like particle (VLP), as used herein, refers to a non-replicative or noninfectious, preferably a non-replicative and non-infectious virus particle, or refers to a non- replicative or non-infectious, preferably a non-replicative and non-infectious structure resembling a virus particle, preferably a capsid of a virus. The term "non-replicative", as used herein, refers to being incapable of replicating the genome comprised by the VLP. The term "non- infectious", as used herein, refers to being incapable of entering the host cell. Preferably a virus-like particle in accordance with the invention is non-replicative and/or non-infectious since it lacks all or part of the viral genome or genome function due to physical, chemical inactivation or due to genetic manipulation. Typically and preferably a virus-like particle lacks all or part of the replicative and infectious components of the viral genome. A virus-like particle in accordance with the invention may contain nucleic acid distinct from their genome. A typical and preferred embodiment of a virus-like particle in accordance with the present invention is a viral capsid such as the viral capsid of the corresponding virus, bacteriophage, preferably RNA-bacteriophage. The terms "viral capsid" or "capsid", refer to a macro molecular assembly composed of viral protein subunits. Typically, there are 60, 120, 180, 240, 300, 360 and more than 360 viral protein subunits. Typically and preferably, the interactions of these subunits lead to the formation of viral capsid or viral-capsid like structure with an inherent repetitive organization, wherein said structure is, typically, spherical or tubular. The term "capsid-like structure" as used herein, refers to a macromolecular assembly composed of viral protein subunits resembling the capsid morphology in the above defined sense but deviating from the typical symmetrical assembly while maintaining a sufficient degree of order and repetitiveness.

[0030] One common feature of virus particle and virus-like particle is its highly ordered and repetitive arrangement of its subunits.

[0031] Virus-like particle of an RNA-bacteriophage: As used herein, the term "virus- like particle of an RNA-bacteriophage" refers to a virus-like particle resembles the structure of an RNA-bacteriophage, being non replicative or non-infectious, and typically and preferably being non replicative and non-infectious. Typically and preferably, the term "virus- like particle of an RNA-bacteriophage" should furthermore refer to a virus-like particle of an RNA-bacteriophage which lacks at least one of the genes, preferably all of the genes, encoding for the replication machinery of the RNA-bacteriophage, and typically and further preferably even at least one of the genes, preferably all of the genes, encoding the protein or proteins responsible for viral attachment to or entry into the host. This definition should, however, also encompass virus-like particles of RNA-bacteriophages, in which the aforementioned gene or genes are still present but inactive, and, therefore, also leading to non- replicative and/or noninfectious virus-like particles of an RNA-bacteriophage. Moreover, the term "virus-like particle of an RNA-bacteriophage" should therefore also encompass in its broadest definition a virus particle of an RNA-bacteriophage, the genome of which has been inactivated by physical or chemical or genetic methods so that the virus particle is not capable of infecting and/or replicating. Preferred VLPs derived from RNA-bacteriophages exhibit icosahedral symmetry and consist of 180 subunits. Within this present disclosure the term "subunit" and "monomer" are interexchangeably and equivalently used within this context. [0032] One, a, or an: when the terms "one", "a", or "an" are used in this disclosure, they mean "at least one" or "one or more" unless otherwise indicated. [0033] Within this application, antibodies are defined to be specifically binding if they bind to the antigen with a binding affinity (Ka) of 10⁶ M^"1 or greater, preferably 10⁷ M^"1 or greater, more preferably 10⁸ M^"1 or greater, and most preferably 10⁹ M^"1 or greater. The affinity of an antibody can be readily determined by one of ordinary skill in the art (for example, by Scatchard analysis.)

[0034] The amino acid sequence identity of polypeptides can be determined conventionally using known computer programs such as the Bestfit program. When using Bestfit or any other sequence alignment program, preferably using Bestfit, to determine whether a particular sequence is, for instance, 95% identical to a reference amino acid sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed. This aforementioned method in determining the percentage of identity between polypeptides is applicable to all proteins, polypeptides or a fragment thereof disclosed in this invention. [0035] Conservative amino acid substitutions, as understood by a skilled person in the art, include isosteric substitutions, substitutions where the charged, polar, aromatic, aliphatic or hydrophobic nature of the amino acid is maintained. Typical conservative amino acid substitutions are substitutions between amino acids within one of the following groups: GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr, Cys; Lys, Arg; and Phe and Tyr.

[0036] In one aspect, the invention provides a composition comprising: (a) a virus-like particle with at least one first attachment site; (b) at least one antigenic peptide with at least one second attachment site, wherein said antigenic peptide comprises: (i) CCR5 PNt domain comprising an amino acid sequence KINVK (SEQ ID NO:25); (ii) a first amino acid comprising a first reactive group and wherein said first amino acid is located at the N-terminal of KINVK; (iii) a second amino acid comprising a second reactive group and wherein said second amino acid is located at the C-terminal of KINVK, wherein said first and said second amino acid does not comprise said second attachment site linking said first attachment site; wherein said first reactive group binds to said second reactive group by at least one covalent bond so that the peptide starting from said first amino acid and ending with said second amino acid is looped; and wherein said virus-like particle and said at least one antigenic peptide are linked through said first and said second attachment site. [0037] Any virus known in the art having an ordered and repetitive structure may be selected as a VLP of the invention. Illustrative DNA or RNA viruses, the coat or capsid protein of which can be used for the preparation of VLPs have been disclosed in WO 2004/009124 on page 25, line 10-21, on page 26, line 11-28, and on page 28, line 4 to page 31, line 4. These disclosures are incorporated herein by way of reference. [0038] Virus or virus-like particle can be produced and purified from virus-infected cell culture. The resulting virus or virus-like particle for vaccine purpose needs to be devoid of virulence. Besides genetic engineering, physical or chemical methods can be employed to inactivate the viral genome function, such as UV irradiation, formaldehyde treatment. [0039] In one preferred embodiment, the VLP is a recombinant VLP. Almost all commonly known viruses have been sequenced and are readily available to the public. The gene encoding the coat protein can be easily identified by a skilled artisan. The preparation of VLPs by recombinantly expressing the coat protein in a host is within the common knowledge of a skilled artisan.

[0040] In one preferred embodiment, the virus-like particle comprises, or alternatively consists of, recombinant proteins or preferably coat proteins, mutants or fragments thereof, of a virus selected form the group consisting of: a) RNA-bacteriophage s; b) bacteriophage; c) Hepatitis B virus, preferably its capsid protein (Ulrich, et al., Virus Res. 50:141-182 (1998)) or its surface protein (WO 92/11291); d) measles virus (Warnes, et al., Gene 160:173-178 (1995)); e) Sindbis virus; f) rotavirus (US 5,071,651 and US 5,374,426); g) foot-and-mouth- disease virus (Twomey, et al., Vaccine 13:1603 1610, (1995)); h) Norwalk virus (Jiang, X., et al., Science 250:1580 1583 (1990); Matsui, S.M., et al., J. Clin. Invest. 87:1456 1461 (1991)); i) Alphavirus; j) retrovirus, preferably its GAG protein (WO 96/30523); k) retrotransposon Ty, preferably the protein pi; 1) human Papilloma virus (WO 98/15631); m) Polyoma virus; n) Tobacco mosaic virus; and o) Flock House Virus p) Cowpea Chlorotic Mottle Virus; q) a Cowpea Mosaic Virus; and r) an Alfalfa Mosaic Virus.

[0041] Assembly of the fragment or mutant of recombinant protein or coat protein into a VLP may be tested, as one skilled in the art would appreciate by expressing the protein in E. co Ii, optionally purifying the capsids by gel filtration from cell lysate, and analysing the capsid formation in an immunodiffusion assay (Ouchterlony test) or by Electron Microscopy (EM) (Kozlovska, T. M.. et al, Gene 757:133-37 (1993)). Immunodiffusion assays and EM may be directly performed on cell lysate. [0042] In one preferred embodiment, the VLP comprises, or consists of, more than one amino acid sequence, preferably two amino acid sequences, of the recombinant proteins, mutants or fragments thereof. VLP comprises or consists of more than one amino acid sequence is referred, in this application, as mosaic VLP.

[0043] The term "fragment of a recombinant protein" or the term "fragment of a coat protein", as used herein, is defined as a polypeptide, which is of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% the length of the wild-type recombinant protein, or coat protein, respectively and which preferably retains the capability of forming VLP. Preferably the fragment is obtained by at least one internal deletion, at least one truncation or at least one combination thereof. The term "fragment of a recombinant protein" or "fragment of a coat protein" shall further encompass polypeptide, which has at least 80%, preferably 90%, even more preferably 95% amino acid sequence identity with the "fragment of a recombinant protein" or "fragment of a coat protein", respectively, as defined above and which is preferably capable of assembling into a virus-like particle.

[0044] The term "mutant recombinant protein" or the term "mutant of a recombinant protein" as interchangeably used in this invention, or the term "mutant coat protein" or the term "mutant of a coat protein", as interchangeably used in this invention, refers to a polypeptide having an amino acid sequence derived from the wild type recombinant protein, or coat protein, respectively, wherein the amino acid sequence is at least 80%, preferably at least 85%, 90%, 95%, 97%, or 99% identical to the wild type sequence and preferably retains the ability to assemble into a VLP.

[0045] In one preferred embodiment, the virus-like particle of the invention is a virus- like particle of a Hepatitis B virus. The preparation of Hepatitis B virus-like particles have been disclosed, inter alia, in WO 00/32227, WO 01/85208 and in WO 01/056905. All three documents are explicitly incorporated herein by way of reference. Other variants of HBcAg suitable for use in the practice of the present invention have been disclosed in page 34-39 WO 01/056905.

[0046] In one further preferred embodiments of the invention, a lysine residue is introduced into the HBcAg polypeptide, to mediate the linking of the antigenic peptide of the invention to the VLP of HBcAg. In preferred embodiments, VLPs and compositions of the invention are prepared using a HBcAg comprising, or alternatively consisting of, amino acids 1-144, or 1-149, 1-185 of SEQ ID NO:20, which is modified so that the amino acids at positions 79 and 80 are replaced with a peptide having the amino acid sequence of Gly-Gly- Lys-Gly-Gly. This modification changes the SEQ ID NO:20 to SEQ ID NO:21. In further preferred embodiments, the cysteine residues at positions 48 and 110 of SEQ ID NO:21, or its corresponding fragments, preferably 1-144 or 1-149, are mutated to serine. The invention further includes compositions comprising Hepatitis B core protein mutants having above noted corresponding amino acid alterations. The invention further includes compositions and vaccines, respectively, comprising HBcAg polypeptides which comprise, or alternatively consist of, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NO:21.

[0047] In one preferred embodiment, the virus-like particle is of a Cowpea Chlorotic

Mottle Virus, a Cowpea Mosaic Virus or an Alfalfa Mosaic Virus. Methods to produce VLP of these viruses have been described in US 2005/0260758 and in WO05067478. [0048] In one preferred embodiment, the virus-like particle of the invention is a virus- like particle of an RNA-bacteriophage, wherein preferably said RNA-bacteriophage is Qβ, AP205, GA or fr, further preferably said RNA-bacteriophage is Qβ.

[0049] In one preferred embodiment of the invention, the virus-like particle of an

RNA-bacteriophage comprises, consists essentially of, or alternatively consists of, recombinant coat proteins, mutants or fragments thereof, of an RNA-bacteriophage. Preferably, the RNA-bacteriophage is selected from the group consisting of a) bacteriophage Qβ; b) bacteriophage Rl 7; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage Mi l; h) bacteriophage MXl; i) bacteriophage NL95; k) bacteriophage f2; 1) bacteriophage PP7 and m) bacteriophage AP205.

[0050] In one further preferred embodiment, the virus-like particle of an RNA- bacteriophage comprises, consists essentially of, or alternatively consists of, recombinant coat proteins, of an RNA-bacteriophage. Preferably, the RNA-bacteriophage is selected from the group consisting of a) bacteriophage Qβ; b) bacteriophage Rl 7; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage Mi l; h) bacteriophage MXl; i) bacteriophage NL95; k) bacteriophage f2; 1) bacteriophage PP7 and m) bacteriophage AP205.

[0051] In one preferred embodiment of the invention, the composition comprises coat protein, mutants or fragments thereof, of RNA-bacteriophages, wherein the coat protein has amino acid sequence selected from the group consisting of: (a) SEQ ID NO:1; referring to Qβ CP; (b) a mixture of SEQ ID NO:1 and SEQ ID NO:2 (referring to Qβ Al protein); (c) SEQ ID NO:3; (d) SEQ ID NO:4; (e) SEQ ID NO:5; (f) SEQ ID NO:6, (g) a mixture of SEQ ID NO:6 and SEQ ID NO:7; (h) SEQ ID NO:8; (i) SEQ ID NO:9; (j) SEQ ID NO: 10; (k) SEQ ID NO:11; (1) SEQ ID NO:12; (m) SEQ ID NO:13; and (n) SEQ ID NO:14. [0052] In one preferred embodiment of the invention, the VLP is a mosaic VLP comprising or alternatively consisting of more than one amino acid sequence, preferably two amino acid sequences, of coat proteins, mutants or fragments thereof, of an RNA- bacteriophage .

[0053] In one very preferred embodiment, the VLP comprises or alternatively consists of two different coat proteins of an RNA-bacteriophage, said two coat proteins have an amino acid sequence of SEQ ID NO: 1 and SEQ ID NO:2, or of SEQ ID NO:6 and SEQ ID NO:7. [0054] In preferred embodiments of the present invention, the virus-like particle of the invention comprises, or alternatively consists essentially of, or alternatively consists of recombinant coat proteins, mutants or fragments thereof, of the RNA-bacteriophage Qβ, fr, AP205 or GA.

[0055] In one preferred embodiment, the VLP of the invention is a VLP of RNA- bacteriophage Qβ. The capsid or virus-like particle of Qβ showed an icosahedral phage-like capsid structure with a diameter of 25 nm and T=3 quasi symmetry. The capsid contains 180 copies of the coat protein, which are linked in covalent pentamers and hexamers by disulfide bridges (Golmohammadi, R. et al., Structure 4:543-5554 (1996)), leading to a remarkable stability of the Qβ capsid. Capsids or VLPs made from recombinant Qβ coat protein may contain, however, subunits not linked via disulfide bonds to other subunits within the capsid, or incompletely linked. The capsid or VLP of Qβ shows unusual resistance to organic solvents and denaturing agents. Surprisingly, we have observed that DMSO and acetonitrile concentrations as high as 30%, and guanidinium concentrations as high as 1 M do not affect the stability of the capsid. The high stability of the capsid or VLP of Qβ is an advantageous feature, in particular, for its use in immunization and vaccination of mammals and humans in accordance of the present invention.

[0056] Further preferred virus-like particles of RNA-bacteriophages, in particular of

Qβ and fr in accordance of this invention are disclosed in WO 02/056905, the disclosure of which is herewith incorporated by reference in its entirety. Particular example 18 of WO 02/056905 gave detailed description of preparation of VLP particles from Qβ. [0057] In another preferred embodiment, the VLP of the invention is a VLP of RNA- bacteriophage AP205. Assembly-competent mutant forms of AP205 VLPs, including AP205 coat protein with the substitution of proline at amino acid 5 to threonine or AP205 coat protein with the substitution of asparigine at amino acid 14 to aspartic acid, may also be used in the practice of the invention and leads to other preferred embodiments of the invention. WO 2004/007538 describes, in particular in Example 1 and Example 2, how to obtain VLP comprising AP205 coat proteins, and hereby in particular the expression and the purification thereto. WO 2004/007538 is incorporated herein by way of reference. AP205 VLPs are highly immunogenic, and can be linked with antigen to typically and preferably generate vaccine constructs displaying the antigen in oriented in a repetitive manner. High antibody titer is elicited against the so displayed antigens showing that linked antigens are accessible for interacting with antibody molecules and are immunogenic.

[0058] In one preferred embodiment, the VLP of the invention comprises or consists of a mutant coat protein of a virus, preferably an RNA-bacteriophage, wherein the mutant coat protein has been modified by removal of at least one lysine residue by way of substitution and/or by way of deletion. In another preferred embodiment, the VLP of the invention comprises or consists of a mutant coat protein of a virus, preferably an RNA- bacteriophage , wherein the mutant coat protein has been modified by addition of at least one lysine residue by way of substitution and/or by way of insertion. The deletion, substitution or addition of at least one lysine residue allows varying the degree of coupling, i.e. the amount of antigen per subunits of the VLP of a virus, preferably of an RNA-bacteriophage, in particular, to match and tailor the requirements of the vaccine.

[0059] In one preferred embodiment, the compositions and vaccines of the invention have an antigen density being from 0.5 to 4.0. The term "antigen density", as used herein, refers to the average number of antigen which is linked per subunit, preferably per coat protein, of the VLP, and hereby preferably of the VLP of an RNA-bacteriophage. Thus, this value is calculated as an average over all the subunits or monomers of the VLP, preferably of the VLP of the RNA-bacteriophage, in the composition or vaccines of the invention. [0060] VLPs or capsids of Qβ coat protein display a defined number of lysine residues on their surface, with a defined topology with three lysine residues pointing towards the interior of the capsid and interacting with the RNA, and four other lysine residues exposed to the exterior of the capsid. Preferably, the at least one first attachment site is a lysine residue, pointing to or being on the exterior of the VLP.

[0061] Qβ mutants, of which exposed lysine residues are replaced by arginines can be used for the present invention. Thus, in another preferred embodiment of the present invention, the virus-like particle comprises, consists essentially of or alternatively consists of mutant Qβ coat proteins. Preferably these mutant coat proteins comprise or alternatively consist of an amino acid sequence selected from the group of a) Qβ-240 (SEQ ID NO: 15, Lysl3-Arg of SEQ ID NO: 1) b) Qβ-243 (SEQ ID NO: 16, AsnlO-Lys of SEQ ID NO:1); c) Qβ-250 (SEQ ID NO:17, Lys2-Arg of SEQ ID NO:1) d) Qβ-251 (SEQ ID NO:18, Lysl6-Arg of SEQ ID NO:1); and e) Qβ-259" (SEQ ID NO:19, Lys2-Arg, Lysl6-Arg of SEQ ID NO:1). The construction, expression and purification of the above indicated Qβ mutant coat proteins, mutant Qβ coat protein VLPs and capsids, respectively, are described in WO 02/056905. In particular is hereby referred to Example 18 of above mentioned application. [0062] In another preferred embodiment of the present invention, the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of mutant coat protein of Qβ, or mutants or fragments thereof, and the corresponding Al protein. In a further preferred embodiment, the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of mutant coat protein with amino acid sequence SEQ ID NO:15, 16, 17, 18, or 19 and the corresponding Al protein.

[0063] Further RNA-bacteriophage coat proteins have also been shown to self- assemble upon expression in a bacterial host (Kastelein, RA. et al., Gene 23:245-254 (1983), Kozlovskaya, TM. et al., Dokl. Akad. Nauk SSSR 287:452-455 (1986), Adhin, MR. et al., Virology 170:238-242 (1989), Priano, C. et al., J. MoI. Biol. 249:283-297 (1995)). In particular the biological and biochemical properties of GA (Ni, CZ., et al., Protein Sci. 5:2485-2493 (1996), Tars, K et al., J. Mol.Biol. 271 :759-773(1997)) and of fr (Pushko P. et al., Prot. Eng. 6:883-891 (1993), Liljas, L et al. J MoI. Biol. 244:279-290, (1994)) have been disclosed. The crystal structure of several RNA bacteriophages has been determined (Golmohammadi, R. et al., Structure 4:543-554 (1996)). Using such information, surface exposed residues can be identified and, thus, RNA-bacteriophage coat proteins can be modified such that one or more reactive amino acid residues can be inserted by way of insertion or substitution. Another advantage of the VLPs derived from RNA-bacteriophages is their high expression yield in bacteria that allows production of large quantities of material at affordable cost.

[0064] In one embodiment, the first amino acid is at the very N-terminus of the antigenic peptide of the invention.

[0065] In one embodiment, the first amino acid is located at the N-terminal of KINVK and not more than 16 amino acids, preferably not more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid away from the first amino acid K of KINVK. In one preferred embodiment, the first amino acid is not more than 16 amino acids, preferably not more than 10, more preferably not more than 5 amino acids away from the first amino acid K of KINVK.

[0066] In one preferred embodiment, the first amino acid is at -16, -15 or -5 position relative to the first K (position 0) of KINVK. In one further preferred embodiment, the first amino acid is a cysteine residue. Typically and preferably the cysteine is generated by insertion, or preferably by substitution of the naturally occurring amino acid residue, normally serine, at that position into cysteine, wherein further preferably the naturally occurring cysteine within the PNt domain will be deleted or preferably substituted, preferably by a serine or an alanine substitution.

[0067] In one very preferred embodiment, the first amino acid is at -2 position relative to the first K (position 0) of KINVK. In one further preferred embodiment, the first amino acid corresponds to, or preferably is, the cysteine residue within SEQ ID NO:22. [0068] In one embodiment, the second amino acid is located at the C-terminal of

KINVK and not more than 14, 12, preferably not more than 10 amino acids, preferably not more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid away from V of KINVK, further preferably not more than 8 amino acids away from amino acid V of KINVK. In one preferred embodiment, the second amino acid is at least 4, preferably at least 5, more preferably at least 6 amino acids away from V of KINVK. In one preferred embodiment, the second amino acid is at least 5 and not more than 10 amino acids away from V of KINVK.

[0069] In one preferred embodiment, the second amino acid is located at +2, +3, +4,

+5, +6, or +7 position relative to amino acid V (position 0) of KINVK. Further preferably the second amino acid is located at +7 position relative to amino acid V (position 0) of KINVK. [0070] In one embodiment, the second amino acid is located at +2 position relative to amino acid V (position 0) of KINVK, further preferably the second amino acid is a cysteine. Typically and preferably the cysteine is generated by insertion, or preferably by substitution of the naturally occurring amino acid residue, normally glutamine, at that position into cysteine.

[0071] In one embodiment, the first reactive group comprises or is a sulfhydryl group, preferably a sulfhydryl group of a cysteine. In one embodiment, the second reactive group comprises or is a sulfhydryl group, preferably a sulfhydryl group of a cysteine. [0072] In one preferred embodiment, the first reactive group and the second reactive group is a sulfhydryl group, preferably a sulfhydryl group of a cysteine residue. In one preferred embodiment, the first amino acid corresponds to, or preferably is, the cysteine residue of SEQ ID NO:22. In one preferred embodiment, the second amino acid is a cysteine residue located at the C-terminal of KINVK and not more than 10 amino acids away from amino acid V of KINVK. The introduction of the cysteine at any one of the +2 to +6 positions relative to amino acid V (position 0) of KINVK can be achieved by insertion or preferably by substitution of any amino acid of QIAAR within SEQ ID NO:22. In one preferred embodiment, the second amino acid is a cysteine residue located at any one of +7, +8, +9 and +10 position relative to amino acid amino acid V (position 0) of KINVK. The cysteine is introduced by insertion. Additional spacing amino acids may be added, preferably alanine and glycine.

[0073] In one preferred embodiment, the first amino acid corresponds to, or preferably is, the cysteine within SEQ ID NO:22 and the second amino acid is a cysteine located at any one of the +7, +8, +9 and +10 position relative to amino acid amino acid V (position 0) of KINVK. In one further preferred embodiment, the CCR5 PNt domain comprises, consists essentially of, or preferably consists of SEQ ID NO:22.

[0074] In one preferred embodiment, the first amino acid corresponds to, or preferably is, the cysteine within SEQ ID NO:22 and the second amino acid is a cysteine located at +7 position relative to amino acid amino acid V (position 0) of KINVK. In one still further preferred embodiment, the antigenic peptide of the invention comprises, consists essentially of, or preferably consists of an amino acid sequence of SEQ ID NO:23.

[0075] In one preferred embodiment, the first amino acid corresponds to, or preferably is, the cysteine within SEQ ID NO:22 and the second amino acid is a cysteine by substituting amino acid Q of QIAAR within SEQ ID NO:22. In one further preferred embodiment, the antigenic peptide of the invention comprises, consists essentially of, or preferably consists of an amino acid sequence of SEQ ID NO:24.

[0076] The side chain of an amino acid which may serve as the first or the second reactive group include thiol (C), amine (K), amido (QQ, arginine (R), carboxylic acid (DE), alcohol (ST), thioether (M), imidazol (K), phenyl (F), phenol (Y), indole (W), and aliphatic (AVILP). [0077] In one preferred embodiment, the first reactive group binds to the second reactive group through at least one intermediate molecule, preferably two, more preferably only one intermediate molecule.

[0078] In one preferred embodiment, one of the two reactive groups is derivatized by at least one, preferably only one, intermediate molecule so that it receives a new functionality which is readily reactive to the other reactive group. For example, the amino group of a lysine may be derivatized by an intermediate molecule, eg, SMPH, so that after derivatization it becomes readily reactive to a sulfhydryl group.

[0079] In one preferred embodiment, both of the two reactive groups are derivatized by at least one, preferably only one, intermediate molecule, respectively, so that after derivatization both are readily to react with each other.

[0080] In one preferred embodiment, the first and the second amino acid are identical.

In one preferred embodiment, the first reactive group and the second reactive group are identical. In one alternative embodiment, the first amino acid and the second amino acid, either after derivatization of one of the two reactive groups or after derivatization of both reactive groups, have the same functionality.

[0081] Thus in one preferred embodiment, the second reactive group is an amino group of a lysine. In one further preferred embodiment, said lysine corresponds to or preferably is the 26^th lysine within SEQ ID NO:22. In one further preferred embodiment, the first reactive group is a sulfhydryl group, preferably a sulfhydryl group of a cysteine residue.

In one further preferred embodiment, the first amino acid corresponds to or preferably is the cysteine within SEQ ID NO:22 and the second amino acid corresponds to or preferably is the

26^th lysine within SEQ ID NO:22.

[0082] In one preferred embodiment, the first reactive group binds to the second reactive group by exclusively non-peptide covalent bond.

[0083] In one preferred embodiment, the first and the second reactive group is a sulfhydryl group, preferably a sulfhydryl group of a cysteine. In one preferred embodiment, the first reactive group binds to the second reactive group by at least one covalent bond, wherein said covalent bond is a disulfide bond. Methods of forming disulfide bond between two sulfhydryl groups are disclosed (see e.g. US8929758; US4518711; US5169833;

WO9109051; WO9108759). Preferably the second attachment site is then a -NH-NH2 group and one way of coupling of the antigenic peptide via -NH-NH2 to the VLP is described in

EXAMPLE 4. [0084] In one embodiment, the intermediate molecule comprises at least two functional reactive sites and wherein the two functional reactive site binds to the two reactive group respectively. In one preferred embodiment, at least one. Preferably both of the bounds between the functional reactive site and the reactive group comprises a thioether bond. [0085] In one preferred embodiment, the first reactive group and the second reactive group comprises or preferably is a sulfhydryl group, preferably a sulfhydryl group of a cysteine, wherein said first reactive group binds to said second reactive group through at least one, preferably only one, intermediate molecule. Preferably the first reactive group and the second reactive group binds to the only one intermediate molecule, respectively. Further preferably at least one of the bounds between the reactive group and the intermediate molecule comprises a thioether bond.

[0086] In one preferred embodiment, the intermediate molecule comprises a halogenoalkane. Halogenoalkanes (also known as haloalkanes or alkyl halides) are compounds containing a halogen atom (fluorine, chlorine, bromine or iodine) joined to one or more carbon atoms in a chain. Provided herein are dihalo-intermediate molecules comprising two halogen atoms, and tri- and tetrahalo-intermediate molecules.

[0087] In one preferred embodiment, the intermediate molecule comprises at least two, preferably two halogen atoms, wherein preferably said two halogen atoms are two Cl atoms, more preferably one Cl atom and one Br atom, even more preferably two Br atoms. [0088] In one preferred embodiment, the intermediate molecule comprises an aromatic compound, wherein preferably said aromatic compound comprises at least two benzylic halogen substituents. Preferably, the aromatic compound comprises at least two benzylic halogen substituents, like for instance halomethyl groups. Suitable examples include, but are not limited, to di(halomethyl)benzene, tri(halomethyl)benzene or tetra(halomethyl)benzene and derivatives thereof. In one further preferred embodiment, the intermediate molecule is a di(halomethyl)benzene (T2), preferably wherein the di(halomethyl)benzene is l,3-bis(bromomethyl)benzene.

[0089] In one preferred embodiment, the intermediate molecule is a tri(halomethyl)benzene or a derivative thereof (T3). Further preferably the tri(halomethyl)benzene is 2,4,6-tris(bromomethyl)mesitylene. In one preferred embodiment, antigenic peptide with two cysteines may be looped through the two cysteines via reaction with T3. The so-looped T3 peptide may react with excessive amount of DTT, which provides a -SH group serving as the second attachment site. [0090] In one preferred embodiment, the intermediate molecule comprises an allylic system. In an allylic system, there are three carbon atoms, two of which are connected through a carbon-carbon double bond. In a preferred embodiment, the formation of a bond between the intermediate molecule and the reactive group substitution occurs via an allylic substitution reaction. In one further preferred embodiment, the intermediate molecule comprises at least one carbon-oxygen double bond (carbonyl group). For example, a scaffold comprises two or more reactive groups comprising the structure - C(O)-CH2-halogen. [0091] The intermediate molecule may comprise poly cyclic aromatic compounds with smaller or larger ring structures. Furthermore, the intermediate molecule may comprise a cyclic molecule with at least one atom other than carbon in the ring structure. A preferred intermediate molecule is meta-dibromo-pyridine. Still furthermore, the intermediate molecule may comprise multiple ring aromatic structure, such as fused-ring aromatic compounds. Still furthermore, the intermediate molecule may comprise multiple aromatic conjugated systems wherein the systems do not share a pair of carbon atoms, e.g. benzene rings are connected directly via a carbon-carbon bond. Further intermediate molecules which can be used for the present invention have been disclosed in WO2004/077062 and this descriptions are incorporated herein by way of reference.

[0092] In one preferred embodiment, the intermediate molecule is selected from a group consisting of: bis-; tris-; or tetra(halomethyl)benzene; bis-; tr.is-; or tetra(halomethyl)pyridine; bis-; tris-; or tetra (halomethyl)pyridazine; bis-; tria-; or tetra(halomethyl)pyrimidine; bis-; tris-; or tetra(halomethyl)pyrazine; bis-; tris-; or tetra(halomethyl)-l,2,3-triazine; bis-; tris-; or tetra(halomethyl)-l,2,4-triazine; bis-; tris-; or tetra(halomethyl)pyrrole,-furan, -thiophene; bis-; tris-; or tetra(halomethyl)imidazole, - oxazole, -thiazol; bis-; tris-; or tetra(halo methyl)- 8H- pyrazole, -isooxazole, -isothiazol; bis-; tris-; or tetra(halomethyl)biphenylene; bis-; tris-; or tetra(halomethyl)terphenylene; 1,8- bis(halomethyl)naphthalene; bis-; tris-; or tetra(halomethyl)anthracene; bis-; tris-; or tetra(2- halomethylphenyl)methane; or, if applicable, another regioisomer thereof. For example, pro video is l,2-bis(halomethyl)benzene; 3,4-bis(halomethyl)pyridine; 3,4- bis(halomethyl)pyridazine; 4,5-bis(halomethyl)pyrimidine; 4,5-bis(halomethyl)pyrazine; 4,5- bis(halomethyl)- 1 ,2,3-triazine; 5 ,6-bis(halomethyl)- 1 ,2,4-triazine; 3 ,4- bis(halomethyl)pyrrole,-furan, -thiophene and other regioisomers, 4,5- bis(halomethyl)imilazole, -oxazole, -thiazol; 4,5-bis(halomethyl)-8H-pyxazole, - isooxazole, - isothiazol; 2,2'-bis(halomethyl)biphenylene; 2,2"- bis(halomethyl) terphenylene; 1, 8- bis(halomethyl) naphthalene l,10-bis(halomethyl)anthracene; bis(2- halomethylphenyl)methane; 1 ,2,8-tris(halomethyl)benzene; 2,3,4-tris(halomethyl)pyridine; 2,8,4- tris(halomethyl)pyrid.azine; 3,4,5-tris(halomethyl)pyrimidine; 4,5,6-tris(halomethyl)- 1,2,8-triazine; 2,8,4-tris(halomethyl)pyrrole, -fliran, -thiophene; 2,4,5- bis(halomethyl)imidazole, -oxazole, -thiazol; 8,4,5-bis(halomethyl)-lH-pyrazole, isooxazole, -isothiazol; 2,4,2'-tris(halomethyl)biphenylene; 2,3',2"- tris(halomethyl)terphenylene; l,8,8-tris(halomethyl)naphthalene 1,8, lθ-tris(halomethyl) anthracene; bis(2-halomethylphenyl) methane; l,2,4,5-tetra(halomethyl)benzene; 1,2,4,5- tetra(halomethyl)pyridine; 2,4,5,6- tetra(halomethyl)pyrimidine; 2,3,4,5- tetra(halomethyl)pyrrole; -furan; -thiophene; 2,2',6,6'-tetra(halomethyl)biphenylene; 2,2",6,6"-tetra(halomethyl) terphenylene 2,8,5,6-tetra(halomethyl)naphthalene and. 2,8,7,8- tetra(halomethyl) anthracene; Bis(2,4-bis(halomethyl)phenyl)methane.

[0093] In one preferred embodiment, the CCR5 PNt domain comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO:22, in which at most three, preferably two, more preferably at most one amino acid has been deleted, inserted or substituted, preferably by conservative substitution. In one preferred embodiment, CCR5 PNt domain comprises, consists essentially of, or preferably consists of an amino acid sequence of SEQ ID NO:22.

[0094] In one preferred embodiment of the invention, the VLP with at least one first attachment site is linked to the antigenic peptide with at least one second attachment site via at least one peptide bond, preferably via exclusively peptide bond, wherein the antigen peptide comprises (i) CCR5 PNt domain comprising an amino acid sequence KINVK (SEQ ID NO:25); (ii) wherein the first amino acid is a cysteine located at the N-terminal of KINVK and wherein the second amino acid is a cysteine located at the C-terminal of KINVK, and wherein the two cysteines binds to each other by a disulfide bond.

[0095] In one preferred embodiment, the first cysteine corresponds to, or preferably is, the cysteine within SEQ ID NO:22. In one preferred embodiment, the second cysteine is located at any one of the +2 to +10 position relative to amino acid V (position 0) of KINVK. Further preferably the second cysteine is located at +7 position relative to amino acid V (position 0) of KINVK.

[0096] In one preferred embodiment, the CCR5 PNt domain comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO:22. In one preferred embodiment, the antigenic peptide of the invention comprises or consists essentially of an amino acid sequence of SEQ ID NO:23, wherein the first cysteine and the second cysteine are linked by a disulfide bond and wherein the first attachment site and the second attachment site are linked by one peptide bond, preferably through the second cysteine (at position 32 of SEQ ID NO:23) to the coat protein of a virus-like particle, preferably to the N-terminus of the coat protein of bacteriophage AP205.

[0097] Gene encoding antigenic peptide is in-frame ligated, either internally or preferably to the N- or the C-terminus to the gene encoding the coat protein of the VLP. Fusion may also be effected by fusing sequences of the antigenic peptide into a mutant of a coat protein where part of the coat protein sequence has been deleted, that are further referred to as truncation mutants. Truncation mutants may have N- or C-terminal, or internal deletions of part of the sequence of the coat protein. For example for the specific VLP HBcAg, amino acids 79-80 are replaced with a foreign epitope. The fusion protein shall retain the ability of assembly into a VLP upon expression which can be examined by electromicroscopy. [0098] Spacer of short amino acid stretch may be added to increase the distance between the coat protein and the antigenice peptide. Glycine and serine residues are particularly favored amino acids to be used in the spacer sequences. Such a spacer confers additional flexibility, which may diminish the potential destabilizing effect of fusing a foreign sequence into the sequence of a VLP subunit and diminish the interference with the assembly by the presence of the foreign epitope.

[0099] The antigenic peptide can be fused to a number of viral coat proteins, by way of examples, to the C-terminus of a truncated form of the Al protein of Qβ (Kozlovska, T. M., et al, Intervirology 39:9-15 (1996)), or being inserted between position 72 and 73 of the CP extension. For example, Kozlovska et al., (Intervirology, 39: 9-15 (1996)) describe QβAl protein fusions where the epitope is fused at the C-terminus of the QβCP extension truncated at position 19. As another example, the antigenic peptide can be inserted between amino acid 2 and 3 of the fr CP (Pushko P. et al., Prot. Eng. 6:883-891 (1993)). Furthermore, the antigenic peptide of the invention can be fused to the N-terminal protuberant β-hairpin of the coat protein of RNA-bacteriophage MS-2 (WO 92/13081). Alternatively, the antigenic peptide can be fused to a capsid protein of papillomavirus, preferably to the major capsid protein Ll of bovine papillomavirus type 1 (BPV-I) (Chackerian, B. et al., Proc. Natl. Acad. Sci.USA 96:2373-2378 (1999), WO 00/23955). Substitution of amino acids 130-136 of BPV- I Ll with the antigenic peptide is also an embodiment of the invention. Further embodiments o fusing antigenic peptide to coat protein, mutants or fragements thereof, to a coat protein of a virus have been disclosed in WO 2004/009124 page 62 line 20 to page 68 line 17 and herein are incorporated by way of reference.

[00100] In another preferred embodiment, the antigenic peptide is fused to either the N- or the C-terminus of a coat protein, mutants or fragments thereof, of RNA-bacteriophage AP205. In one further preferred embodiment, the fusion protein further comprises a spacer, wherein said spacer is positioned between the coat protein, fragments or mutants thereof, of AP205 and the antigenic peptide. Preferably said spacer composed of less than 20, even more preferably less than 15, more preferably less than 12, 10, still more preferably less than 8, less than 5 amino acids (see WO2006/032674 for detail).

[00101] In one preferred embodiment, the antigenic peptide is fused at the N-terminus of the coat protein of fr. In one further preferred embodiment, the CCR5 PNt domain has an amino acid sequence of SEQ ID NO:22, wherein the first cysteine is the cysteine within SEQ ID NO:22 and wherein the second cysteine is added at the C-terminal of CCR5 PNt domain. [00102] In one preferred embodiment, the antigenic peptide of the invention is fused to the N-terminus of the coat protein of AP205. In one further preferred embodiment, the CCR5 PNt domain has an amino acid sequence of SEQ ID NO:22, wherein the first cysteine is the cysteine within SEQ ID NO:22 and wherein the second cysteine is added at the C-terminal of CCR5 PNt domain.

[00103] In one preferred embodiment, the antigenic peptide is fused to the N-terminus of coat protein of AP205 or fr through a spacer comprising at least one amino acid, preferably comprises less than 20, even more preferably less than 15, more preferably less than 12, 10, still more preferably less than 8, less than 7, 6, 5 or 4 amino acids.

[00104] In one preferred embodiment, the composition comprises or alternatively consists essentially of a virus-like particle with at least one first attachment site linked to at least one antigenic peptide of the invention with at least one second attachment site via at least one non-peptide bond, preferably the non-peptide bond is a covalent bond. Preferably the first attachment site does not comprise or is not sulfhydryl group of cysteine. Further preferably the first attachment site does not comprise or is not sulfhydryl group. In one preferred embodiment, the first attachment site comprises, or preferably is, an amino group, preferably the amino group of a lysine residue. In another preferred embodiment, the second attachment site comprises, or preferably is, a sulfhydryl group, preferably a sulfhydryl group of a cysteine. [00105] In a very preferred embodiment, the at least one first attachment site is an amino group, preferably an amino group of a lysine and the at least one second attachment site is a sulfhydryl group, preferably a sulfhydryl group of a cysteine.

[00106] In one preferred embodiment, the antigenic peptide of the invention further comprises an amino acid linker, wherein said amino acid linker comprises at least one second attachment site. The linker is associated to the antigenic peptide by way of at least one covalent bond, preferably, by at least one, typically one peptide bond. In a further preferred embodiment, the linker comprises a sulfhydryl group, preferably of a cysteine residue. In another preferred embodiment, the amino acid linker is a cysteine residue. [00107] The selection of linkers has been disclosed in WO2005/108425 Al, page 32-33, which is incorporated herein by way of reference. In one preferred embodiment, the amino acid linker is selected from the group consisting of: (a) C; (b) GC; (c) GGC; (d) GSC; (e) GGC-CONH2; (f) GC-CONH2; (g) C-CONH2; and (h) GSC-CONH2.

[00108] In one preferred embodiment, the second attachment site comprises or is a sulfhydryl group, preferably a sulfhydryl group of a cysteine. In one preferred embodiment, the amino acid linker is fused to the C-terminal of the PNt domain. In one further preferred embodiment, the second attachment site is located at the C-terminal of the second amino acid of the invention.

[00109] In one preferred embodiment, the at least one antigenic peptide with at least one second attachment site of the invention comprises or consists essentially of or consists of a peptide having an amino acid sequence selected from the group consisting of: (a) MDYQVSSPIYDINYYTSEPCQKINVKQIAARCC (SEQ ID NO:27) ; (b) MDYQVSSPIYDINYYTSEPC QKINVKQIAARCGSC (SEQ ID NO:26); (C) MDYQVSSPIYDINYYTSEPCQKINVKCC (SEQ ID NO:29); and (d) MDYQVSSPIY DINYYTSEPC QKINVKCSGGSC (SEQ ID NO:28). In one very preferred embodiment, the at least one antigenic peptide with at least one second attachment site of the invention comprises or consists essentially of or consists of MDYQVSSPIYDINYYTSEPC QKINVKQIAARCGSC (SEQ ID NO:26).

If the antigenic peptide with at least one second attachment site comprises at least three, preferably consists of three, cysteine residues, the one cysteine, which serves as the second attachment site and which is preferably located at the very C-terminus of the antigenic peptide, shall be protected by commonly known method in the art, for example by acetamidomethyl group, before the peptide reacts with the intermediate molecule. [00110] In one preferred embodiment of the invention, the antigenic peptide of the invention is linked to the VLP by way of chemical cross-linking, typically and preferably by using a heterobifunctional cross-linker. In preferred embodiments, the hetero-bifunctional cross-linker contains a functional group which can react with the preferred first attachment sites, preferably with the amino group, more preferably with the amino groups of lysine residue(s) of the VLP, and a further functional group which can react with the preferred second attachment site, i.e. a sulfhydryl group, preferably of cysteine artificially added to, preferably at the C-terminal of, the antigenic peptide, and optionally also made available for reaction by reduction. Several hetero-bifunctional cross-linkers are known to the art. These include the preferred cross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross-linkers available for example from the Pierce Chemical Company, and having one functional group reactive towards amino groups and one functional group reactive towards sulfhydryl groups. The above mentioned cross-linkers all lead to formation of an amide bond after reaction with the amino group and a thioether linkage with the sulfhydryl groups. Another class of cross-linkers suitable in the practice of the invention is characterized by the introduction of a disulfide linkage between the antigenic peptide and the VLP upon coupling. Preferred cross-linkers belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce). [00111] Linking of the antigenic peptide of the invention to the VLP by using a hetero- bifunctional cross-linker according to the preferred methods described above, allows coupling of the antigenic peptide to the VLP in an oriented fashion. Other methods of linking the antigenic peptide to the VLP include methods wherein the antigenic peptide is cross-linked to the VLP, using the carbodiimide EDC, and NHS. The antigenic peptide may also be first thiolated through reaction, for example with SATA, SATP or iminothiolane. The antigenic peptide, after deprotection if required, may then be coupled to the VLP as follows. After separation of the excess thiolation reagent, the antigenic peptide is reacted with the VLP, previously activated with a hetero-bifunctional cross-linker comprising a cysteine reactive moiety, and therefore displaying at least one or several functional groups reactive towards cysteine residues, to which the antigenic peptide can react, such as described above. Optionally, low amounts of a reducing agent are included in the reaction mixture. In further methods, the antigenic peptide is attached to the VLP, using a homo-bifunctional cross-linker such as glutaraldehyde, DSG, BM[PEO]4, BS3, (Pierce) or other known homo-bifunctional cross-linkers with functional groups reactive towards amine groups or carboxyl groups of the VLP.

[00112] In other embodiments of the present invention, the composition comprises or alternatively consists essentially of a virus-like particle linked to antigenic peptide via chemical interactions, wherein at least one of these interactions is not a covalent bond. Such interactions include but not limited to antigen-antibody interaction, receptor-ligand interaction. Linking of the VLP to the antigenic peptide can be effected by biotinylating the VLP and expressing the antigenic peptide as a streptavidin- fusion protein. [00113] One or several antigen molecules, i.e. antigenic peptide, can be attached to one subunit of the VLP, preferably of RNA-bacteriophage, preferably through the exposed lysine residues, if sterically allowable. A specific feature of the VLPs of RNA-bacteriophage and in particular of the VLP of RNA-bacteriophage Qβ is thus the possibility to couple several antigenic peptides per subunit. This allows for the generation of a dense antigen array. [00114] In a very preferred embodiment of the invention, the antigenic peptide is linked via a cysteine residue, having been added to the C-terminus of the antigenic peptide, to lysine residues on the surface of the VLPs of RNA-bacteriophages, and in particular to the VLP of RNA-bacteriophages Qβ.

[00115] As described above, four lysine residues are exposed on the surface of the VLP of Qβ coat protein. Typically and preferably these residues are derivatized upon reaction with a cross-linker molecule. In the instance where not all of the exposed lysine residues can be coupled to an antigen, the lysine residues which have reacted with the cross-linker are left with a cross-linker molecule attached to the ε-amino group after the derivatization step. This leads to disappearance of one or several positive charges, which may be detrimental to the solubility and stability of the VLP. By replacing some of the lysine residues with arginines, as in the disclosed Qβcoat protein mutants, we prevent the excessive disappearance of positive charges since the arginine residues do not react with the preferred cross-linkers. Moreover, replacement of lysine residues by arginine residues may lead to more defined antigen arrays, as fewer sites are available for reaction to the antigen.

[00116] Accordingly, exposed lysine residues were replaced by arginines in the following Qβcoat protein mutants: Qβ-240 (Lysl3-Arg; SEQ ID NO: 15), Qβ-250 (Lys 2-Arg, Lysl3-Arg; SEQ ID NO:17), Qβ-259 (Lys 2-Arg, Lysl6-Arg; SEQ ID NO:19) and Qβ-251; (Lysl6-Arg, SEQ ID NO:18). In a further embodiment, we disclose a Qβ mutant coat protein with one additional lysine residue Qβ-243 (Asn 10-Lys; SEQ ID NO: 16), suitable for obtaining even higher density arrays of antigens.

[00117] In one aspect, the invention provide an antigenic peptide comprising: (i) CCR5

PNt domain comprising an amino acid sequence KINVK (SEQ ID NO:25); (ii) a first amino acid comprising a first reactive group and wherein said first amino acid is located at the N- terminal of KINVK; (iii) a second amino acid comprising a second reactive group and wherein said second amino acid is located at the C-terminal of KINVK, wherein said first reactive group binds to said second reactive group by at least one covalent bond so that the peptide starting from said first amino acid and ending with said second amino acid is looped.

[00118] The preferred nature and location of the first and the second amino acid and the preferred CCR5 PNt domain have been described throughout this application.

[00119] In one preferred embodiment, the antigenic peptide of the invention comprises, consists essentially of, or consists of an amino acid sequence of SEQ ID NO:22.

[00120] In one preferred embodiment, the first amino acid is a cysteine. In one preferred embodiment, the second amino acid is a cysteine. In one preferred embodiment, the first and the second amino acid is a cysteine.

[00121] In one preferred embodiment, the first amino acid is cysteine, wherein preferably said first amino acid corresponds to, or preferably is, the cysteine within SEQ ID

NO:22. In one preferred embodiment, the second amino acid is a cysteine residue located at any one of +5, +6, +7, +8, +9 and +10 position relative to amino acid amino acid V (position

0) of KINVK. In one further preferred embodiment, second amino acid is cysteine, wherein preferably second amino acid is located +7 position relative to amino acid V (position 0) of

KINVK.

[00122] In one preferred embodiment, the antigenic peptide of the invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of: (i) SEQ ID NO:23 and (ii) SEQ ID NO:24. In one very preferred embodiment, the antigenic peptide comprises or preferably consists of amino acid sequence of SEQ ID

NO:23.

[00123] In one preferred embodiment, the antigenic peptide further comprising at least one, preferably one, intermediate molecule, wherein said intermediate molecule comprises at least two, preferably two, functional reactive sites, wherein said at least two, preferably two, functional reactive sites bind to said first reactive and said second reactive group respectively, wherein preferably at least one of the bounds between the functional reactive site and reactive group comprises a thioether bond.

[00124] In one preferred embodiment, the intermediate molecule comprises a halogenoalkane, wherein preferably said intermediate molecule comprises at least two, preferably two halogen atoms, wherein preferably said two halogen atoms are two Cl atoms, more preferably one Cl atom and one Br atom, even more preferably two Br atoms. Preferably the intermediate molecule comprises an aromatic compound, wherein preferably said aromatic compound comprises at least two benzylic halogen substituents. [00125] In one preferred embodiment, the intermediate molecule is a halomethylarene, preferably selected from the group consisting of bis(bromomethyl)benzene, tris(bromomethyl)benzene and tetra(bromomethyl)benzene, or a derivative thereof. Further preferably the intermediate molecule is a di(halomethyl)benzene, preferably wherein said di(halomethyl)benzene is l,3-bis(bromomethyl)benzene.

[00126] The utility of the antigenic peptide lies preferably in that it mimics the tertiary structure of the CCR5 PNt domain. Thus the antigenic peptide of the invention may be used as antigenic site to raise antibodies or as a bait for screening useful drugs binding to it. Furthermore the antigenic peptide may be useful in the treatment or prevention of HIV- infection.

[00127] In one aspect, the invention provides a vaccine composition comprising the composition of the invention.

[00128] In one preferred embodiment, the vaccine composition further comprises at least one, preferably one, adjuvant. The administration of the at least one adjuvant may hereby occur prior to, contemporaneously or after the administration of the inventive composition. The term "adjuvant" as used herein refers to non-specific stimulators of the immune response or substances that allow generation of a depot in the host which when combined with the vaccine and pharmaceutical composition, respectively, of the present invention may provide for an even more enhanced immune response. Examples of the at least one adjuvant include and preferably consist of complete and incomplete Freund's adjuvant, aluminum hydroxide, aluminium salts, and modified muramyldipeptide. Further adjuvants are mineral gels such as aluminum hydroxide, surface active substances such as lyso lecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Cory neb acterium parvum. Such adjuvants are also well known in the art. Further adjuvants that can be administered with the compositions of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS- 18, CRL1005, Aluminum salts (Alum), MF-59, OM-174, OM-197, OM-294, and Virosomal adjuvant technology. Still further adjuvant include immunostimulatory nucleic acid, preferably the immunostimulatory nucleic acid contains one or more modifications in the backbone, preferably phosphorothioate modifications. The modification is to stabilize the nucleic acid against degradation.

[00129] In another preferred embodiment, the vaccine composition is devoid of adjuvant.

[00130] An advantageous feature of the present invention is the high immunogenicity of the composition, even in the absence of adjuvants. Thus, the administration of the vaccine of the invention to a patient will preferably occur without administering at least one adjuvant to the same patient prior to, contemporaneously or after the administration of the vaccine. VLP has been generally described as an adjuvant. However, the term "adjuvant", as used within the context of this application, refers to an adjuvant not being the VLP used for the inventive compositions, rather in addition to said VLP.

[00131] The invention further discloses a method of immunization comprising administering the vaccine of the present invention to an animal or a human, preferably to a human. The vaccine may be administered by various methods known in the art, but will normally be administered by injection, infusion, inhalation, oral administration, or other suitable physical methods. The conjugates may alternatively be administered intramuscularly, intravenously, transmucosally, transdermally, intranasally, intraperitoneally or subcutaneously. Components of conjugates for administration include sterile aqueous (e.g., physiological saline) or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption.

[00132] Vaccines of the invention are said to be "pharmacologically acceptable" if their administration can be tolerated by a recipient individual. Further, the vaccines of the invention will be administered in a "therapeutically effective amount" (i.e., an amount that produces a desired physiological effect).

[00133] In one aspect, the invention provides a pharmaceutical composition comprising the composition as taught in the present invention and an acceptable pharmaceutical carrier. When vaccine of the invention is administered to an individual, it may be in a form which contains salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the conjugate. Examples of materials suitable for use in preparation of pharmaceutical compositions are provided in numerous sources including REMINGTON'S PHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co., (1990)). [00134] The invention teaches a process for producing the composition of the invention comprising the steps of: (a) providing a VLP with at least one first attachment site; (b) providing at least one antigenic peptide with at least one second attachment site, and wherein said antigenic peptide comprises: (i) CCR5 PNt domain comprising an amino acid sequence KINVK (SEQ ID NO:25); (ii) a first amino acid comprising a first reactive group and wherein said first amino acid is located at the N-terminal of KINVK; (iii) a second amino acid comprising a second reactive group and wherein said second amino acid is located at the C- terminal of KINVK, wherein said first and said second amino acid does not comprise said second attachment site linking said first attachment site; wherein said first reactive group binds to said second reactive group by at least one covalent bond so that the peptide starting from said first amino acid and ending with said second amino acid is looped; (c) linking said VLP and said at least one antigenic peptide to produce said composition, wherein said at least one antigenic peptide and said VLP are linked through said at least one first and said at least one second attachment site.

[00135] In one aspect, the present invention provides a method of preventing and/or treating, preferably treating, HIV infection, wherein the method comprises administering the inventive composition or the inventive vaccine composition, respectively, to a human. [00136] In another aspect, the invention provides for the use of the composition for the manufacture of a medicament for prevention and/or treatment of HIV infection in human.

EXAMPLES EXAMPLE 1

Synthesis of peptides P36 and P37

[00137] Peptides P36 and P37 were synthesized by solid-phase peptide synthesis using a 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy (RinkAmide) resin (BACHEM) on a Syro-synthesizer (MultiSynTech). All Fmoc-amino acids were purchased from Orpegen Pharma or Senn Chemicals with side-chain functionalities protected as N-t-Boc (KW), O-t-Bu (DESTY), N-Trt (HNQ), S-Trt (C), S(Acm) (C), or N-Pbf (R) groups. A coupling protocol using a 6.5-fold excess of HBTU/HOBt/amino acid/DIPEA (1 :1 :1 :2) in NMP with a 30 minute activation time using double couplings was employed. Acetylation of peptides was performed by reacting the resin with NMP/ AC₂CVDIE A (10:1 :0.1, v/v/v) for 30 min at room temperature. Acylated peptides were cleaved from the resin by reaction with TFA (15 ml/g resin) containing 13.3% (w) phenol, 5% (v) thioanisole, 2.5% (v) 1,2-ethanedithiol, and 5% (v) ImIIiQ-H₂O for 2-4 hrs at RT. The crude peptides were purified by reversed-phase high performance liquid chromatography (RP-HPLC), either on a "DeltaPack" (25x100 or 40x210 mm inner diameter, 15 um particle size, 100 A pore size; Waters) or on a "XTERRA" (19 x 100 mm inner diameter, 5 um particle size (Waters) RP- 18 preparative Ci 8 column with a lineair AB gradient of 1-2% B/min. where solvent A was 0.05% TFA in water and solvent B was 0.05% TFA in ACN. The correct primary ion molecular weights of the peptides was confirmed by electron-spray ionization mass spectrometry on a Micromass ZQ (Micromass) or a VG Quattro II (VG Organic) mass spectrometer. [00138] The peptides synthesized are the following:

P36: AC-MDYQVSSPIYDINYYTSEPC(SH) QKINVKC(SH) SGGSC(Acm)-CONH₂ (SEQ ID NO:28)

P37: AC-MDYQVSSPIYDINYYTSEPC(SH)QKINVKQIAARC(SH)GSC(ACm)-CONH₂ (SEQ ID NO:26)

[00139] P36 and P37 peptides without amino -terminal acetyl-group are synthesized as they may better mimic the PNt domain with N-terminal free methoinine.

EXAMPLE 2 Synthesis of T2-P36-SH and T2-P37-SH

[00140] Peptides P36 and P37 were cyclized onto a T2-scaffold via reaction with 1.05 equivalent of 1,3- (bisbromomethyl)benzene in 25% ACN/75% ammonium bicarbonate (20 mM, pH 7.8) for 3 hours at room temperature. The solutions were freeze-dried and the crude peptides were purified by RP-HPLC (for conditions see above) and freeze-dried. For removal of the Acm protective group the peptide constructs were treated with excess (10 equiv.) of I₂ in a mixture of MeOH/DMSO (9:1, v/v) at 1 mM (final concentration) for 15 min. at room temperature, followed by destruction of excess of I₂ with vitC (200 mM). The reaction mixtures were then diluted with 9 volumes Of H₂O and filtered over a RP Cis-cartridge (Sep- Pak® Vac 3cc for HPLC-extraction, Waters Corporation). The peptide-constructs were then collected by elution with ACN/H2O (6 mL, 1 :1 v/v) followed by removal of the solvent by freeze-drying. Subsequently, the peptide-constructs T2-P36-SH and T2-P37-SH were purified by RP-HPLC and freeze-dried (3x) from ACNZmUIiQ-H₂O solution in order to ensure complete removal of traces of TFA and/or ammonium bicarbonate. 1,3- (bisbromomethyl)benzene (T2) was purchased from Sigma-Aldrich. The final products are the following:

T2

T2-P36-SH: AC-MDYQVSSPIYDINYYTSEPCQKINVKCSGGSC(SH)-CONH₂

T2

T2-P37-SH: AC-MDYQVSSPIYDINYYTSEPCQKINVKQIAARCGSC(SH)-CONH₂

EXAMPLE 3 Coupling of antigenic peptide P36, P37 to the virus-like particle of QB

[00141] 2 g/1 virus-like particle of Qβ were derivatised with 1.14 mM SMPH (Pierce) for 30 minutes at 25°C and then dialysed against 20 mM phosphate pH7.4. 0.284 mM peptide P36 (from 5 mM stock in DMSO) and 1 g/1 derivatised Qβ VLPs were incubated for two hours at 25°C. The coupling products were analysed by SDS-page.

[00142] 2 g/1 virus-like particle of Qβ were derivatised with 1.29 mM SMPH (Pierce) for 30 minutes at 25°C and then dialysed against 20 mM MES pH6. 0.178 mM peptide P37 (from 5 mM stock in DMSO) and 1 g/1 derivatised Qβ VLPs were incubated for two hours at 25°C. The coupling products were analysed by SDS-PAGE as shown in FIG. 1.

EXAMPLE 4

Coupling of peptide R8, R9 to virus-like particle of QB

[00143] Peptide R8 and R9 were chemically synthesized. The two cysteines within the peptide were linked by one disulfide bond. The -NH-NH2 group at the very C-terminus of the peptide was used as the second attachment site for coupling to VLP. R8

MDYQVSSPIYDINYYTSEPCQKINVKCG-NH-MK (S-S disulfide bridged) (SEQ ID

NO:30)

R9

MDYQVSSPIYDINYYTSEPCQKINVKQIAAPvCG-NH-MK (S-S disulfide bridged) (SEQ

ID NO:31)

[00144] 0.7 g/1 virus-like particle of Qβ were derivatised with 0.5 mM SFB

(Succinimidyl 4-formylbenzoate dissolved in DMSO, Novabiochem) in 0.1 M phosphate pH7.4, 0.15 M NaCl for 150 minutes at 25°C and then dialysed against 10 mM Hepes pH8. 0.24 mM peptide R8 (from 5 mM stock in DMSO) and 0.66 g/1 derivatised Qβ VLPs were incubated over night at 25°C. The coupling products were analysed by SDS-page. [00145] 0.7 g/1 virus-like particle of Qβ were derivatised with 0.5 mM SFB in 0.1 M phosphate pH7.4, 0.15 M NaCl for 150 minutes at 25°C and then dialysed against 10 mM Hepes pH8. 0.188 mM peptide R9 (from 5 mM stock in DMSO) and 0.66 g/1 derivatised Qβ VLPs were incubated over night at 25°C. The coupling products were analysed by SDS- PAGE.

EXAMPLE 5 Fusing antigenic peptide to the N-terminus of AP205 coat protein

[00146] The construction of Construct 378-2 (with a short GSGG spacer at the N- terminus of the AP205 coat protein) and Construct 382-2 (with a long GSGTAGGGSGS spacer at the N-terminus of AP205) has been described in detail in EXAMPLE 1 of

WO2006/032674.

[00147] The antigenic peptide with sequence of SEQ ID NO:23 is fused to the coat protein of AP205 via either the short spacer or the longer spacer.

[00148] E.coli JM109 cells are transformed with the corresponding AP205 fusion protein plasmids. For expression, the overnight culture is diluted at 1:50 in M9 medium supplemented with casaminoacids (Difco) and containing 20 mg/1 Ampicillin and growth of the culture carried out at 37 ⁰C with vigorous aeration for 14-20 hours.

[00149] Cells are lysed by ultrasonication in lysis buffer (50 mM Tris, 5 mM EDTA 0.1 %

Tween 20, pH 8.0) supplemented with 5 μg/ml PMSF. The lysate is clarified by centrifugation, and the pellet washed with lysis buffer containing 1 M urea. The pooled supernatants are purified over a Sepharose CL-4B column in NET buffer. Eluted fractions containing the VLPs are pooled, concentrated using an Amicon centrifugal filter unit, and purified over a Sepharose 6B column in NET buffer. The fractions containing VLPs are pooled, concentrated with a centrifugal filter unit and dialyzed against 10 mM Hepes pH 7.5, Particle assembly and display of the antigenic peptide is demonstrated by analysis of purified VLPs by SDS-PAGE and EM.

EXAMPLE 6 Mouse immunisation with QB-P 16, QB-P36 and QB-P37

[00150] BaIbC mice were primed with 50 μg Qβ VLP-P36 on day 0 subcutaneously in 0.2 ml 20 mM phosphate pH 7.4. Mice were further boosted with the same vaccine on day 14 and day 28. BaIbC mice injected with Qβ VLP only were used as control. The CC-QB and the CC-

P36 antibody titers were checked by ELISA at day 21 and day 35. QB-P16 immunization followed the same regimen.

[00151] The serum IgG titers against coated RNAse-P36 were between 2'500 - 8'00O at days 21 and 35.

[00152] BaIbC mice were primed with 50 μg Qβ VLP-P37 on day 0 subcutaneously in 0.2 ml 20 mM MES pH7.4. Mice were further boosted with the same vaccine on day 14 and day

58 BaIbC mice injected with Qβ VLP only were used as control. The CC-QB and the CC-P37 antibody titers were checked by ELISA at day 65.

[00153] The serum IgG titers against coated RNAse-P37 peptide were between 20O00 -

100'000 at day 65.

EXAMPLE 7 Rabbit immunisation with QB-P 16, QB-P36 and QB-P37

[00154] New Zealand White rabbits were primed with 100 μg Qβ-P36 peptide on day 0 intradermic in 0.2 ml PBS. After boosting with the same vaccine on days 0, 21, 42, 70, the CC- QB and the CC-CCR5 antibody titers were checked by ELISA at days 0, 21, 49, 63, 80. [00155] The serum IgG titers against coated RNAse-P36 peptide were around 1500 at day 86. EXAMPLE 8 Purification of polyclonal mouse and rabbit IgG

[00156] Serum from five immunised mice obtained from EXAMPLE 6 or 9 ml of rabbit serum obtained from EXAMPLE 7 were centrifuged for five minutes at 14O00 rpm. The supernatant was loaded on a column of 3.3 ml prewashed protein G and protein A sefarose. The column was washed with PBS and eluted with 100 mM glycine pH2.8. 1 ml fractions were collected in tubes previously provided with 120 μl 1 M Tris pH8. Peak fractions absorbing at 280 nm were pooled. The eluted antibodies were dialysed against PBS buffer.

EXAMPLE 9 FACS staining of cellular CCR5 with polyclonal IgG

[00157] In order to check whether the antigenic peptide used for immunisation can induce antibodies that bind to CCR5, we have performed a FACS (fluorescence activated cell sorting) staining of cell-surface exposed CCR5. CEM.NKR-CCR5 is a CCR5 -expressing variant of the CEM.NKR, a human cell line that naturally expresses CD4 (Trkola et al., J. Virol, 1999, page 8966).

[00158] CEM.NKR-CCR5 cells were grown in RPMI 1640 culture medium (with 10% FCS, glutamine, and antibiotics). Cells were pelleted and resuspended in phosphate-buffered saline (PBS) containing 1% fetal calf serum (FCS). 1 g/1 human γ-globulin (Jackson Immuno Research) was added as a blocking agent and incubated for 20 minutes. 0.1 ml cells in the concentration of 2.3xlO⁵ cells/well were plated and then pelleted in a V-bottom 96-well plate. The cells were then resuspended with 0.1 ml CC-CCR5 polyclonal antibodies obtained from EXAMPLE 8 (1 mg/1 purified IgG or 1/50 diluted serum; dilutions with 1% FCS /PBS). After 20 minutes on ice, cells were washed twice in 1% FCS/PBS and stained for 20 minutes on ice in 1% FCS/PBS with either 0.4 mg/1 PE-goat-α-mouse-immunoglobulin (Pharmingen) or with 1/800 diluted PE-donkey-α-rabbit-IgG (Jackson Immuno Research). After two washes in 1% FCS/PBS, cells were resuspended in 0.5 mg/1 propidium iodide, 1% FCS/PBS. [00159] 5'0OO - 10 '000 live cells (propidium iodide negative) were analysed by flow cytometry for binding of PE labelled antibodies. The geometric mean of each staining was determined using the "Flow Jo" flow cytometry software. [00160] Sera of days 21 and 35 from three out of five mice immunized with Qβ VLP-P36 positively stained CEM.NKR cells, whereas α-Qβ sera did not stain CEM.NKR cells. Sera of day 65 from 7 out of 9 mice immunized with Qβ VLP-P37 positively stained CEM.NKR cells. Monoclonal antibodies 2D7 (Pharmingen) or 45531 (R&D systems) were used as positive controls. The data were shown in FIG. 2. Similar positive data of FACS staining were also obtained for sera of 21 days of mice immunized with P16 (data not shown).

EXAMPLE 10

Stimulated primary CD8 depleted PBMC (for HIV neutralisation assays)

[00161] Buffy coats obtained from three healthy blood donors were depleted of CD8⁺ T cells using Rosette Sep cocktail (StemCell Technologies Inc.) and PBMC isolated by Ficoll- Hypaque centrifugation. Cells were adjusted to 4x10⁶ per ml in culture medium (RPMI 1640, 10% FCS, 10 U/ml IL-2, glutamine and antibiotics), divided into three parts and stimulated with either 5 μg/ml phytohemagglutinin (PHA), 0.5 μg/ml PHA or anti-CD3 MAb OKT3 as described (Rusert P. et al, Virology 326: 113-129). After 72 h, cells from all three stimulations were combined (referred to as 3 -way stimulated PBMC) and used as source of stimulated CD4⁺ T cells for infection and virus isolation experiments.

EXAMPLE 11 HIV-Neutralisation assay

[00162] A HIV neutralisation assay with stimulated CD8-depleted PBMC was carried out essentially as described (Trkola et al., 1998, J. Virol. 72, 396).

[00163] The sources of the R5 viruses (CCR5 co-receptor specific strains: JR-FL and

SF162) have been described (O'Brien et al., Nature 1990, 348, page 69; and Shioda et al.,

Nature 1991, 349, page 167). The HIV-I inoculums were adjusted to contain approximately

1,000 to 4,000 TCID5o/ml in assay medium (TCID50: 50% tissue culture infective dose).

Aliquots (60 μl) were incubated with serial dilutions of purified polyclonal rabbit IgG or control antibody 2D7 (25 ng/ml - 25 μg/ml; Pharmingen) for 1 h at 37°C.

[00164] Briefly, cells were incubated with serial dilutions of purified polyclonal rabbit IgG or control antibody 2D7 (25 μg/ml - 25 ng/ml; Pharmingen) in 96-well culture plates for Ih at

37°C. Then virus inoculum (100 TCID₅₀; 50% tissue culture infective dose; Trkola et al., J. Virol, 1999, page 8966) was added and plates cultured for 4-14 days. Either JR-FL or SF162 R5 virus was used for infection. The total infection volume was 200 μl. On day 6 post infection, the supernatant medium was assayed for the HIV-I p24 antigen production by using an immunoassay, as described (Moore et al, 1990. Science 250, page 1139). The calculated inhibitory doses refer to the concentration of antibodies used. p24 antigen production in the absence of testing antibodies was designated as 100%, and the p24 antigen production in antibody-containing cultures was calculated relative to this. The antibody concentrations causing 50%, 70% and 90% reduction in p24 antigen production were determined by linear regression analysis. If the appropriate degree of inhibition was not achieved at the highest or lowest antibody concentration, a value of > or < was recorded and these upper or lower limits were used for statistical analysis.

[00165] Alternatively R5 tropic virus stocks of RHPA Env pseudotyped virus carrying a luciferase reporter gene were prepared by transfecting 10 cm dishes seeded with 293-T cells with 15 μg of the backbone plasmid (pNLluc-AM) and 5 μg of the envelope clone and 40 μg of PEI (linear, 25 kDa, Polysciences, Inc.). Virus stocks were titrated on TZM-bl cells as described (Huber M et al, (2006) PLoS Medicine 3: e441). Neutralization activity of MAbs against pseudotyped virus carrying the patient derived and in vitro selected envelope genes was evaluated on CD8 depleted activated PBMC obtained from EXAMPLE 10 by preincubating the cells for Ih with serial dilutions of purified antibodies obtained from EXAMPLE 8. Then, 10000 - 20000 TCID₅₀/ml of the virus in assay medium (RPMI1640, 10% FCS, 100 U IL-2, 2μg/ml polybrene) was added and incubated for 48h. The antibody concentration causing 50% (IC50), 70% (IC70) and 90% (IC90) reduction in luciferase reporter gene production after 48h was determined by regression analysis. The monoclonal antibody PAl 4 was used the positive control. The result is shown in FIG. 3. While 90% neutralization was not achieved with the concentration 100ug/ml of Qβ antibodies, the highest concentration measured, antibodies raised against peptide P 16, P36 and P37 neutralized 90% virus infection within a concentration comparable to the positive control.

EXAMPLE 12 Synthesize peptide with T3/DTT

[00166] Antigenic peptide with two cysteines, such as: XXXXC(SH)XXXXC(SH)XXXX(X represents any amino acid except cysteine) may also be looped through the two cysteines by T3as the following:

[00167] After synthesize, antigenic peptide was reacted with ~3 equivalent of 2,4,6- tris(bromomethyl)mesitylene in 50% ACN/50% ammonium bicarbonate (20 mM, pH 7.8) for 1-5 min. at room temperature, followed by reaction with excess (20-50 equiv.) of 1,4- dithiothreitol (DTT). The solvents were removed by freeze-drying and the crude T3/DTT peptide was purified by RP-HPLC (for conditions see above) followed by removal of solvent via freeze-drying. 2,4,6-tris(bromomethyl)mesitylene (T3) and 1,4-dithiothreitol (DTT) were purchased from Sigma-Aldrich. DTT provides an additional -SH group which serves as the second attachment site for coupling to VLP.

[00168] FIG. 4 showed P16 (SEQ ID NO:32) peptide, which was synthesized according to the above described method.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising:

(a) a virus-like particle with at least one first attachment site;

(b) at least one antigenic peptide with at least one second attachment site, wherein said antigenic peptide comprises:

(i) CCR5 PNt domain comprising an amino acid sequence KINVK (SEQ ID NO:25):

(ii) a first amino acid comprising a first reactive group and wherein said first amino acid is located at the N-terminal of KINVK;

(iii) a second amino acid comprising a second reactive group and wherein said second amino acid is located at the C-terminal of KINVK, wherein said first and said second amino acid does not comprise said second attachment site linking said first attachment site; wherein said first reactive group binds to said second reactive group by at least one covalent bond so that the peptide starting from said first amino acid and ending with said second amino acid is looped; and wherein said virus-like particle and said at least one antigenic peptide are linked through said first and said second attachment site.

2. The composition of claim 1, wherein said virus-like particle is a virus-like particle of an RNA-bacteriophage, wherein preferably said RNA-bacteriophage is Qβ or AP205.

3. The composition of claim 1, wherein said virus-like particle is a virus-like particle of an RNA-bacteriophage comprises a coat protein, a fragment or a mutein thereof, of an RNA- bacteriophage.

4. The composition of any one of the preceding claims, wherein said first reactive group is a sulfhydryl group, wherein preferably said sulfhydryl group is a sulfhydryl group of the cysteine within SEQ ID NO:22.

5. The composition of any one of the preceding claims, wherein said second reactive group is a sulfhydryl group, wherein preferably said sulfhydryl group is a sulfhydryl group of a cysteine.

6. The composition of any one of the preceding claims, wherein said second amino acid is located +7 position relative to amino acid V (position 0) of KINVK, wherein preferably .said antigenic peptide comprises or preferably consists of an amino acid sequence of SEQ ID NO:23.

7. The composition of any one of the claims 1-5, wherein said second amino acid is located +2 position relative to amino acid V (position 0) of KINVK, wherein preferably said antigenic peptide comprises or preferably consists of an amino acid sequence of SEQ ID NO:24.

8. The composition of any one of the preceding claims, wherein said first reactive group binds to said second reactive group by at least one covalent bond, wherein said covalent bond is a disulfide bond.

9. The composition of any one of the preceding claims, wherein said first reactive group binds to said second reactive group through at least one, preferably one, intermediate molecule, wherein said intermediate molecule comprises at least two functional reactive sites and wherein said two functional reactive site binds to said two reactive group respectively.

10. The composition of claim 9, wherein at least one, preferably both of the bounds between said functional reactive site and reactive group comprises a thioether bond.

11. The composition of claim 9 or 10, wherein said intermediate molecule comprises a halogenoalkane.

12. The composition of any one of the claims 9-11, wherein said intermediate molecule is a di(halomethyl)benzene, preferably wherein said di(halomethyl)benzene is 1,3- bis(bromomethyl)benzene.

13. The composition of any one of the preceding claims further comprises an amino acid linker and wherein said amino acid linker comprises at least one second attachment site, wherein preferably said amino acid linker is fused to the C-terminal of the antigenic peptide, wherein preferably said second attachment site is located at the C-terminal of said second amino acid.

14. The composition of any one of the preceding claims, wherein said second attachment site comprises or is a sulfhydryl group, preferably a sulfhydryl group of a cysteine.

15. The composition of any one of the preceding claims, wherein said CCR5 PNt domain comprises, consists essentially of, or preferably consists of an amino acid sequence of SEQ ID NO:22.

16. The composition of any one of the preceding claims, wherein said antigenic peptide with at least one second attachment site consists of an amino acid sequence as set forth in SEQ ID NO:26.

17. The composition of any one the preceding claims, wherein said first attachment site comprises or is an amino group, preferably an amino group of a lysine.

18. The composition of any one of the preceding claims, wherein said virus-like particle and said antigenic peptide are linked by at least one peptide bond.

19. The composition of any one of the preceding claims, wherein said virus-like particle and said antigenic peptide are linked by at least one covalent bond, wherein preferably said covalent bond is a non-peptide bond.

20. An antigenic peptide comprising:

(i) CCR5 PNt domain comprising an amino acid sequence KINVK (SEQ ID NO:25):

(ii) a first amino acid comprising a first reactive group and wherein said first amino acid is located at the N-terminal of KINVK; (iii) a second amino acid comprising a second reactive group and wherein said second amino acid is located at the C-terminal of KINVK, wherein said first reactive group binds to said second reactive group by at least one covalent bond so that the peptide starting from said first amino acid and ending with said second amino acid is looped.

21. The antigenic peptide of claim 29 further comprising one intermediate molecule, wherein said intermediate molecule comprises at least two functional reactive sites, wherein said at least two functional reactive sites bind to said first reactive and said second reactive group respectively.

22. The antigenic peptide of any one of the claims 20-21, wherein said intermediate molecule comprises an aromatic compound, wherein preferably said aromatic compound comprises at least two benzylic halogen substituents.

23. The antigenic peptide of any one of the claims 20-22, wherein said intermediate molecule is a di(halomethyl)benzene, preferably wherein said di(halomethyl)benzene is 1 ,3-bis(bromomethyl)benzene.

24. The antigenic peptide of any one of the claims 20-23, wherein said first reactive group is a sulfhydryl group, wherein preferably said sulfhydryl group is a sulfhydryl group of the cysteine within SEQ ID NO:22.

25. The antigenic peptide of any one of the claims 20-24, wherein said second amino acid is cysteine, wherein preferably second amino acid is located +7 position relative to amino acid V (position 0) of KINVK, wherein further preferably said antigenic peptide comprises or preferably consists of an amino acid sequence of SEQ ID NO:23.

26. The antigenic peptide of any one of the claims 20-24, wherein said second amino acid is located +2 position relative to amino acid V (position 0) of KINVK, wherein preferably said antigenic peptide comprises or preferably consists of an amino acid sequence of SEQ ID NO:24.

27. A vaccine composition comprising the composition of any one of the preceding claims.

28. The vaccine composition of claim 27 comprising an adjuvant.

29. A pharmaceutical composition comprising:

(a) the composition of any one of the claims 1-19;

(b) an acceptable pharmaceutical carrier.

30. A method of producing the composition of any one of the claims 1-19 comprising:

(a) providing a VLP with at least one first attachment site;

(b) providing at least one antigenic peptide with at least one second attachment site, and wherein said antigenic peptide comprises:

(i) CCR5 PNt domain comprising an amino acid sequence KINVK (SEQ ID NO:25):

(iii) a second amino acid comprising a second reactive group and wherein said second amino acid is located at the C-terminal of KINVK, wherein said first and said second amino acid does not comprise said second attachment site linking said first attachment site; wherein said first reactive group binds to said second reactive group by at least one covalent bond so that the peptide starting from said first amino acid and ending with said second amino acid is looped;

(c) linking said VLP and said at least one antigenic peptide to produce said composition, wherein said at least one antigenic peptide and said VLP are linked through said at least one first and said at least one second attachment site.

31. A method of treating HIV infection comprising the step of administering the composition of any one of the claims 1-19, the vaccine composition of any one of claims 27-28, or the pharmaceutical composition of claim 29 into a HIV infected patient.

32. Use of the composition of any one of the claims 1-19 for the manufacture of a medicament for the treatment of HIV infection.