WO2004052395A1

WO2004052395A1 - L2-peptide of the human papillomavirus associated with virus-like particles

Info

Publication number: WO2004052395A1
Application number: PCT/EP2003/014040
Authority: WO
Inventors: Ralph Biemans; Brigitte Desiree Alberte Colau
Original assignee: Glaxosmithkline Biologicals Sa
Priority date: 2002-12-09
Filing date: 2003-12-05
Publication date: 2004-06-24
Also published as: GB0228715D0; AU2003290012A1

Abstract

The invention relates to HPV peptides in physical association with an HPV VLP.

Description

L2 -PEPTIDE OF THE HUMAN PAPILLOMAVIRUS ASSOCIATED WITH VIRUS-LIKE PARTICLES

The present invention relates to vaccines against Human papillomavirus (HPV)

Papillomaviruses are small DNA tumour viruses, which are highly species specific. So far, over 100 individual human papillomavirus (HPN) genotypes have been described. HPNs are generally specific either for the skin (e.g. HPN-1 and -2) or mucosal surfaces (e.g. HPN-6 and -11) and usually cause benign tumours (warts) that persist for several months or years. Such benign tumours may be distressing for the individuals concerned but tend not to be life threatening, with a few exceptions.

Some HPVs are also associated with cancers. The strongest positive association between an HPV and human cancer is that which exists between HPN- 16 and HPN- 18 and cervical carcinoma. Cervical cancer is the most common malignancy in developing countries, with about 500,000 new cases occurring in the world each year. For a review on the prospects for prophylactic and therapeutic vaccination against HPN- 16 see Cason J., Clin. Immunother. 1994; 1(4) 293-306 and Hagenesee M.E., Infections in Medicine 1997 14(7) 555-556,559-564.

Although minor variations do occur, all HPNs genomes described have at least eight early genes, El to E8 and two late genes LI and L2. In addition, an upstream regulatory region harbours the regulatory sequences that appear to control most transcriptional events of the HPN genome.

HPN LI based vaccines are disclosed in WO94/00152, WO94/20137, WO93/02184 and WO94/05792. Such a vaccine can comprise the LI antigen as a monomer, a capsomer or a virus like particle. Methods for the preparation of VLPs are well known in the art, and include VLP disassembly-reassembly approaches to provide enhanced homogeneity, for example as described in WO9913056 and US6245568. Such particles may additionally comprise L2 proteins. L2 based vaccines are described, for example, in WO93/00436. Other HPV vaccines are based on the Early proteins, such as E7 or fusion proteins such as L2-E7. The protection afforded by fragments of HPV proteins has been studied in some cases.

Fragments of the L2 gene have been experimentally tested in mice studies. Kawana et al (Vaccine, 19, 2001, 1496 - 1502) disclose an L2 peptide of 12 amino acids used for intranasal administration, said to represent a cross neutralising epitope. Further studies have been carried out in human subjects (Kawana et al Vaccine 21 (2003) 4256 - 4260)

Roden et al disclose use of large (>200 amino acid) L2 fragments, which are shown to be cross neutralising.

US patent 6380157 discloses a method of therapeutically treating a papillomavirus tumor or lesion, said method comprising administering a peptide of at least about 127 amino acids of the L2 protein.

Varsani et al, 20^th international HPV conference, Paris, October 4-9, 2002, Poster 449 disclose HPV -16 VLPs into which an L2 peptide has been cloned.

Despite recent advances in research there is still a need for an effective vaccine against cervical cancer or other HPV related illness.

The present invention relates to immunogenic compositions and vaccines against human papillomavirus.

Statement of Invention

In a first aspect the present invention relates to an immunogenic composition comprising an HPV peptide in physical association with an HPV virus like particle (VLP) or suitable carrier.

The invention further relates to use of an immunogenic composition as defined above in the preparation of a vaccine for the prevention or treatment of cervical cancer or genital warts. The invention further relates to a method of preventing and/or treating cervical cancer or genital warts, the method comprising delivering to an individual at risk of such diseases an effective amount of an immunogenic composition as defined above.

In a related aspect the invention relates to a vaccine composition comprising an HPV peptide in physical association with an HPV virus like particle (VLP) or suitable carrier.

The invention also relates to a method of vaccine production, the method comprising association of the peptide of the invention with an HPV VLP or suitable carrier.

The invention further relates to use of a vaccine composition as defined above in the preparation of a medicament for the prevention or treatment of cervical cancer or genital warts.

The invention further relates to a method of preventing and/or treating cervical cancer or genital warts, the method comprising delivering to an individual at risk of such disease an effective amount of a vaccine as defined above.

The invention further relates to a polynucleotide encoding the peptides and/or VLPs of the invention, and uses thereof in vaccine production and prevention and/or treatment as outlined above.

The invention particularly relates to an HPV peptide chemically linked to an HPV VLP or carrier.

Detailed description

Any suitable peptide may be used in the present invention for association with the HPV VLP or carrier. Peptides may be derived from the HPV LI or L2 proteins, or the HPV El, E2, E3, E4, E5, E6 or E7 proteins. Peptides are preferably immunogenic peptides and such peptides can be identified standard techniques well known in the art - for example, delivery of peptides into model animals or humans by suitable delivery routes and measurement of antibody and/or cellular immune responses by, for example, ELISA or cytokine analysis/measurement respectively. Suitable methods are given in Example 1.

Examples of preferred peptides from HPV proteins include those disclosed in, for example; Muderspach et al (Clinical Cancer Research vol 6, 3406 - 3416 September 2000), relating to an E7 peptide; Williams et al (Journal of virology August 2002 p7418 - 7429, vol 76, no 15) relating to HPV 11 peptides; Welters et al (Cancer Research 63, 636 - 641, Feb 2003) relating to E6 peptides; Steele et al (Journal of Virology June 2002, p6027 - 6306) relating to LI, E4 and E6 peptides; Zwaveling et al (Journal of immunology 2002, 169, p350 - 358) relating to E7 peptides; and de Jong et al (Cancer Research 62, 472 - 479 January 15 2002), relating to E2 peptides. The teaching of all documents and peptides used therein is herein incorporated by reference.

Most preferred peptides are derived from HPV L2 protein. The present invention is herein described in detail in respect of an L2 peptide by way of example. The teaching in respect of L2 can, however, equally be applied to other HPV peptides, and reference to 'L2' can be take an as a reference to a peptide from any other suitable HPV peptide, preferably for example LI, El, E2, E3, E4, E5, E6, E7, unless otherwise apparent from the context.

For the avoidance of doubt, the means to physically associate peptides with VLP/caπϊer taught herein relates to all peptides, and HPV peptides of the invention can have none, or one, or more of the preferred features described for L2 peptides hereinbelow. HPV peptides may be derived from proteins from any HPV type, as appropriate, with peptides from oncogenic cancer types (16, 18, 31,33,35,39,45,51,52,56,58,59,66,68) or genital warts types (6, 11) being preferred.

The L2 peptide of the invention may be any suitable immunogenic L2 peptide. L2 peptides may be tested for immunogenicity by standard techniques well known in the art - for example, injection of peptides into model animals or humans and measurement of antibody and/or cellular immune responses by, for example, ELISA or cytokine analysis/measurement respectively. Suitable methods are given in Example 1.

Preferably the L2 peptide is a cross reactive peptide, that is, able to elicit an immune response which recognises an L2 protein or L2 peptide from another HPV genotype. Preferably the peptide is cross-reactive with 1 or 2 or more other genotypes, preferably a genotype associated with causation of cervical cancer, and most preferably is cross-reactive with at least one of HPV 16 or HPV 18, preferably both HPV 16 and HPV 18.

Preferably the L2 peptide is able to provoke an immune response against homologous HPV infection, that is, against HPV infection by the HPV type from which the sequence is derived.

Preferably the L2 peptide is able to provide cross protection, and suitably comprises a cross neutralising epitope, preferably for one or more of HPV types associated with cervical cancer or genital warts, preferably one or more of HPV 16, 18, 6, 11 , 31 or 45.

Cross protection suitably occurs when an L2 peptide is capable of generating a protective immune response against infection/disease caused by an HPV type which is not the same type as that from which the L2 peptide sequence was derived or obtained. Preferably the L2 peptide is capable of generating a protective immune response against infection by 2 (or more) different HPV genotypes, preferably also being able to provoke a homologous protective immune response.

Cross protection may be assessed by comparing incidence of infection and/or disease for a group of HPV types (infection being incident or persistent infection) in individuals vaccinated with a given L2 peptide vs a non vaccinated group. Complete cross protection against a type, or group of types, is not required in the present invention - indeed, any level of cross protection provides a benefit. Preferably the level of cross protection observed is such that the vaccinated group has 5% less infection and/or disease than a comparable non vaccinated group, more preferably up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65% up to 70%, up to 80%, up to 90% or even up to 1 0% less infection and/or disease.

Cross protection may be assessed by detecting the presence of nucleic acid specific for various HPV types in the vaccinees and control group. Detection may be carried out, for example, using techniques as described in WO03014402, and references therein, particularly for non-specific amplification of HPV DNA and subsequent detection of DNA types using a LiPA system as described in WO 99/14377, and in Kleter et al, [Journal of Clinical Microbiology (1999), 37 (8): 2508-2517], the whole contents of which are herein incorporated by reference. Any suitable method can, however, be used for the detection of HPV DNA in a sample, such as type specific PCR using primers specific for each HPV type of interest. Suitable primers are known to the skilled person, or can be easily constructed given that the sequences of the different HPV types are known.

Suitably cross protection is observed in the male and/or female population, preferably women who are seronegative for HPV infection, or seronegative for HPV 16 and 18, preferably adolescent women pre-sexual activity.

Cross protection (as assessed by protection seen in a vaccinated group vs a control group) is preferably seen against oncogenic types, such as any one of the group of high risk cancer types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 or 68 or, collectively, groups of high risk cancer types such as any 2,3,4,5,6,7,8, 9,10, 11, 12, 13, or indeed all, of these high risk cancer types. All possible combinations of 2,3,4,5,6,7,8,9,10, 11, 12 and 13 of these high risk cancer types are specifically contemplated, and as such there are various different 'groups' of VLP types individualised herein, for which the level of cross protection can be analysed in comparison to a placebo group.

Preferably the L2 peptide is derived from HPV 16 and provides cross protection against incident or persistent infection and/or disease caused by HPV infection from other HPV types, preferably one or more of the group of HPV types HPV 31, 33, 35, 52 and 58, or one of more of the group of types HPV 31, 35, 58. Also preferred is an L2 peptide derived from HPV 18 and which provides cross protection against incident or persistent infection and/or disease caused by HPV infection from other HPV types, preferably one or more of the group of HPV types HPV 31, 33, 35, 52 and 58, or one of more of the group of types HPV 31, 35, 58.

The L2 peptide preferably comprises the L2 sequence of amino acids 108 - 120 derived from the HPV 16 genotype (having the amino acid sequence 'LVEETSFIDAGAP' as disclosed in Kawana 2001 et al, supra, the entire contents of which are herein incorporated by reference), or comprises an equivalent L2 sequence from another HPV genotype. Equivalent sequences are those peptides which correspond to the 'LVEETSFIDAGAP' HPV 16 peptide by virtue of sequence identity or relative position within the L2 protein. Preferably the equivalent is a functional equivalent of the HPV 16 L2 108 - 120 peptide, suitably capable of providing cross protection, suitably to one or more of HPV types 6, 11, 31, 45 and 18, most preferably capable of providing cross protection against one or more oncogenic cancer types HPV 16, 18, 31,33,35,39,45,51,52,56,58,59,66,68.

Suitable examples of peptides are given in Kawana et al, (Journal of Virology 1999, . p6188 - 6190), hereby incorporated by reference. In particular it is preferred that the L2 peptide comprises the HPV 16, 6,11, 33, 58, 18 or 2a L2 peptide sequences as disclosed therein (see Kawana supra, Fig 1). Also preferred are L2 peptides comprising the consensus sequence of the L2 peptide - namely LXEX GAX, where X is any amino acid. Preferably the sequence is L - V/I - E - E/D - T/S - S/A- F/I/N - I/N - X - A/S - G - A -P/S where 7' indicates a choice of amino acids.

L2 peptides may be synthesised for use in the invention using techniques well known in the art, and may suitably include amino acid residues that facilitate conjugation to carriers or VLPs, for example. Preferred is a construct comprising spacer and coupling residues such as: peptide-GGC-ΝH₂ , wherein the glycines are spacer residues and cysteine is suitable for use in a coupling reaction

L2 peptides may be tested for cross-reactivity by standard techniques. By way of example, peptides to be tested can be mixed with sera from patients infected with different HPV strains, to check for cross-reactivity. Alternatively phage display libraries may be used to screen for peptides which are cross-reactive with sera from individuals infected with different HPV genotypes. In another approach ELISA assays may be used to detect antibodies reactive against different L2 sequences. The cross neutralising response against different HPV types may also be analyzed by using a pseudovirions neutralisation assay - see for example Combita et al, Journal of Virology, July 2002, p6480 - 6486.

The term 'peptide', as used herein, refers to any 2 or more amino acids, although peptides are suitably at least 5, 6,7,8,9,10, 11,12,13,14, 15, 20 or more amino acids in length. Preferably the L2 peptides of the invention are short peptides of less than 100 amino acids, suitably less than 50 amino acids, preferably less than 40 amino acids, 30 amino acids and most preferably less than 20 amino acids. Less preferred is the full length L2 protein. The L2 peptide portion itself may be combined with other sequences, such as additional epitopes as discussed below, which may then increase the overall peptide length. It is preferred that the total length of construct including the L2 peptide be less than 200 amino acids, preferably less than 150 amino acids and < most preferably less than 100 amino acids in length.

Optionally the vaccine of the invention comprises multiple L2 peptides of the invention, preferably at least one being a cross-reactive L2 peptide, and preferably 2 or more being cross-reactive.

The invention also relates to mutants and variants of the above preferred L2 peptide, such as deletion, addition, inversion or substitution mutants which retain cross- reactivity. Preferred variants include modifications to the L2 peptide so as to optimise binding between the antigen and MHC molecules. Examples of this approach are described in Kawana et al, supra, and are well known in the art.

L2 peptides of the invention may optionally be branched or circularised peptides.

Preferred are L2 peptides that are delivered in combination with a source of CD8 and/or TH1 epitopes. This may be achieved by mixing of the L2 peptide with a suitable source of such epitopes, or most preferably the epitopes may be fused to the peptide. Suitable epitopes include, for example, the amino acid sequences QYΓKANSKFIGITE, PGINGKAIHLVNNESSE, FNNFTVSFWLRVPKVSASHLE from tetanus toxin, the Measles F epitope LSEIKGVIV-HRLEGV or the PADRE sequence (see Benmohamed et al Immunology, 2002, 106, 113 - 121, incorporated herein by reference). Methods to synthesise or link the L2 peptide with other epitopes are well known in the art.

Also preferred are L2 hpopeptides, in which the L2 peptide is conjugated to a lipid. Examples of suitable lipids and production techniques are given in Gras-Masse (Molecular immunology 38, [2001] 423 - 431), Wiesmuller et al (Int. J. Peptide Protein Res. [1992] 25 - 260) and Benmohamed supra, the contents of which are herein incorporated by reference. In essence, the lipid portion acts to provide an adjuvanting effect to the peptide. The lipid portion suitably helps the peptide to interact with and be delivered across the cell membrane.

Preferably the L2 peptide is directly linked to the lipid portion, preferably through a linkage via a lysine or glycine residue on the peptide. If necessary the L2 peptide can be generated with a C terminal or N terminal linker comprising a lysine or glycine, in order to facilitate lipid attachment.

Suitable lipids include palmitic acid derivatives, suitably straight chain derivatives, which are disclosed in Benmohamed supra and references contained within, all of which are incorporated by reference.

Further preferred are lipopeptide combinations in which the L2 peptide component is lipidated and provided in combination with a peptide having a TH1 and or CD8 epitope. Preferably the L2 lipopeptide is conjugated or formed as a fusion peptide with the peptide having a TH1 and/or CD8 epitope.

Suitable epitopes may also be provided by conjugation of the L2 peptide with a suitable HPV peptide or polypeptide, such as an early antigen from HPV.

Reference generally to L2 peptides herein, accordingly, is taken to include all above possibilities such as; L2 peptides with cross reactive or cross protective activity, L2 mutants and variants; L2 hpopeptides; L2 peptides, hpopeptides, mutants and variants mixed or conjugated to a source of CD8 and/or THl epitopes, or attached to DNA or RNA sequences, and combinations thereof, unless otherwise apparent from the context. All combinations are herein individually and separately disclosed. Preferably L2 peptides retain the ability to stimulate the immune response when encapsulated or conjugated, for example.

As stated above, reference to L2 peptides includes reference to other HPV peptides, suitably immunogenic peptides.

The L2 peptide of the invention is physically associated with an HPV VLP or carrier.

HPV VLPs are well known in the art, and may be Ll-only VLPs or comprise additional proteins.

VLPs may comprise full length LI protein.

Where the L2 peptide is used in conjunction with a VLP, then it is preferred that the LI protein used to form the VLP is a truncated LI protein. Preferably the truncation removes a nuclear localisation signal. Preferably the truncation is a C terminal truncation. Preferably the C terminal truncation removes less than 50 amino acids, more preferably less than 40 amino acids. Most preferably the C terminal truncation removes 34 amino acids from HPV 16 and 35 amino acids from HPV 18.

Truncated LI proteins are suitably functional LI protein derivatives. Functional LI protein derivatives are capable of raising an immune response (if necessary, when suitably adjuvanted), said immune response being capable of recognising a VLP consisting of the full length LI protein and/or the HPV type from which the LI protein was derived.

When used in the invention VLPs may also comprise other functional protein derivatives, including mutants of the full length or truncated HPV LI proteins such as deletion, substitution, or insertion mutants. Suitable derivatives also include codon optimised sequences. The LI protein or derivative may also be a fusion protein, such as the fusion of the LI protein with L2 or an early protein. The LI protein or functional protein derivative is suitably able to form a VLP, and VLP formation can be assessed by standard techniques such as, for example, electron microscopy and dynamic laser light scattering.

Whether the LI protein is truncated or not, it is preferred that the LI still retains a DNA and/or RNA binding capability such as is found in the full-length molecule. This may be as a result of a naturally occurring LI region or a DNA/RNA binding domain that is engineered into the LI sequence. Such a region can assist in the preparation of a VLP mixture with a polynucleotide encoding an L2 peptide, for example, by permitting the interaction of the LI protein with a polynucleotide encoding an L2 peptide such that the polynucleotide becomes linked to or encapsulated within the HPV VLP.

The physical association between the L2 peptide and the VLP in the present invention requires that the L2 peptide is combined with the VLP in some way which is more than simple mixing of L2 peptide and VLP. Preferably the L2 peptide and VLP or carrier are not in simple admixture. Physical association may include chemical association. In particular the L2 peptide may be chemically crosslinked, directly or indirectly via a linker, to the VLP or carrier, which provides a physical association. Alternatively LI capsids may be generated from LI protein or LI protein derivatives, and then VLP formation allowed to occur in the presence of the L2 peptide, such that the peptide is encapsulated within the VLP.

The HPV peptide and VLP and/or carrier are thus physically associated when the components are conjugated, such as by chemical crosslinking, linked by encapsulation, by ionic or covalent interaction. Preferably there is a covalent bond between the peptide and the VLP or carrier.

Preferably the L2 peptide of the invention is conjugated to a VLP, suitably using technology as outlined in Example 1 or equivalent.

In one embodiment of the invention the L2 peptide may be attached to a DNA or RNA sequence which can itself associate with the VLP, preferably with the LI protein, suitably through an ionic or covalent interaction. In this way the L2 peptide can be physically associated with the VLP.

In addition a VLP comprising encapsulated polynucleotide such as DNA or RNA may be used in combination with an L2 peptide.

Also preferred is the combination of a circularised L2 peptide linked to a VLP with a linker, preferably a linear peptide linkage or other suitable linkage.

Preferably the L2 peptide does not form a structural part of the VLP itself. In particular we prefer that the L2 peptide is not in the form of a chimaera with the LI protein or peptide which forms the VLP. This is the case even if the VLP is made up from elements of LI and L2 proteins or peptides, for example being an LI chimaera with a fragment of L2. The present invention specifically then contemplates an additional L2 component - the L2 peptide of the invention, physically associated with the VLP. Thus, the L2 peptide of the invention preferably does not form an integral part of the shell of the VLP.

Where the L2 peptide is associated with a VLP then preferably there is no interference between the L2 peptide and the VLP, such that both components remain immunogenic. Preferably the combination of L2 peptide and VLP is capable of provoking a protective immune response against the HPV infection and/or disease, suitably against infection/disease caused by the HPV type equivalent to the protein type (s) found in the VLP, and more preferably to other types which do not have a protein or peptide component in the vaccine. Interference may be assessed by, for example, the methods given in example 1.

It is not necessary for there to be no interference between L2 peptide and HPV VLP, and some reduction in immune response generated by the VLP/L2 peptide combination may be tolerated in comparison to that induced by the individual components, with the proviso that the whole composition is immunogenic. Preferably there is no interference in the immune response generated against either component and most preferably there is synergy between components, such that the overall immune response is greater or broader than that of the individual components added together, for example at the level one or more of the magnitude, speed of response, duration of response or any other factor.

When the L2 peptide is formulated with a carrier, then the carrier is suitably an ISCOM, virosome, proteosome, liposome, or microparticle. The peptide may be added to a preformed ISCOM, proteosome, liposome, virosome or microparticle or used to prepare a mixed ISCOM/virosome/microparticle/ proteosome/liposome where possible. Suitable microparticles, ISCOMS, proteosomes, liposomes and virosomes, along with methods for preparing them, are well known in the art.

An L2 peptide of the invention formulated with a carrier may also be combined with a VLP, for example.

The L2 peptide of the invention is preferably physically associated with the carrier.

It is generally preferred that the L2 peptide is associated with the outside of the VLP or carrier, and not contained or encapsulated within it.

The invention also extends to cover polynucleotides such as DNA and RNA which encode the peptides and proteins of the invention. By way of example, DNA encoding the both the LI and L2 peptide, preferably also encoding a suitable CD8 and/or THl epitope is preferred. The invention further covers vectors comprising said polynucleotides and cells comprising said vectors, for example yeast or insect cells in which the polynucleotides may be expressed.

The L2 peptide and VLP and/or carrier of the invention preferably form an immunogenic compound, capable of provoking an immune response.

The composition of the invention preferably generates an immune response in a human or animal subject against 1, 2 or more HPV genotypes, preferably any 1, 2 or more selected from the group of HPV 6, 11, 16, 18, 31, 45, 52, 53, 58, 33, 35, 56 and 59. The composition of the invention preferably provides protection against infection and/ or disease from 1, 2 or more HPV genotypes, preferably any 1, 2 or more selected from HPV 6, 11, 16, 18, 31, 45, 52, 53, 58, 33, 35, 56 and 59. Preferably the composition provides protection against at least HPV 16 or 18, and more preferably against both HPV 16 and 18.

Preferably the composition comprises an HPV 18 VLP and an L2 peptide comprising a sequence derived from HPV 16 L2, preferably chemically conjugated together.

Suitably the composition is a vaccine composition.

The vaccine of the invention may be used to treat or prevent HPV infection and/or disease. For example the vaccine may be used therapeutically to reduce viral load and/or infections that lead to cervical carcinoma or CIN III sequelae. The invention thus relates to use of the vaccine of the invention in the therapeutic treatment of diseases related to HPV infection and in prophylaxis of infection or disease. Preferred is the use of the vaccine of the invention in prophylaxis of infection and/or disease. The term 'infection', as used herein suitably relates to incident infection and/or persistent infection.

Optionally the vaccine may also be formulated or co-administered with other HPV antigens such as early antigens or non-HPV antigens. Suitably these non HPV antigens can provide protection against other diseases, most preferably sexually transmitted diseases such as herpes simplex virus, chlamydia and HIV. We particularly prefer that the vaccine comprises gD or a truncate thereof from HSV. In this way the vaccine provides protection against both HPV and HSV.

For all vaccines of the invention, it is preferred that the vaccine is used for the vaccination of adolescent girls aged 10-15, preferably 10-13 years. The vaccine is also preferably suitable for administration to a paediatric population, 0-10 years old. The vaccine may also be administered to women following an abnormal pap smear or after surgery following removal of a lesion caused by HPV. Thus the vaccine is preferably suitable for both a seronegative population as a prophylactic vaccine and/or a seropositive population in a therapeutic setting. Preferably the vaccine is delivered in a 2 or 3 dose regime, for example in a 0, 1 month regime or 0,1 and 6 month regime respectively. Suitably the vaccination regime incorporates a booster injection after 5 to 10 years, preferably 10 years. Other regimes, with 4 or more doses, may also be used.

Preferably the vaccine is a liquid vaccine formulation, although the vaccine may be lyophilised and reconstituted prior to administration.

The vaccines of the invention may comprise a suitable adjuvant or imunostimulant such as, but not limited to, detoxified lipid A from any source and non-toxic derivatives of lipid A, saponins and other reagents capable of stimulating a THl type response.

It has long been known that enterobacterial lipopolysaccharide (LPS) is a potent stimulator of the immune system, although its use in adjuvants has been curtailed by its toxic effects. A non-toxic derivative of LPS, monophosphoryl lipid A (MPL), produced by removal of the core carbohydrate group and the phosphate from the reducing-end glucosamine, has been described by Ribi et al (1986, Immunology and Immunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY, p407-419) and has the following structure:

A further detoxified version of MPL results from the removal of the acyl chain from the 3-position of the disaccharide backbone, and is called 3-O-Deacylated monophosphoryl lipid A (3D-MPL). It can be purified and prepared by the methods taught in GB 2122204B, which reference also discloses the preparation of diphosphoryl lipid A, and 3-O-deacylated variants thereof.

A preferred form of 3D-MPL is in the form of an emulsion having a small particle size less than 0.2μm in diameter, and its method of manufacture is disclosed in WO 94/21292. Aqueous formulations comprising monophosphoryl lipid A and a surfactant have been described in WO9843670A2.

The bacterial lipopolysaccharide derived adjuvants to be formulated in the compositions of the present invention may be purified and processed from bacterial sources, or alternatively they may be synthetic. For example, purified monophosphoryl lipid A is described in Ribi et al 1986 (supra), and 3-O-Deacylated monophosphoryl or diphosphoryl lipid A derived from Salmonella sp. is described in GB 2220211 and US 4912094. Other purified and synthetic lipopolysaccharides have been described (Hilgers et al, 1986, Int. Arch. Allergy. Immunol, 79(4):392-6; Hilgers et al, 1987, Immunology, 60(l):141-6; and EP 0 549 074 Bl). A particularly preferred bacterial lipopolysaccharide adjuvant is 3D-MPL.

Accordingly, the LPS derivatives that may be used in the present invention are those immunostimulants that are similar in structure to that of LPS or MPL or 3D-MPL. In another aspect of the present invention the LPS derivatives may be an acylated monosaccharide, which is a sub-portion to the above structure of MPL.

Saponins are taught in: Lacaille-Dubois, M and Wagner H. (1996. A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp 363- 386). Saponins are steroid or triterpene glycosides widely distributed in the plant and marine animal kingdoms. Saponins are noted for forming colloidal solutions in water which foam on shaking, and for precipitating cholesterol. When saponins are near cell membranes they create pore-like structures in the membrane which cause the membrane to burst. Haemolysis of erythrocytes is an example of this phenomenon, which is a property of certain, but not all, saponins.

Saponins are known as adjuvants in vaccines for systemic administration. The adjuvant and haemolytic activity of individual saponins has been extensively studied in the art (Lacaille-Dubois and Wagner, supra). For example, Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), and fractions thereof, are described in US 5,057,540 and "Saponins as vaccine adjuvants", Kensil, C. R., CritRev Ther Drug Carrier Syst, 1996, 12 (l-2):l-55; and EP 0 362 279 Bl. Particulate structures, termed Immune Stimulating Complexes (ISCOMS), comprising fractions of Quil A are haemolytic and have been used in the manufacture of vaccines (Morein, B., EP 0 109 942 Bl; WO 96/11711; WO 96/33739). The haemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been described as potent systemic adjuvants, and the method of their production is disclosed in US Patent No.5,057,540 and EP 0 362 279 Bl. Other saponins which have been used in systemic vaccination studies include those derived from other plant species such as Gypsophila and Saponaria (Bomford et al, Vaccine, 10(9):572-577, 1992).

An enhanced system involves the combination of a non-toxic lipid A derivative and a saponin derivative particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in WO 96/33739.

A particularly potent adjuvant formulation involving QS21 and 3D-MPL in an oil in water emulsion is described in WO 95/17210 and is a preferred formulation.

Accordingly in one embodiment of the present invention there is provided a vaccine adjuvanted with detoxified lipid A or a non-toxic derivative of lipid A, more preferably adjuvanted with a monophosphoryl lipid A or derivative thereof.

Preferably the vaccine additionally comprises a saponin, more preferably QS21.

Preferably the formulation additionally comprises an oil in water emulsion. The present invention also provides a method for producing a vaccine formulation comprising mixing an L2 peptide of the present invention together with a pharmaceutically acceptable excipient, such as 3D-MPL.

Additional components that are preferably present in an adjuvanted vaccine formulation according to the invention include non-ionic detergents such as the octoxynols and polyoxyethylene esters as described herein, particularly t- octylphenoxy polyethoxyethanol (Triton X-100) and polyoxyethylene sorbitan monooleate (Tween 80); and bile salts or cholic acid derivatives as described herein, in particular sodium deoxycholate or taurodeoxycholate. Thus, a particularly preferred formulation comprises 3D-MPL, Triton X-100, Tween 80 and sodium deoxycholate, which may be combined with an L2 antigen preparation to provide a suitable vaccine.

In one preferred embodiment of the present invention, the vaccine comprises a vesicular adjuvant formulation comprising cholesterol, a saponin and an LPS derivative. In this regard the preferred adjuvant formulation comprises a unilamellar vesicle comprising cholesterol, having a lipid bilayer preferably comprising dioleoyl phosphatidyl choline, wherein the saponin and the LPS derivative are associated with, or embedded within, the lipid bilayer. More preferably, these adjuvant formulations comprise QS21 as the saponin, and 3D-MPL as the LPS derivative, wherein the ratio of QS21:cholesterol is from 1 :1 to 1 :100 weight/weight, and most preferably 1 :5 weight/weight. Such adjuvant formulations are described in EP 0 822 831 B, the disclosure of which is incorporated herein by reference.

Suitably the vaccines of the invention are used in combination with aluminium, and are suitably adsorbed or partially adsorbed onto aluminium adjuvants. Suitably the adjuvant is an aluminium salt, preferably in combination with 3D MPL, such as aluminium phosphate and 3D MPL. Aluminium hydroxide, optionally in combination with 3D MPL is also preferred.

The vaccine may also comprise aluminium or an aluminium compound as a stabiliser.

The vaccines of the invention may be provided by any of a variety of routes such as oral, topical, subcutaneous, musosal (typically intravaginal), intraveneous, intramuscular, intranasal, sublingual, intradermal and via suppository. However, mucosal administration is most preferred, such as intranasal or intravaginal

The dosage of the vaccine components will vary with the condition, sex, age and weight of the individual, the administration route and HPV of the vaccine. The quantity may also be varied with the number of VLP types. Suitably the delivery is of an amount of vaccine suitable to generate an immunologically protective response. Where VLPs are used in the invention, suitably each vaccine dose comprises 1-100 μg of each VLP, preferably 5-80μg, more preferably 5- 30 μg each VLP, most preferably 5-20 μg of each VLP with 5μg, 6μg, lOμg, 15 μg or 20μg especially preferred.

Any appropriate dose of L2 peptide may be used, such as suitably, lμg -lg, preferably l-1000μg, preferably 10- 500μg, preferably 20 - 200μg, most preferably 50 - 100 μg.

The invention also relates to a method of preventing or treating cervical cancer or genital warts, the method comprising administration of an effective amount of an L2 peptide composition of the invention to an individual in need of such treatment. When the vaccine of the invention comprises or consists of a mixture of HPV 16, HPV 18, HPV 31 and HPV 45 VLPs as specifically disclosed in co-pending application GB0206360 (WO03/077942) then preferably the L2 peptide is not that specifically disclosed in K. Kawana et al Vaccine 19, (2001) pl496-1502.

When the vaccine comprises or consists of a chimaeric VLP as specifically disclosed in co-pending application GB0206359 (WO03/078455), then preferably the L2 peptide is not that disclosed in K. Kawana et al Vaccine 19, (2001) pl496-1502.

The suitability of a vaccine containing an L2 peptide associated with a VLP may be assessed, for example, by immunisation of mice in the following assay protocol:

Method

Groups of 10 Balb/C mice are immunised intramuscularly three or four times at 2 weeks intervals with the VLP/L2 peptide based formulations. On day 14 following the last immunisation, the specific L2 antibody response induced by vaccination was monitored by ELISA. The cross neutralising response against different HPV types may be analyzed by using a pseudovirion neutralisation assay - see Combita et al, supra. Cytokine production (IL5/IFN) is monitored on spleen cells taken 14 days after the last immunization.

Example 1

Groups of 10 Balb/C mice were immunised intramuscularly according to the protocol outlined below with the VLP/L2 peptide based formulations. On day 14 following the last immunisation, the specific L2 and VLP antibody responses induced by vaccination were monitored by ELISA, and cytokine production was assessed.

The analysis of the immune response was carried out as follows: Anti-VLPs serology (lg response)

Quantitation of anti-VLP18 antibody was performed by ELISA using VLP 18 as the coating antigen. Antigen and antibody solutions were used at 50 μl per well. Antigen were diluted at a final concentration of 0.5 μg/ml in PBS and were adsorbed overnight at 4°C to the wells of 96-wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). The plates were then incubated for 1 hr at 37°C with PBS containing 1% bovine serum albumin. Sera (starting at 1/400) in buffer containing PBS + 0.1% Tween20 + 1% BSA (saturation buffer without serum) were added to the VLP-s coated plates and incubated for 1 hr 30 min at 37°C. The plates were washed four times with PBS 0.1% Tween20 and biotin-conjugated anti-mouse lg (from Dako) diluted at 1/1000 in saturation buffer without serum was added to each well and incubated for 1 hr 30 at 37°C. After a washing step, streptavidin-horseradish peroxysade complex (Dako, UK) diluted 1/5000 in saturation buffer without serum was added for an additional 30 min at 37°C. Plates were washed as above and incubated for 20 min at room temperature with a solution of o-phenylenediamine (Sigma) 0.04% H₂O₂ 0.03% in 0.1% Tween20, 0.05M citrate buffer pH 4.5. The reaction was stopped with H SO₄ 2N and read at 490/630 nm. Elisa titers were calculated from a reference by SoftMaxPro (using a four parameters equation) and expressed in EU/ml.

Anti-L2 peptide serology (lg response)

Measure of anti-L2 antibody was performed by ELISA using the L2 peptide as coating antigen. Antigen and antibody solutions were used at 50 μl per well. Antigens were diluted at a final concentration of 1 μg/ml in PBS and were adsorbed overnight at 4°C to the wells of 96-wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). The plates were then incubated for 1 hr at 37°C with PBS containing 1% Bovine Serum Albumin or Gloria 5%. Sera (starting at 1/10) in buffer containing PBS + 0.1% Tween20 + 1% BSA (saturation without serum) were added to the L2 coated plates and incubated for 1 hr 30 min at 37°C. The plates were washed four times with PBS 0.1%) Tween20 and biotin-conjugated anti-mouse lg (Dako, UK) diluted at 1/1000 in saturation buffer without serum was added to each well and incubated for 1 hr 30 at 37°C. After a washing step, streptavidin-horseradish peroxydase Amdex (Amersham, UK) or streptavidin-POD (Dako, UK) was added for an additional 30 min at 37°C. Plates were washed as above and incubated for 20 min at room temperature with a solution of o-phenylenediamine (Sigma) 0.04% H₂O₂ 0.03% in 0.1% Tween20, 0.05M citrate buffer pH 4.5. The reaction was stopped with H₂SO₄ 2N and read at 490/620 nm. Elisa titers were calculated from a reference by SoftMaxPro (using a four parameters equation) and expressed in EU/ml.

Cytokine production

Three weeks after the fourth immunisation, mice were sacrificed, spleens were removed aseptically and pooled. Cell suspension were prepared in RPMI 1640 medium (Gibco) containing additives (sodium pyruvate ImM, MEM non essential amino acids, Pen/Strep, Glutamine and β-2 mercaptoethanol) and 5% foetal calf serum. Cells were cultured at a final concentration of 5.10⁶ cells/ml, in 1 ml per flat- bottomed 24 wells plates with a concentration of 1 μg of L2 peptide. Supernatants were harvested 96 hrs later and frozen until tested for the presence of IFN-γ and IL-5 using mice CBA kits from BD Biosciences (Pharmingen).

The L2 peptide was conjugated to the HPV 18 VLP using the following technology: Conjugation of 2 peptide on HPN18

The coupling reagent is a selective heterobifunctional crosslinker, one end of the compound activating amino group of the protein carrier by a succinimidyl ester and the other end coupling sulfhydryl group of the peptide by a maleimido group (3). The reactional scheme is described in Figure 1 : Activation of the carrier by reaction between lysine and succinimidyl ester followed by the coupling between activated protein and the peptide cysteine by reaction with the maleimido group.

The maleimide group is most selective for sulfhydryl groups when the pH of the reaction mixture is kept between 6.5 and 7.5. At pH 7, the rate of reaction of maleimides with sulfhydryl is 1000-fold faster than with amines. A stable thioether linkage between the maleimide group and the reacted sulfhydryl is formed which cannot be cleaved under physiological conditions.

In order to conjugate the peptide L2 (amino acid 108-120 of HPV16) on HPV 18 using the maleimide chemistry, this L2 peptide was synthesised by Νeosystem (Strasbourg, France) with three additional amino acids in C- terminal: two glycines used as spacer and one cysteine used for the coupling

(AC-LVEETSFΓDAGAPGGC-ΝH₂).

The conjugation of L2 peptide on HPV LI VLP was realised as follows:

The VLPs HPV18 are in a 20 mM Na₂HPO₄/NaH₂PO .2H₂O, 500 mM NaCl pH 6.0, pH was adjusted at 6.86 by addition of 0.3M NaOH.

About 3 mg of HPV 18 (756 μg/ml, Bradford dosage) was incubated at room temperature with a large excess of heterobifunctional cross-linking reagent GMBS (N-[γ-maleimidobutyryloxy]succinimide ester) (220 fold-molar excess of GMBS) dissolved in DMSO (3.96 mg in 50 μl) with stirring. After 60 minutes with stirring, the activated VLPs was purified by gel filtration (PD10 column from Amersham or Toyopearl HW-40, XK 16/40, elution with 20 mM phosphate, 500 mM NaCl pH 6.0) in order to remove excess of reagent and GMBS by-products. After purification, an estimation of the number of maleimide groups added on HPV18 was done using the Ellman's reaction: five maleimides groups were added on the VLPs. Then, L2 peptide (equivalent to 12.5 fold-molar excess) was dissolved in 100 μl DMSO and added to the activated VLPs solution. pH was adjusted at pH 6.8 by addition of 0.3M NaOH. The conjugation reaction was allowed to continue for one hour at room temperature and followed by a quenching step with a cysteine hydrochlorhydre solution (4 mg/ml) during 30 minutes at the same temperature. In order to remove excess peptide, the conjugate solution was dialysed against 20 mM Na₂HPO₄/NaH₂PO₄.2H₂O, 500 mM NaCl pH 6.0 in a dialysis cassette (10,000 MWCO, Pierce). The L2-VLPs solution was then filtered through a sterile 0.22 μm membrane. Protein content was estimated by a Lowry dosage. Analysis of the conjugate by SDS-PAGE followed by a coomassie blue staining showed that the major product was seen as a protein of high molecular weight vs. 53 kDa for the untreated VLPs.

The L2 peptide conjugated to the HPV 18 VLP provokes a clear immune response in comparison to the control groups - see Figure 2 and Figure 3 in which the antibody response against L2 and the VLP is assessed 14 days post injection II or injection III. The response to the VLP component is slightly reduced post II, but not to a level that - would be expected to reduce any protective effect. Data for IFN-γ and IL-5 production are also presented in Figures 4 and 5, showing that the L2 peptide enhances the production of cytokines when compared to VLPs alone.

Claims

1 An immunogenic composition comprising an HPV peptide in physical association with an HPV virus like particle (VLP) or suitable carrier.

2 A composition according to claim 1 wherein the VLP or carrier is associated with the peptide by chemical crosslinking, encapsulation, ionic or covalent interaction.

3 A composition according to claim 1 or 2 wherein the L2 peptide is in physical association with a VLP.

4 A composition according to any preceding claim wherein the L2 peptide is cross reactive.

5 A composition according to any preceding claim wherein the L2 peptide is cross protective.

6 A composition according to any preceding claim wherein the peptide is selected from one of the following list: an LI peptide, an L2 peptide, an El peptide, an E2 peptide, an E3 peptide, an E4 peptide, an E5 peptide, an E6 peptide and an E7 peptide.

7 A composition according to claim 6 wherein the peptide is an L2 peptide

8 A composition according to claim 7 wherein the L2 peptide comprises amino acids 108 - 120 derived from the HPV 16 genotype, having the sequence 'LVEETSFIDAGAP', or is a functional equivalent thereof.

A composition according to any preceding claim comprising an adjuvant or immunostimulant.

10 A composition according to any preceding claim which is a vaccine composition. Use of a composition according to any of claims 1 -9 in the preparation of a vaccine for the prevention or treatment of cervical cancer or genital warts.

A method of preventing or treating cervical cancer or genital warts, the method comprising delivering to an individual at risk an effective amount of a vaccine according to claim 10.

A method of vaccine production, the method comprising physically associating a peptide with an HPV VLP or suitable carrier by conjugation.