Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0011—Cancer antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5258—Virus-like particles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55505—Inorganic adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/70—Multivalent vaccine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/80—Vaccine for a specifically defined cancer
  • A61K2039/892—Reproductive system [uterus, ovaries, cervix, testes]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20023—Virus like particles [VLP]

Definitions

• the present invention relates to human papillomavirus (HPV) vaccines.
• Papillomaviruses are small DNA tumour viruses, which are highly species specific. So far, over 100 individual human papillomavirus (HPV) genotypes have been described. HPVs are generally specific either for the skin (e.g. HPV-I and -2) or mucosal surfaces (e.g. HPV-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.
• HPVs human papillomavirus
• HPVs are also associated with cancers, known as oncogenic HPV types.
• the strongest positive association between an HPV and human cancer is that which exists between HPV- 16 and HPV- 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.
• HPV types which can cause cancer are types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 (referred to as "oncogenic-HPV types").
• Types 16 and 18 are those which have the highest association with cervical cancer.
• Types 31 and 45 are the types with the next highest association with a cancer risk (Munoz N, Bosch FX, de Sanjose S et al. International Agency for Research on Cancer Multicenter Cervical Cancer Study Group. N Engl J Med 2003; 348: 518-27.)
• VLPs HPV virus like particles
• Animal studies have shown that VLPs produce no cross protection against infection for other HPV types - see, for example Suzich, J.
• HPV 16, 18 VLP vaccine can provide cross protection against infection by HPV types other than 16 and 18. Statistically significant protection was observed against certain groups of HPV types. However, the level of cross protection against individual types within groups was not disclosed.
• the present invention relates to a multivalent HPV vaccine, the vaccine comprising an Ll protein or immunogenic fragment thereof from at least 3 different oncogenic HPV types, those types including HPV 16 and HPV 18, wherein the vaccine does not comprise an Ll protein or immunogenic fragment thereof from an HPV type selected from the list consisting of HPV 31, HPV 45, HPV 52 or any combination thereof.
• the present invention further relates to use of a composition comprising an Ll protein or immunogenic fragment thereof from HPV 16 and HPV 18 in the manufacture of a medicament for prevention of infection and/or disease by one or more of the group consisting of HPV 31, HPV 45 and HPV 52.
• the present invention further relates to use of a composition comprising an Ll protein or immunogenic fragment thereof from HPV 16 and HPV 18 in the manufacture of a medicament for prevention of cyto logical abnormalities, and/or reduction of the frequency of c ytological abnormalities, and/or prevention of CIN lesions (ASCUS, CEN 1, CIN 2, CIN , cervical cancer) in an individual, the abnormalities or lesions caused by at least one HPV type other than HPV 16 or HPV 18, suitably being caused by HPV type 31, or 45, or 52, or a combination thereof.
• the invention further relates to a method of prevention and/or treatment of HPV infection and/or disease, the method comprising delivering to an individual in need thereof an effective amount of a composition comprising an Ll protein or immunogenic fragment thereof from at least 3 different oncogenic HPV types, those types including HPV 16 and HPV 18, wherein the vaccine does not comprise an Ll protein or immunogenic fragment thereof from an HPV type selected from the list consisting of HPV 31, HPV 45, HPV 52 or any combination thereof.
• the invention further relates to use of a multivalent composition in the manufacture of a medicament for the prevention and/or treatment of HPV infection and/or disease, the multivalent composition comprising an Ll protein or immunogenic fragment thereof from at least 3 different oncogenic HPV types, those types including HPV 16 and HPV 18, wherein the vaccine does not comprise an Ll protein or immunogenic fragment thereof from an HPV type selected from the list consisting of HPV 31, HPV 45, HPV 52 or any combination thereof, and wherein the medicament provides protection against infection and/or disease caused by the omitted HPV type.
• the invention also relates to a method for manufacture of a vaccine, the method comprising combining an Ll protein or immunogenic fragment thereof from at least 3 different oncogenic HPV types, those types including types HPV 16 and HPV 18, wherein the vaccine does not comprise an Ll protein or immunogenic fragment thereof from an HPV type selected from the list consisting of HPV 31, HPV 45, HPV 52 or any combination thereof.
• Cross protection may be considered as the protection afforded by a vaccine containing one HPV type against infection (incident or persistent) and/or disease caused by a different HPV type.
• Cross protection may be assessed by considering the vaccine efficacy (V.E.), wherein the V.E. is the % improvement in protection against infection or disease by the vaccine compared to a placebo group for a given type. Infection may be incident or persistent infection.
• Disease may be abnormal cytology, ASCUS, CINl, CIN2, CIN3 or cervical cancer related to HPV infection. Infection may be assessed by PCR, for example. Disease may be assessed by, for example, histological examination or analysis of biomarkers such as pi 6.
• HPV 16 and HPV 18 Ll containing vaccines against certain other HPV types allows Ll components from these HPV types to be omitted from a vaccine comprising HPV 16 and HPV 18 while still providing a vaccine which provides some protection against incident and/or persistent infection and/or disease related to those omitted types.
• HPV types 16 found in 53.5% of cervical cancer
• types 45 (6.7%) and 31 (2.9%) are the next most significant in terms of their frequency in cervical cancers (Munoz et al. supra).
• HPV 33 (2.6%) is next, followed by HPV 52 (2.3%).
• types 31 and 45 would generally be included by the skilled person after types 16 and 18 from a statistical perspective.
• HPV types potentially allows inclusion of Ll protein from other HPV types, or indeed antigens from other viruses or pathogens, into a vaccine in a scenario where the total amount of antigen in a vaccine may be limited, for example by physical, chemical, regulatory or other constraints.
• HPV types include oncogenic HPV types such as HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.
• the present invention relates to a multivalent HPV vaccine, the vaccine comprising an Ll protein or immunogenic fragment thereof from at least 3 different oncogenic HPV types, those types including HPV 16 and HPV 18, wherein the vaccine does not comprise an Ll protein or immunogenic fragment thereof from an HPV type selected from the list consisting of HPV 31, HPV 45, HPV 52 or any combination thereof.
• the vaccine does not contain an Ll protein or immunogenic fragment thereof from HPV 31.
• the vaccine is capable of providing protection against incident and /or persistent HPV infection by HPV 31.
• the vaccine of the invention does not contain an Ll protein or immunogenic fragment thereof from HPV 45. In one aspect of the invention the vaccine is capable of providing protection against incident and /or persistent HPV infection by HPV 45. In one aspect of the invention the vaccine does not contain an Ll protein or immunogenic fragment thereof from HPV 52.
• the vaccine is capable of providing protection against incident and /or persistent HPV infection by HPV 52. In one aspect of the invention the vaccine of the invention does not contain an
• Ll protein or immunogenic fragment thereof from HPV 31 and 45 Ll protein or immunogenic fragment thereof from HPV 31 and 45.
• the vaccine is capable of providing protection against incident and /or persistent HPV infection by both HPV 31 and 45.
• the vaccine does not contain an Ll protein or immunogenic fragment thereof from HPV 31 and 52.
• the vaccine is capable of providing protection against incident and /or persistent HPV infection by both HPV 31 and 52.
• the vaccine of the invention does not contain an Ll protein or immunogenic fragment thereof from HPV 45 and 52. hi one aspect of the invention the vaccine is capable of providing protection against incident and /or persistent HPV infection by both HPV 52 and 45.
• the vaccine is capable of providing protection against incident and /or persistent HPV infection by HPV 31 and HPV 45 and HPV52.
• the vaccine is capable of protection against persistent infection.
• the vaccine is capable of protection against incident infection.
• Example 1 Incident and persistent cervical infection are defined in Example 1.
• a vaccine comprising HPV 16 Ll and HPV 18 Ll proteins (for example, as described in example 1) provides protection against cytological abnormalities caused by certain other oncogenic HPV types such as HPV 52, and is significantly protective with respect to such abnormalities caused by a group of HPV high risk types (defined as 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). Cytological abnormalities are suitably detected by the well known Pap smear technique.
• the invention further relates to use of a combination of an Ll protein or immunogenic fragment thereof from HPV 16 and HPV 18 in the preparation of a composition for the prevention of cytological abnormalities or reduction of the frequency of cytological abnormalities in an individual caused by other (non HPV 16, HPV 18) HPV types, suitably oncogenic HPV types, and in the prevention of histologically-confirmed CIN lesions (CEST 1, CIN 2, CESf 3) and cervical cancer associated with infection by HPV types which are not HPV 16 or 18. Said use is in addition to the prevention or reduction of such events caused by the HPV types in the vaccine, HPV 16 and 18.
• the prevention of cytological abnormalities, reduction of the frequency of cytological abnormalities or prevention of histological-confirmed CEST lesions is prevention against those abnormalities or lesions caused by types not included in the combination, suitably selected from the list of HPV 31, HPV 45 and HPV 52, or is prevention against those abnormalities or lesions caused by the group of 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Said use is in addition to the prevention or reduction of such events caused by the HPV types in the vaccine, HPV 16 and 18.
• composition comprising HPV 16 and HPV 18 for use as above is the multivalent HPV vaccine of the invention, the vaccine comprising an Ll protein or immunogenic fragment thereof from at least 3 different oncogenic HPV types, those types including HPV 16 and HPV 18, wherein the vaccine does not comprise an Ll protein or immunogenic fragment thereof from an HPV type selected from the list consisting of HPV 31, HPV 45, HPV 52 or any combination thereof.
• the vaccine of the invention comprises Ll or immunogenic fragment from
• HPV 16, HPV 18 and at least one other oncogenic HPV type are those types associated with a risk of cervical cancer and those oncogenic types that might be included in the vaccine of the invention in addition to HPV 16 and HPV 18 include, but are not limited to, HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, with the proviso that the vaccine does not comprise all of HPV 31, 45 and 52.
• the vaccine of the invention suitably comprises an HPV 33 Ll protein or immunogenic fragment thereof.
• the vaccine of the invention suitably comprises an HPV 58 Ll protein or immunogenic fragment thereof.
• the vaccine of the invention suitably comprises an HPV 59 Ll protein or immunogenic fragment thereof.
• the vaccine of the invention suitably comprises an HPV 16 Ll protein or immunogenic fragment thereof, HPV 18 Ll protein or immunogenic fragment thereof, HPV 33 Ll protein or immunogenic fragment thereof and HPV 58 Ll protein or immunogenic fragment thereof.
• Ll proteins or protein fragments from additional HPV types can be included in the vaccine of the invention, such as skin types (in particular HPV 5 and 8) and types associated with genital warts, such as HPV 6 and 11. Types 6 and 11 are not considered oncogenic types herein.
• the vaccine may include an HPV early antigen, for example an antigen selected from the list consisting of HPV El, E2, E3, E4, E5, E6, E7, or E8.
• the vaccine may lack an HPV early antigen, for example an antigen selected from the list consisting of HPV El, E2, E3, E4, E5, E6, E7, or E8.
• the vaccine of the invention is trivalent (contains an HPV Ll or fragment thereof from 3 different oncogenic HPV types).
• the vaccine is tetravalent.
• the vaccine is pentavalent.
• the vaccine is heptavalent.
• the vaccine is septavalent.
• the vaccine is octavalent. Higher order valancies are also contemplated herein.
• the vaccine is at least tetravalent, pentavalent, heptavalent, septavalent or octavalent with respect to oncogenic HPV types.
• the combination of HPV components within the vaccine does not significantly impact the immunogenicity of any one HPV component.
• there is no biologically relevant interference between HPV antigens in the combination of the invention such that the combined vaccine of the invention is able to offer effective protection against infection or disease caused by each HPV genotype represented in the vaccine.
• the immune response against a given HPV type in the combination is at least 50 % of the immune response of that same HPV type when measured individually, preferably 100% or substantially 100%.
• the combined vaccine of the invention preferably stimulates an immune response which is at least 50% of that provided by a combined HPV 16 / HPV 18 vaccine.
• the immune response generated by the vaccine of the invention is at a level in which the protective effect of each HPV type is still seen.
• the immune response may suitably be measured, for example, by antibody responses, in either preclinical or human experiments. Measurement of antibody responses is well known in the art, and disclosed in (for example) WO03/077942.
• the invention relates to use of a composition comprising HPV 16 and HPV 18 Ll protein, or immunogenic fragment thereof, in the manufacture of a medicament for prevention of infection by HPV 31.
• the invention further relates to use of a composition comprising HPV 16 and
• the invention further relates to use of a composition comprising HPV 16 and
• HPV 18 Ll protein, or immunogenic fragment thereof in the manufacture of a medicament for prevention of infection by HPV 52.
• the invention relates to use of a vaccine comprising HPV 16 Ll proteins, or immunogenic fragment thereof, in the preparation of a medicament for the prevention of infection and/or disease caused by HPV 31, or HPV 52, or any combination thereof.
• the invention relates to use of a vaccine comprising HPV 18 Ll proteins, or immunogenic fragment thereof, in the preparation of a medicament for the prevention of infection and/or disease caused by HPV 45.
• the composition for said use may lack an antigenic component from the HPV type for which cross protection is provided.
• the composition for said use may comprise such an antigenic component, e.g. the Ll protein or fragment thereof from said cross protected type, hi the latter case the use of the composition comprising
• HPV 16 and HPV 18 Ll protein, or immunogenic fragment thereof provides both cross protection (e.g. against HPV 31, 45 and 52) and homologous protection (e.g. against
• composition in one aspect is a multivalent composition comprising Ll proteins or immunogenic fragments thereof from HPV 16, HPV 18 and at least one other oncogenic HPV type, wherein an Ll protein or immunogenic fragment thereof from one or more HPV types selected from the group consisting of HPV 31, HPV 45, and HPV 52 is omitted from the vaccine and wherein the vaccine provides protection against infection caused by the omitted HPV type.
• suitable immunogenic fragments of HPV Ll include truncations, deletions, substitution, or insertion mutants of Ll .
• Such immunogenic fragments are suitably capable of raising an immune response (if necessary, when adjuvanted), said immune response being capable of recognising an Ll protein such as a virus like particle, from the HPV type from which the Ll protein was derived.
• a suitable immunogenic fragment of HPV 16 is capable of cross protection against at least one of HPV 31 and HPV 52, and in an aspect of the invention, capable of cross protection against both.
• a suitable immunogenic fragment of HPV 18 is capable of cross protection against HPV 45.
• Cross protection obtainable by immunogenic fragments of HPV 16 and/or HPV 18 can be assessed by trials in humans, for example as outlined in Example 1.
• different vaccines according to the present invention can be tested using standard techniques, for example as in Example 1 , or in standard preclinical models, to confirm that the vaccine is immunogenic.
• Suitable immunogenic Ll fragments include truncated Ll proteins.
• the truncation removes a nuclear localisation signal.
• the truncation is a C terminal truncation.
• the C terminal truncation removes fewer than 50 amino acids, such as fewer than 40 amino acids.
• the Ll is from HPV 16 then in another aspect the C terminal truncation removes 34 amino acids from HPV 16 Ll .
• the Ll is from HPV 18 then in a further aspect the C terminal truncation removes 35 amino acids from HPV 18 Ll.
• Truncated Ll Proteins are described in US 6,060,324, US 6,361,778, and US 6,599,508 incorporated herein by reference.
• the HPV 16 amino acid sequence is the following sequence: (SEQ ED NO: 1)
• the invention relates to virus like particles consisting only of HPV 16 Ll having the amino sequence above, and to compositions containing such VLPs.
• HPV 16 sequence may also be that disclosed in WO9405792 or US6649167, for example, suitably truncated. Suitable truncates are truncated at a position equivalent to that shown above, as assessed by sequence comparison.
• HPV 18 amino acid sequence is the following sequence: (SEQ ID NO: 2)
• the invention relates to virus like particles consisting only of HPV 18
• HPV 18 sequence is disclosed in WO9629413, which may be suitably truncated. Suitable truncates are truncated at a position equivalent to that shown above, as assessed by sequence comparison.
• Other HPV 16 and HPV 18 sequences are well known in the art and may be suitable for use in the present invention.
• Suitable truncations of HPV 31, HPV 45 and HPV 52 may also be made, suitably removing equivalent C terminal portions of the Ll protein to those described above as assessed by sequence alignment.
• Truncated Ll proteins are disclosed in, for example, WO9611272 and US6066324, herein incorporated by reference.
• the Ll protein or fragment of the invention may optionally be in the form of a fusion protein, such as the fusion of the Ll protein with L2 or an early protein.
• the HPV Ll protein is suitably in the form of a capsomer or virus like particle
• HPV VLPs may be used in the present invention.
• HPV VLPs and methods for the production of VLPs are well known in the art.
• VLPs typically are constructed from the Ll and optionally L2 structural proteins of the virus, see for example WO9420137, US5985610, W09611272. US6599508B1, US6361778B1, EP 595935. Any suitable HPV VLP may be used in the present invention which provides cross protection, such as an Ll or Ll + L2 VLP.
• the VLP is an Ll -only VLP.
• the vaccine comprises HPV 16 and HPV 18 Ll only VLPs, suitably in combination with an Ll VLP selected from HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, with the proviso that the vaccine does not comprise VLPs from all of HPV 31, 45 and 52.
• VLP formation can be assessed by standard techniques such as, for example, electron microscopy and dynamic laser light scattering.
• the VLP may comprise full length Ll protein.
• the Ll protein used to form the VLP is a truncated Ll protein, as described above.
• VLPs may be made in any suitable cell substrate such as yeast cells or insect cells e.g. baculovirus cells, and techniques for preparation of VLPs are well known in the art, such as WO9913056, US 6416945B1 , US 6261765B1 and US6245568, and references therein, the entire contents of which are hereby incorporated by reference.
• VLPS are suitably made by disassembly and reassembly techniques, which can provide for more stable and/or homogeneous papillomavirus VLPs. For example, McCarthy et al, 1998 "Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Virus like Particles in Vitro" J.
• Virology 72(1):33-41 describes the disassembly and reassembly of recombinant Ll HPV 11 VLPs purified from insect cells in order to obtain a homogeneous preparation of VLPs.
• WO9913056 and US6245568 also describe disassembly/reassembly processes for making HPV VLPs.
• HPV VLPS are made as described WO9913056 or US6245568.
• HPV Ll the invention may be combined with an 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.
• an 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.
• enterobacterial lipopolysaccharide is a potent stimulator of the immune system, although its use in adjuvants has been curtailed by its toxic effects.
• LPS enterobacterial lipopolysaccharide
• MPL monophosphoryl lipid A
• 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 3-0-deacylated variants thereof.
• a suitable form of 3D-MPL is in the form of an emulsion having a small particle size less than 0.2 ⁇ m 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.
• 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.
• 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.
• 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.
• ICOMS Immune Stimulating Complexes
• Quil A 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 QS 17 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.
• 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 3 D-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 vaccine adjuvanted with detoxified lipid A or a non-toxic derivative of lipid A more suitably adjuvanted with a monophosphoryl lipid A or derivative thereof.
• the vaccine additionally comprises a saponin, for example QS21.
• the vaccine formulation comprises an oil in water emulsion.
• the present invention also provides a method for producing a vaccine formulation comprising mixing an Ll peptide of the present invention together with a pharmaceutically acceptable excipient, such as 3D-MPL.
• a 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.
• the vaccine comprises a vesicular adjuvant formulation comprising cholesterol, a saponin and an LPS derivative
• the adjuvant formulation suitably comprises a unilamellar vesicle comprising cholesterol, having a lipid bilayer suitably comprising dioleoyl phosphatidyl choline, wherein the saponin and the LPS derivative are associated with, or embedded within, the lipid bilayer.
• 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 in one aspect, a ratio of 1 :5 weight/weight.
• Such adjuvant formulations are described in EP 0 822 831 B, the disclosure of which is incorporated herein by reference.
• the vaccines of the invention are used in combination with aluminium, and are suitably adsorbed or partially adsorbed onto aluminium adjuvants.
• the adjuvant is an aluminium salt, which may be in combination with 3D MPL, such as aluminium phosphate and 3D MPL. Aluminium hydroxide, optionally in combination with 3D MPL is also suitable.
• the vaccine comprises the combination of HPV VLPs with an aluminium salt or with an aluminium salt + 3D MPL. Aluminium hydroxide is suitable as the aluminium salt.
• the vaccine may also comprise aluminium or an aluminium compound as a stabiliser.
• the adjuvant may be a combination of an oil-in-water emulsion adjuvant and 3D MPL.
• the oil-in-water emulsion comprises a metabolisable oil, a sterol and an emulsifying agent.
• the vaccines of the invention may be provided by any of a variety of routes such as oral delivery (e.g. see WO9961052 A2) , topical, subcutaneous, mucosal
• the vaccine may also be formulated or co-administered with other HPV antigens or non-HPV antigens.
• these non-HPV antigens can provide protection against other diseases, such as sexually transmitted diseases such as herpes simplex virus, EBV, chlamydia and HIV.
• the vaccine comprises gD or a truncate thereof from HSV. In this way the vaccine provides protection against both HPV and HSV.
• 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.
• the delivery is of an amount of vaccine suitable to generate an immunologically protective response.
• each vaccine dose comprises 1-100 ⁇ g of each VLP, in one aspect 5-80 ⁇ g, in another aspect 5- 30 ⁇ g each VLP, in a further aspect 5-20 ⁇ g of each VLP, in a yet further aspect 5 ⁇ g, 6 ⁇ g, lO ⁇ g, 15 ⁇ g or 20 ⁇ g.
• the vaccine can comprise HPV Ll protein components, preferably as virus like particles, in different amounts.
• HPV 16 and HPV 18 VLPs may be provided at a higher dose than other oncogenic types, such as HPV 33 or 58.
• HPV 16 and HPV 18 Ll only VLPs are provided at 20 ⁇ g per dose for human use.
• Other HPV VLPs may be used at a lower dose, such as 15 or 1 O ⁇ g per dose for human use.
• the vaccine is used for the vaccination of adolescent girls aged 10-15, such as 10-13 years. However, older girls above 15 years old and adult women may also be vaccinated.
• the vaccine may also be administered to women following an abnormal pap smear or after surgery following removal of a lesion caused by HPV, or who are seronegative and DNA negative for HPV cancer types.
• the vaccine of the invention may be used in men.
• 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.
• the vaccination regime incorporates a booster injection after 5 to 10 years, such as 10 years.
• the vaccine is a liquid vaccine formulation, although the vaccine may be lyophilised and reconstituted prior to administration.
• the vaccines of the invention comprise certain HPV components as laid out above.
• the vaccine consists essentially of, or consists of, said components.
• 'vaccine' refers to a composition that comprises an immunogenic component capable of provoking an immune response in an individual, such as a human, optionally when suitably formulated or adjuvant.
• a vaccine suitably elicits a protective immune response against incident infection, or persistent infection, or cytological abnormality such as ASCUS, CESTI, CIN2 , CIN3, or cervical cancer caused by one or more HPV types.
• the mixture comprised, per 0.5 ml dose, 20 ⁇ g of HPV-16 Ll VLP, 20 ⁇ g of HPV-18 Ll VLP and was adjuvanted with 500 ⁇ g of aluminum hydroxide and 50 ⁇ g of 3D MPL.
• the placebo group was injected with 500 ⁇ g of aluminum hydroxide alone.
• V.E. The vaccine efficacy (V.E.) against certain cancer HPV types was assessed, wherein the V.E. is the % improvement in protection against infection or disease by the vaccine compared to a placebo group.
• Cross protection was assessed by detecting the presence of nucleic acid specific for various oncogenic types in the vaccinees and control group. Detection was carried out 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 specifically 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 oncogenic HPV types are known.
• the primary objective of this study was to assess vaccine efficacy in the prevention of infection with HPV-16, HPV-18, or both (HPV-16/18), between months 6 and 18 in participants who were initially shown to be seronegative for HPV-16/18 by ELISA and negative for HPV-16/18 DNA by PCR.
• Women eligible for the initial phase included healthy women aged 15-25 years, who had had no more than six sexual partners, no history of an abnormal Pap test or ablative or excisional treatment of the cervix, and no ongoing treatment for external condylomata; and who were cytologically negative, seronegative for HPV- 16 and HPV- 18 antibodies by ELISA, and HP V-DNA-negative by PCR for 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) no more than 90 days before study entry.
• Each dose of the bivalent HPV- 16/18 virus-like particle vaccine contained 20 Pg of HPV-16 Ll virus-like particle and 20 Pg of HPV- 18 Ll virus-like particle.
• Each type of virus-like particle was produced on Spodoptera frugiperda Sf-9 and Trichoplusia ni Hi-5 cell substrate with AS04 adjuvant containing 500 Pg aluminum hydroxide and 50 fg 3- deacylated monophosphoryl lipid A (MPL, Corixa, Montana, USA) provided in a monodose vial.
• the placebo contained 500 Pg of aluminum hydroxide per dose, and was identical in appearance to the HPV- 16/18 vaccine.
• Health-care providers obtained cervical specimens with a cervical brush and spatula (washed in PreservCyt, Cytyc Corporation, Boxborough, MA, USA) for cytology and HPV DNA testing at screening and months 6, 12, and 18.
• PreservCyt Cytyc Corporation, Boxborough, MA, USA
• the central histology laboratory made an initial diagnosis from the formalin-fixed tissue specimens for clinical management.
• a panel of three pathologists made a subsequent consensus diagnosis for HPV- 16 and HPV- 18 associated lesions with the CIN system. This consensus diagnosis also included review of the sections taken at the time of microdissection for PCR detection of lesional HPV DNA.
• HPV DNA isolated from the cytology specimen MagneticNaPure Total Nucleic Acid system, Roche Diagnostics, Almere, Netherlands
• cervical biopsy specimen proteinase K extraction
• SPFlO broad-spectrum primers that amplify a 65 bp region of the Ll gene.
• a line probe assay (LiPA Kit HPV INNO LiPA HPV genotyping assay, SPF-IO system version 1, Innogenetics, Gent, Belgium, manufactured by Labo Biomedical Products, Rijswijk, Netherlands) detected 25 HPV genotypes (6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, and 74).
• HPV- 16 and HPV- 18 PCR Any specimen that was positive by DNA enzyme immunoassay was tested by type-specific HPV- 16 and HPV- 18 PCR.
• HPV- 16 type-specific PCR primers amplified a 92 bp segment of the E6/E7 gene and HPV-18 type-specific PCR primers amplified a 126 bp segment of the Ll gene.
• MF Baay, WG Quint, J Koudstaal et ah Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas. J Clin Microbiol 34 (1996), pp.
• HPV-DNA test results were concealed from investigators during the study and cyto logical and histological diagnoses were only revealed for clinical management purposes. Analyses included HPV- 16/18 DNA results for cervical specimens and combined cervical and self-obtained cervicovaginal specimens.
• the intention-to-treat and according-to-protocol cohorts are shown in the figure, in which the reasons for exclusion from analyses are listed in rank order; women who met more than one exclusion criterion were only counted once according to the highest ranking criterion.
• Vaccine efficacy was defined as 1 minus the ratio between these two proportions; 95% CIs measured the precision of the efficacy estimates, p values were calculated with the two-sided Fisher's exact test. Corresponding rates were expressed as the numbers of cases with the outcome divided by the numbers of participants at risk.
• the according-to-protocol 18-month cohort included enrolled women who received three scheduled doses of vaccine and complied with the protocol as described in the figure.
• Immunogenicity was assessed in a subset of the according-to-protocol safety cohort, which included women with serology results at months 0, 7, and 18, who received all three doses of study vaccine or placebo according to schedule, complied with the blood sampling schedule, and did not become positive for HPV-16/18-DNA during the trial. Seropositivity rates between the vaccine and placebo groups were compared with Fisher's exact test (p ⁇ 0-001 judged significant). Geometric mean titres were compared with ANOVA and Kruskal-Wallis test. Block randomisation and statistical analyses were done with SAS version 8.2 (SAS Institute, Cary, North Carolina).
• Table B is a midpoint analysis with data taken from all patients at the timepoint at which at least 50% of the cohort were 18 months after their first vaccination.
• Table C gives the final results, all data being from subjects at 18 months post first vaccination (month 0). In the ATP group all patients received 3 doses of vaccine at 0, 1 and 6 months and were seronegative at 6 months.
• Table B demonstrates that HPV 16 and HPV 18 provide statistically significant cross protection against the group of high risk cancer types 31,33,35,39,45,51,52,56,58,59, 66 and 68.
• Table C demonstrates that, except for the HPV- 18 related types (which show a very strong trend), there is statistically significant cross-protection against the groups of: HPV 31, 35, 58; HPV 31, 33, 35, 52, 58; and the 12 high risk (non HPV- 16/18) types evaluated. Further analysis was carried out on the specific cross protection against specific types.
• Vaccine efficacy was assessed against infections and diseases related to the 12 high risk cancer types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, HPV-16 phylogenetic-related types (the groups of; 31, 35, and 58; 31, 33, 35, 52 and 58) and HPV-18 phylogenetic related types (45 and 59).
• HPV-16 related HPV-16 phylogenetically related types 35, 31, 58 without considering other HPV types
• HPV-16 related* HPV-16 phylogenetically related types 35, 31, 58, 33, 52 without considering other HPV types
• HPV- 18 related HPV- 18 phylogenetically related types 45, 59 without considering other HPV types
• HPV-16 and/or HPV-18 related HPV-16 and/or HPV- 18 phylogenetically related types 35, 31, 58, 45, 59 without considering other HPV types
• HPV-16 and/or HPV-18 related* HPV-16 and/or HPV-18 phylogenetically related types 35, 31, 58,
• N number of subjects in specific cohort
• % Vaccine efficacy is 1 — (A/B) x 100, adjusted for relative size of vaccine and placebo group, wherein
• A % women in vaccine group with incident infection, persistent infection or cytological abnormality, as appropriate for the table
• B % women in placebo group with incident infection, persistent infection or cytological abnormality, as appropriate for the table.

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