BRPI0610032A2 - use of a human papillomavirus l1 protein or immunogenic fragment thereof from a first type of hpv, vaccination program for protection against hiv infection and / or disease, method for preventing hpv infection and / or disease, vaccine composition and kit - Google Patents

Abstract

USE OF A L1 HUMAN PAPILLOMAVIRUS PROTEIN OR IMMUNOGENIC FRAGMENT OF THE SAME AS A FIRST TYPE OF HPV, VACCINATION PROGRAM FOR THE PROTECTION AGAINST THE INFECTION AND / OR HPV DISEASE, METHOD FOR THE PREVENTION OF THE INFECTION AND THE PREVENTION OF THE INFECTION AND THE PREVENTION OF THE INFECTION. VACCINE, E, KIT. A method for preventing HPV infection and / or disease, the method comprising releasing a first HPV vaccine that comprises an L1 protein or immunogenic fragment thereof from at least HPV 16 and HPV 18 and a second HPV vaccine. HIPV that does not comprise the L1 components of HIPV 16 and HIPV 18 of the first vaccine and that the second vaccine comprises an L1 protein or immunogenic fragment thereof from at least one other type of oncogenic HPV, where the first and second vaccines can be released in any order and the release is separated by an appropriate time interval.

Description

"USE OF A HUMAN PAPILOMAVIRUS PROTEIN II OR IMMUNOGENIC FRAGMENT OF THE SAME OF A FIRST HPV5 TYPE Vaccination PROGRAM FOR HPV INFECTION AND / OR DISEASE, METHOD FOR PREVENTING INFECTION AND / OR HPV PREVENTION VACCINE, AND, KIT "

The present invention relates to the human papillomavirus (HPV) vaccine.

Background of the invention

Papillomaviruses are small DNA tumor viruses that are highly species specific. To date, more than 100 individual human papillomavirus (HPV) genotypes have been described. HPVs are generally specific to skin surfaces (eg HPV-I and -2) or mucosal (eg HPV-6 and -11) and usually cause benign tumors (warts) that persist for several months or years. Such benign tumors can be distressing to the individuals involved but tend not to be life threatening, with a few exceptions.

Some HPVs are also associated with cancers, known as oncogenic HPV types. The strongest positive association between HPV and human cancer is 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 worldwide each year.

Other types of HPV that can cause cancer are types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Types 16 and 18 are those that have the highest association with cancer. cervical. Types 31 and 45 are the types with the highest contiguous association with a cancer risk (Munoz N, Bosch FX of Sanjose S et al. International Agency for Research on Multicenter Cervical Cancer Study Group. N Engl J Med 2003; 348: 518-27.) HPV virus equivalent particles (VLPs) have been suggested as potential vaccines for the treatment of HPV. Animal studies have shown that VLPs do not produce any cross-protection against infection with other HPV types - see, for example, Suzich, JA, et al., Proc Natl Acad Sci, 92: 11553-11557, 1995 and Breitburd, Seminars in Cancer Biology, vol 9, 1999, pp 431-445.

W02004 / 056389 discloses that an HPV 16, 18 VLP vaccine can provide cross-protection against infection with HPV types other than 16 and 18. Statistically significant protection has been observed against certain groups of HPV types.

There is still a need for a vaccine that protects against multiple types of HPV.

Invention Report

The present invention relates to a vaccination program for protection against HPV infection and / or disease, the program comprising releasing a first HPV vaccine comprising an L1 protein or immunogenic fragment thereof, of at least HPV 16 and HPV 18. and a second HPV vaccine that does not comprise the HPV 16 and HPV 18 L1 components of the first vaccine and that the second vaccine comprises an L1 protein or immunogenic fragment thereof from at least one other type of oncogenic HPV, wherein the first and The second vaccines may be released in any order and the release is separated by an appropriate interval.

The present invention further relates to a vaccination program for protection against HPV infection or disease, the program comprising the release of a first HPV vaccine comprising an L1 protein or immunogenic fragment thereof from at least HPV 16 and HPV 18 and after a suitable interval, a second HPV vaccine that does not comprise HPV 16 and HPV 18 L1 components of the first vaccine and that the second vaccine comprises an L1 protein or immunogenic fragment thereof from at least one other type of oncogenic HPV.

The invention further relates to a method for preventing HPV infection and / or disease, the method comprising releasing a first HPV vaccine comprising an L1 protein or immunogenic fragment thereof from at least HPV 16 and HPV 18 and a second HPV vaccine that does not comprise HPV 16 and HPV 18 L1 components of the first vaccine and that the second vaccine comprises an L1 protein or immunogenic fragment thereof of at least one other type of oncogenic HPV, wherein the first and the second vaccines may be released in any order and the release is separated by an appropriate time interval.

The invention also relates to a method for preventing HPV infection and / or disease, the method comprising releasing a first HPV vaccine comprising an L1 protein or immunogenic fragment thereof from at least HPV 16 and HPV 18 and thereafter a suitable range of a second HPV vaccine that does not comprise HPV 16 and HPV 18 L1 components of the first vaccine and that the second vaccine comprises an L1 protein or immunogenic fragment thereof, of at least one other type of oncogenic HPV .

The invention also relates to vaccine compositions of the invention itself.

The invention also relates to kits comprising the first and second vaccine compositions of the invention.

In one aspect the invention relates to the use of a protein

HPV L1 or immunogenic fragment thereof from a first HPV type in the preparation of a medicament for enhancing the immune response generated by an HPV L1 protein or immunogenic fragment thereof from a second different HPV type. In one aspect, the ll protein or fragment thereof of the second HPV type is in the form of a virus equivalent particle and is used to enhance the response generated by the ll protein or fragment thereof from the first HPV type also in the form of a virus equivalent particle.

The invention also relates to the use of HPV LI, preferably in the form of VLPs, of non-HPV 16 strains of HPV in enhancing the response to HPV 16.

The invention also relates to the use of L1, preferably in the form of VLPs, of non-HPV 18 strains of HPV in enhancing the response to HPV 18.

In one aspect, strains to enhance the immune response are those for which some level of cross-protection is observed in Example 1, such as HPV 31, HPV 45, and HPV 52.

Figures

Figures 1 and 5 illustrate antibodies generated to HPV 16 with different onset and booster regimes.

Figures 2 and 6 illustrate antibodies generated to HPV 18 with different onset and booster regimes.

Figures 3 and 7 illustrate antibodies generated to HPV 31 with different onset and booster regimes.

Figures 4 and 8 illustrate antibodies generated to HPV 45 with different onset and booster regimes.

Figure 9 illustrates a summary of antibody responses in Figures 1 to 8.

Figures 10 and 14 illustrate the% of HPV16 L1-specific T cells in the CD4 + population with different onset and booster regimens.

Figures 11 and 15 illustrate the% of HPV18 L1-specific T cells in the CD4 + population with different onset and booster regimens.

Figures 12 and 16 illustrate the% of HPV31 L1-specific T cells in the CD4 + population with different onset and booster regimens.

Figures 13 and 17 illustrate the% of HPV45 ll-specific T cells in the CD4 + population with different onset and booster regimens.

Figure 18 illustrates a summary of the MIC data in Figures 10-13.

Detailed Description

The general existence of cross-protection produced by HPV 16 and HPV 18 against both incident and persistent infection, as assessed for certain groups of HPV types, was disclosed in W02004 / 056389.

Surprisingly, we found that cross-protection against certain types of HPV (non-HPV 16, HPV 18) (as judged by the effectiveness of an HPV 16 and HPV 18 vaccine against these types) is higher than against certain other HPV types ( non-HPV 16, HPV 18). Cross-protection may be considered as protection produced by a vaccine containing one type of HPV against infection (incident or persistent) and / or disease caused by a different type of HPV. Cross-protection can be assessed by considering the efficacy of the vaccine (V.E.), where V.E. is the% improvement in protection against vaccine infection or disease compared to a placebo group by a given type.

The infection can be incident or persistent infection. The disease may be abnormal cytology, ASCUS, CIN1, CIN2, CIN3 or HPV infection-related cervical cancer. Infection can be assessed by PCR, for example. The disease can be assessed by histological examination or biomarker analysis such as ρ 16.

Some level of cross-protection is considered present when cross-protection is statistically significantly higher than that given by placebo. Suitably, the cross-protection level is greater than 0% and up to 20%, up to 40%, up to 60%, up to 70%, up to 80% or higher than 80%, as measured by the vaccine efficacy of a HPV against infection or disease caused by a type of HPV not present in the vaccine compared to a placebo.

Such a finding has potential implications for vaccine design and vaccination program design.

For example, the types for which cross protection is observed may be used as a reciprocal start or reinforcement, after an appropriate interval, as well as providing homologous protection and may allow the number of vaccinations for any given HPV type to be reduced.

The present invention relates to a vaccination program for protection against HPV infection or disease, the program comprising the release of a first HPV vaccine comprising an L1 protein or immunogenic fragment thereof from at least HPV 16 and HPV 18. and a second HPV vaccine that does not comprise HPV 16 and HPV 18 L1 components of the first vaccine and that the second vaccine comprises an L1 protein or immunogenic fragment thereof of at least one other type of oncogenic HPV, wherein the first and second Vaccines can be released in any order and the release is separated by an appropriate interval.

A suitable range can be 7 days, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4, months, 5 months, 6 months, 1 year. Vaccination programs can then be 0, 1 month or 0, 2 months or 0, 4 months or 0, 6 months, for example. In general, a suitable range is one in which the booster effect of the second released vaccine can be observed, for example by measuring antibody titres or cell mediated immunity. In general, a suitable range is one in which the primary immune response was elicited by the release of a vaccine, as measured by serum antibody titers, for example prior to the release of a second vaccine. In one aspect, a suitable range is just after the peak of the primary response, typically the antibody response. In one aspect this interval is from 2 to 26 weeks after the initial vaccination, suitably from 2 to 22 weeks, from 2 to 18 weeks, from 2 to 14 weeks, from 2 to 12 weeks, from 2 to 10 weeks, from 2 to 8 weeks, from 2 to 6 weeks and in an aspect 1 month after the initial vaccination.

Oncogenic HPV types include HPV 31, 33, 35, 39, 45, 51,52, 56, 58, 59, 66 and 68.

Suitably, cross-protection is observed between at least one component of the first vaccine and one component of the second vaccine. As such, the first vaccine suitably comprises a protein ll or immunogenic fragment thereof from HPV 16 and HPV 18 and the second vaccine suitably comprises a protein ll or immunogenic fragment thereof from HPV 31 or HPV 45 or HPV 52.

In one aspect a first vaccine comprises L1 protein or immunogenic fragment thereof from HPV 16 and HPV 18. In a further aspect the first vaccine includes L1 protein or immunogenic fragment thereof from additional HPV types, such as one or more of the HPV types 31 , 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68. In one aspect the HPV type is HPV 33.

In another aspect, the second vaccine excludes at least the HPV 16 and 18 antigens from the first vaccine and comprises an L1 protein or immunogenic fragment thereof of an HPV type which has been shown to have a cross-protective interaction with HPV 16 and / or HPV 18. herein, suitably HPV 31 and / or 45 and / or 52. The second vaccine may include additional types such as one or more of HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.

In one aspect the second vaccine comprises protein ll or immunogenic fragment thereof of HPV 31 and / or HPV 45. In a further aspect the second vaccine includes protein ll or immunogenic fragment thereof of type 52. In a further aspect the second vaccine includes L1 protein or immunogenic fragment thereof, type 58.

Accordingly, the invention relates to a vaccine composition comprising an HPV 31 ll protein or immunogenic fragment thereof but excluding at least HPV 16 and HPV 18 ll protein or immunogenic fragment thereof.

The invention also relates to a vaccine composition comprising an HPV 45 L1 protein or immunogenic fragment thereof but excluding an L1 protein or immunogenic fragment thereof from at least HPV 16 and HPV 18.

The invention also relates to a vaccine composition comprising an HPV 52 L1 protein or immunogenic fragment thereof but excluding an L1 protein or immunogenic fragment thereof from at least HPV 16 and HPV 18.

In another aspect the second vaccine excludes all HPV ll-containing antigens from the first vaccine. Suitably the first and second vaccines share non-identical L1 proteins or identical complete protein fragments. For the avoidance of doubt, there may be regions within the L1 proteins or L1 fragments used in the first and second vaccines that are similar or identical. However the use of identical antigens, the antigen being total or a fragment thereof, both in the first and second vaccines is not preferred in this regard.

In one aspect, the first and second vaccines do not share an HPV ll protein or immunogenic fragment thereof of the same HPV type.

In one aspect, the components of the first vaccine contain non-phylogenetically and closely related HPV species such as HPV 16 and HPV 18.

In one aspect the components of the second vaccine contain non-phylogenetically and closely related HPV species, such as HPV 31 and HPV 45.

Non-phylogenetic and closely related types are from different species groups as evaluated by de Villiers et al. Virology. June 2004, 20; 324 (1): 17-27.

Suitably the HPV 16 and / or HPV 18 components in the first vaccine component protect against HPV infection and / or disease caused by at least one type of HPV in the second vaccine.

Suitably the first and second vaccines of the invention comprise HPV ll VLPs from the following HPV types:

first vaccine a combination of HPV 16, HPV 18 and HPV 33. The first vaccine may additionally contain HPV 58.

second vaccine is a combination of HPV 31, HPV 45 and optionally HPV 52 and / or HPV 58.

Suitably, the second released vaccine enhances the immune response against at least one component in the first released vaccine. Booster can be appropriately measured, for example, by antibody titers, using standard methods in the art, such as ELISA.

In one aspect, an HPV L1 protein or immunogenic fragment thereof is used to enhance previously elevated cross-reactive antibodies to a different HPV type.

In one aspect, the invention relates to the use of protein L1 or immunogenic fragment thereof from one HPV type in enhancing the immune response against a different second HPV type.

In one aspect, the invention relates to the use of protein L1 or immunogenic fragment thereof from an HPV type in enhancing the immune response against a homologous HPV type.

In one aspect, the invention relates to the use of protein L1 or immunogenic fragment thereof from one HPV type in enhancing the immune response against a different second HPV type, wherein the second type is phylogenetically related to the first type. Phylogenetic relationships between HPV types are well known in the art (see, for example, de Villers et al. Virology. 2004 Jun 20; 324 (1): 17-27). In this publication, papillomaviruses can be seen included in distinct species that are phylogenetically related. For example, in species 16 types 16, 31, 33 are found. In species 7 types 18 and 45 are found.

In another aspect, the invention relates to the use of HPV 31 L1 protein or immunogenic fragment thereof in the preparation of a medicament for enhancing an immune response to (generated by) an HPV 16 vaccine comprising an L1 protein or fragment thereof.

In another aspect, the invention relates to the use of HPV 52 L1 protein or immunogenic fragment thereof in the preparation of a medicament for enhancing an immune response to an HPV 16 vaccine comprising an L1 protein or fragment thereof.

In another aspect, the invention relates to the use of HPV 45 L1 protein or immunogenic fragment thereof in the preparation of a medicament for enhancing an immune response to an HPV 18 vaccine comprising an L1 protein or fragment thereof.

In another aspect, the invention relates to the use of HPV 18 L1 protein or immunogenic fragment thereof in the preparation of a medicament for enhancing an immune response to an HPV 45 vaccine comprising an L1 protein or fragment thereof.

In another aspect, the invention relates to the use of HPV 16 L1 protein or immunogenic fragment thereof in the preparation of a medicament for enhancing an immune response to an HPV 31 vaccine comprising an L1 protein or fragment thereof.

In another aspect, the invention relates to the use of the HPV 16 L1 protein or immunogenic fragment thereof in the preparation of a medicament for enhancing an immune response to an HPV 52 vaccine comprising a protein L1 or fragment thereof.

The present relates in one aspect to a vaccination program for protection against HPV infection or disease, the program comprising the release of a first HPV vaccine comprising an L1 protein or immunogenic fragment thereof from at least HPV 16. and HPV 18 and after a suitable interval a second HPV vaccine that does not comprise HPV 16 and HPV 18 L1 components of the first vaccine and that the second vaccine comprises an L1 protein or immunogenic fragment thereof of at least one other HPV type oncogenic. In this aspect of the invention, components of the first and second vaccines as defined above may be released in reverse order. By way of example, the first HPV to be released may comprise HPV 31 and HPV 45 antigens, while the second vaccine to be released comprises HPV 16 and HPV 18 antigens.

Vaccines of the invention may further comprise, within the limits of the invention, antigens from other HPV types, such as other oncogenic antigenic types (e.g. HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68.), skin types (eg types 5, 8) and genital wart types (6, 11).

Appropriately, each vaccine is capable of protection against persistent infection for the HPV types present in the vaccine.

Appropriately, each vaccine is capable of protection against incident infection for HPV types present in the vaccine.

Incident and persistent cervical infection are defined in

Example 1

Appropriately, each vaccine is capable of protection against cytological abnormalities related to HPV infection (eg ASCUS, CIN 1, CIN2, CIN3, cervical cancer), suitably caused by types not present in the vaccine, such as HPV 31, 45 or 52. .

Proteins L1 or protein fragments of additional HPV types may be included in the vaccine of the invention, such as skin types (in particular HPV 5 and 8) and genital wart associated types such as HPV 6 and 11. Types 6 and 11 are not considered oncogenic types here.

In one aspect the vaccine may comprise HPV ll protein components, preferably as virus equivalent particles, in different amounts. In one aspect, HPV 16 and HPV 18 VLPs may be delivered at a higher dose than other oncogenic types, such as HPV 33 or 58. In one aspect, HPV 16 and HPV 18 ll-only VLPs are provided. at 20 μg per dose for human use. Other HPV VLPs may be used at a lower dose, such as 15 or 10 μg per dose for human use.

In one aspect of the invention, the vaccine may include an early HPV antigen, for example an antigen selected from the list consisting of HPV E1, E2, E3, E4, E5, E6, E7 or E8. In an alternative aspect the vaccine may require an early HPV antigen, for example an antigen selected from the list consisting of HPV E1, E2, E3, E4, E5, E6, E7 or E8.

In one aspect, a vaccine component of the invention is trivalent (contains one HPV L1 or fragment thereof of 3 different types of oncogenic HPV). In an additional aspect the vaccine is tetravalent. In an additional aspect the vaccine is pentavalent. In an additional aspect the vaccine is hexavalent. In an additional aspect the vaccine is heptavalent. In an additional aspect the vaccine is octavalent. Higher order valences are also contemplated here. In additional aspects the vaccine is at least tetravalent or at least pentavalent or at least hexavalent or at least heptavalent or at least octavalent with respect to oncogenic HPV types. In a further aspect the combined vaccines of the invention include L1 protein or immunogenic fragments thereof of 4, 5, 6, 7, 8, 9 or 10 different types of HPV.

Preferably, the combination of HPV components within the vaccine does not significantly bind the immunogenicity of any HPV component. In particular it is preferred that there is no biologically relevant interference between HPV antigens in the combination of the invention, such that the combined vaccine of the invention is capable of providing effective protection against infection for each HPV genotype represented in the vaccine. Suitably, the immune response against a given HPV type in combination is at least 50% of the immune response of that same HPV type when measured individually, preferably 100% or substantially 100%. For responses to HPV 16 and HPV 18, the combined vaccine of the invention preferably stimulates an immune response that is at least 50% of that provided by a combined HPV 16 / HPV 18 vaccine. Suitably the immune response generated by the vaccine of the invention is at a level at which the protective effect of each HPV type is still seen. The immune response may suitably be measured, for example, by antibody responses in preclinical or human experiments. Measurement of antibody responses is well known in the art and disclosed (for example) in W003 / 077942.

Where the vaccine or composition of the invention comprises an immunogenic L1 fragment, then suitable HPV L1 immunogenic fragments include L1 truncations, deletions, substitution or insertion mutants. Such immunogenic fragments are suitably capable of creating an immune response (if necessary, when aided), said immune response being capable of recognizing an L1 protein such as a virus equivalent particle of the HPV type from which the L1 protein was derived.

In one aspect, a suitable immunogenic fragment of HPV 16 is capable of cross-protection against at least one of HPV 31 and HPV 52 and in one aspect of the invention capable of cross-protection against both.

In another aspect, a suitable immunogenic HPV 18 fragment is capable of cross-protection against HPV 45.

The cross protection obtainable by HPV 16 and / or HPV 18 immunogenic fragments can be assessed by human experiments, for example, as summarized in Example 1.

Similarly, different vaccines according to the present invention may be tested using standard techniques, for example as in Example 1 or standard preclinical models, to confirm that the vaccine is immunogenic.

Suitable immunogenic L1 fragments include truncated L1 proteins. In one aspect, truncation removes a nuclear localization signal. In another aspect the truncation is a C-terminal truncation. In a further aspect, the C-terminal truncation removes less than 50 amino acids, such as less than 40 amino acids. Where L1 is from HPV 16 then in another aspect C-terminal truncation removes 34 amino acids from HPV 16. Where L1 is from HPV 18 so in an additional aspect C-terminal truncation removes 35 amino acids from HPV 18

In one aspect the HPV 16 sequence is as follows:

(SEQID NO: 1)

MSLWLPSEAWYLPPVPVSKVVSnEBEYVARTNIYYÁGTSRLLAVGHPYFPIKKPNNNfCl 60

LVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGVCISGH 120

PLLNKLDDTENASAYAANAGVDNRECISMDYKQlXJLCLIGCKPPiGEHWGKGSPCnvlVAV 180

NPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPD YIKMVSE 240

PYGDSLFFYLRRHQMFVRHLFNRAGAVGEKVPDDLYIKGSGSTANLASSNYFPTPSGSMV 300

TSDAQIFNKPYWLQRAQGHWGICWGNQLFVIVVDTTRSTNMSLCAAISTSETTYKNTNF 360

KEYLRHGEEYDLQFtFQLCKmTADVMTYCHSMNSTILEDWNFGLQPPPGGTLEDTYRF 420

VTSQAIACQKHTPPAPKEDPLKKYTFWEVNLICEKFSADLDQFPLGRKFLLQ 471

In another aspect, the invention relates to virus equivalent particles consisting only of HPV 16 L1 having the above amino sequence and compositions containing such VLPs.

The HPV 16 sequence may also be that disclosed in W09405792 or US6649167, for example, appropriately truncated. Proper locks are locked to a position equivalent to that shown above as assessed by sequence comparison.

In one aspect the HPV 18 sequence is as follows: (SEQ ID NO: 2)

MALWRPSDNTVYLPPPSVARVWTDDYVmTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ 60

DIPKVSAYQYRVFRVQLPDPNKFGLPDNSIYNPETQRLVWACVGVEIGRGQPLGVGLSGH 120

PFYNKLDDTESSHAATSNVSEDVRDNVSVDYKQTQLCiLGCAPAJGEHWAKGTACKSRPL ISO

SQGDCPPLELKNTVLEDGDMVDTXjYGAMDFSTLQDmCEVPLDICQSlCKYPDYLQMSAD 240

PYGDSMFFCLRREQLFARHFWNRAGTMGDTVPPSLYÍKGTGMRASPGSCVYSPSPSGSIV 300

TSDSQLFNKPYWLHlCAQGHN ^ GVCWVmQLFVWVDTTRSTNLTICASIQSPVPGQYDATK 360

FKQYSRHVE £ YDU5F] FQLCTITLTADVMSYifSMNSSILEDWNFGVPPP'rTSLVDTYR 420

FVQSVAITCQKDAAPAENKDPYDKLKF \ WVDLKEKFSLDLDQYPLGRJCFLVQ 472

In another aspect, the invention relates to virus equivalent particles consisting only of HPV 18 L1 having the above amino sequence and compositions containing such VLPs.

An alternative HPV 18 sequence is disclosed in W09629413, which may be properly locked. Proper locks are locked to 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.

Where the L1 protein is from another type of HPV then the C-terminal locks made by HPV 16 and HPV 18 can be used based on DNA or protein sequence alignments.

Appropriate locks of, for example, HPV 31, HPV 45, HPV 52, HPV 58, HPV 33 may also be done in one aspect by removing the equivalent C-terminal portions of the L1 protein from those described above as assessed by the alignment of sequence. Serotype 31 Truncated L1 Nucleotide Sequence (SEQ ID NO: 5)

ATGTCTCTGTGGCGGCCTAGCGAGGCTACTGTCl'ACtTftCCACCTGTCCCAGTGTCTZ ^ ^^ CGGATGAATATGTAACACGAACCAACATATATTATCACKCaGGC Gri'G'rAAGCA 70 14 0 210 tatagggtatttagggttcgtttaccagatccaaacaaatttggatttcctgatacatctttttataatc TCCAtATrATTCCATACCTAAATCTGACAATCCTAAAAAAATAGTTGTACCAAAGGTGTC ^ 280 ^ .3 C7TGAAÁCTCAA CG CTTAGTTTGGGCCTGIXrFTGGTT SO TATT AGTGGTCATCCATTATTAAATAAATTTGATG ACAC GGCACTGATAATAGGGAATGTATATCAATGGATT 420 ^ 490 ^ 630 CACCTAlTGGAGAGCATTGGGíyFAAAGGTAGTCCT TCCkTTAGMTTAAAAAATTCAGTTATACAAGATGGGGAm TTTACTGCTTTACAAGACACTAAAAGTAATGITCerTTGCAC 560 ^ 700 ^ 770 ATTATCTTAAAATCGTTGCTGAGCCATATGGCGATACATTATTTTITT TGTAAGGCATTTTTrr AATAGATCAOTCACGGTtXJtrrGAATCGCT '40 TCCGGTTCAACAGCrACTrTAGCTAACAGTACATA 010 ATGCACAAATTTTTAATAAACCATATrt ^ ^ ^ OH ATCC 1120 AATTTATAlWCAGrTATGOVkAA CAATCAGtTAlTTGITACTGTGGTAGATACCACACGTAGTÂCCA10S0 AAGAGTGATACTACATTTAAAAGTAGTAATTTrAAAGAGTATTTAAGACATGGT ^ 1190 ^ TTTGTCACCTCACAGGCCATTACATGTCAAAAAACrGCCCCCCAAAAGCCCAAGGAAGATCCAT TGCrrATTTIXXIAAGATTGGAATTTTGGATTGACCACACCTCCCTCAGGTT 1260 1330 AAAG ATTATGTATTCTGGGAGGTfAATCTAAAAGAAAAGlT ^ 1400 CAAATTTTTATTACAGTAA 1419

In another aspect, the invention relates to virus equivalent particles consisting only of HPV 31 L1 having the amino sequence encoded by the above sequence and compositions containing such VLPs.

Serotype 45 Truncated Ll Nucleotide Sequence (SEQ ID NO: 6)

ATGGCTTTGTGGCGCCCrÂGTGACAGTAC 7O

CreAtGATTOTGTGTCTCGCACAA 14O

TCCATATTTTAGGGTTGTOCCTAGTGGTGCAGCTA 210

mmGGGTGl ^ TAGftGTAGCTTTACeCGATC 280

CTCAAACACAACGTTTGGTTTCGGCATGTGTACTAT 350

CCTAAGTCCCCATCCATTTTATAATAAATTGGATC 420

ACGCAGGATGTTAGGGATAA1Xj1'GTCAGTTGATTATAAGCAAA 490 CTCCTATTGGTGAGÇAÇIX ^ GCCAACGGCACAC ^^ 560

TCCTTTGGAACTTAAAAACACCATTATTGAGGATGGTGATATGGTC 630

TTTAGTACACTGCAGGATACAAAGTGCGACXm-CCATTAGACAlT ^ 700

ATTATTTGCAAATGTCTGCTCATCCCTATGGGGATTCTATGT ^ 770

TGCAAGACATTTTTGGAATAGGGCAGGTGTTATGGGTGACACAGT ^ 840

ACTA ^ GCTAATAlOCGTGAAACCCCrcGC 910

CTTCTGA TTCTCAftTTATirTAATAftCCCATATT TO 980

rrGGCATAATmGTTGTTTGTTACTGTAGTCXJACACTACCCGCAGTACTAATrTAACATTATGTGCCTCT 1050

ACACAAAATCCTGTGCCAGGTACATATGATCCTACTAAGCTTAAGCACTATAGTAGACATGT <3GAGGAAT 1120

ATQ ATTTAC ^ TITATTTTTCAGTTGI ^ ACT ^ 1190

TATGAATAGmGlVttTm ^ AAAATTtKAftrTC 1260

ACATATCGTTTTGTGCAATCAGtTGCTGTTACCTGTCAAAAGGATACÍ (^ 1330

CATATGATAAATTAAAGTT ^ GGACTGTTGACCTAÂA ^ 1400 CCTTGGTCGAAAGTTTTTAGTTCAGTAA

In another aspect, the invention relates to virus equivalent particles consisting only of HPV 45 L1 having the amino sequence encoded by the above sequence and to compositions containing such VLPs.

The L1 protein or fragment of the invention may optionally be in the form of a fusion protein, such as the fusion of L1 protein with L2 or a premature protein.

The HPV L1 protein is suitably in the form of a capsomer or virus equivalent particle (VLP). In one aspect HPV VLPs may be used in the present invention. HPV VLPs and methods for producing VLPs are well known in the art. VLPs are typically constructed from L1 and optionally L2 virus structural proteins, see for example W09420137, US5985610, W09611272, US6599508B1, US6361778B1, EP 595935. Any suitable HPV VLP may be used in the present invention which provides cross protection such as as a VLP Ll or from L1 + L2.

Suitably the VLP is an Ll-only VLP.

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 L1 protein. In one aspect the L1 protein used to form the VLP is a locked L1 protein as described above.

VLPs can be made on any suitable cell substrate, such as yeast cells or insect cells, for example baculovirus cells, and techniques for preparing VLPs are well known in the art, such as W09913056, US 6416945B1, US 6261765B1 and US6245568 and references therein, the total contents of which are hereby incorporated by reference.

VLPS are suitably manufactured by disassembly and reassembly techniques, which may take into account more stable and / or homogeneous papillomavirus VLPs. For example, McCarthy et al, 1998 "Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Virus Iike Particles in Vitro" J. Virology 72 (1): 33-41, describe disassembly and reassembly of purified recombinant HPV 11 L1 VLPs. insect cells to obtain a homogeneous preparation of VLP's. W09913056 and US6245568 also describe disassembly / reassembly processes for the manufacture of HPV VLPs.

In one aspect, HPV VLPs are manufactured as described in W09913056 or US6245568.

The HPV L1 of the invention may be combined with an adjuvant or immunostimulant such as, but not limited to, detoxified lipid A from any source and non-toxic lipid A derivatives, saponins and other reagents capable of stimulating a TH1-type response.

Enterobacterial lipopolysaccharide (LPS) has long been known to be a potent stimulator of the immune system, although its use in adjuvants has been cut off by its toxic effects. A non-toxic LPS derivative, monophosphoryl A lipid (MPL), produced by the removal of the core carbohydrate group and the terminal reducing glycosamine phosphate, 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:

<formula> formula see original document page 20 </formula>

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

A suitable form of 3 D-MPL is in the form of an emulsion that has a small particle size less than 0.2 μηι in diameter and its manufacturing method is disclosed in WO 94/21292. Aqueous formulations comprising monophosphoryl A lipid and a surfactant have been described in W09843670A2.

Bacterial lipopolysaccharide derived adjuvants to be formulated in the compositions of the present invention may be purified and processed from bacterial sources or alternatively may be synthetic. For example, purified monophosphoryl A lipid is described in Ribi et al 1986 (supra) and 3-O-deacylated monophosphoryl or diphosphoryl A lipid derived from Salmonella sp. are described in GB 22202 Ile 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 (1): 141-6; and EP 0 549 074 B1). In one aspect, the 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 those 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 of the above MPL structure.

Saponins are shown in: Lacaille-Dubois, M and Wagner H. (1996. A review of the biological and pharmacological activities of saponins. Phytomedicine vol 2 pp 363386). Saponins are triterpene steroids or glycosides widely distributed in the plant and marine animal kingdoms. Saponins are noted for the formation of colloidal solutions in frothing water and precipitation of cholesterol. When saponins are close to cell membranes they create membrane pore equivalent structures that cause the membrane to rupture. Red blood cell hemolysis 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. Adjuvant and hemolytic activity of individual saponins have been extensively studied in the art (Lacaille-Dubois and Wagner, supra). For example, Quil A (derived from Quillaja Saponaria Molina tree bark from South America) and fractions thereof are described in US 5,057,540 and "Saponins as vaccine adjuvants", Kensil, CR, Crit Rev Ther Drug Carrier Syst. 1996, 12 (1-2): 1-55; and EP 0 362 279 B1. Particulate structures, called Immune Stimulating Complexes (ISCOMS), which comprise Quil A fractions are hemolytic and have been used in the manufacture of vaccines (Morein, B., EP 0 109 942 BI; WO 96/11711; WO 96/33739). Hemolytic saponins QS21 and QS 17 (purified Quil A HPLC fractions) 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 B1. Other saponins that have been used in systemic vaccination studies include those derived from another plant species, such as Gypsophila and Saponaria (Bomford et al., Vaccine, 10 (9): 572-577, 1992).

An improved 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 reacogenic composition where 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 use of these adjuvant forms an aspect of the invention.

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

In one aspect, the vaccine additionally comprises a saponin, for example QS21.

In one aspect, the formulation further 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 3 D-MPL.

Additional components that may be included in a vaccine formulation according to the invention include nonionic detergents such as octoxynols and polyoxyethylene esters as described herein, particularly t-octylphenoxy polyethoxyethanol (Triton X-100) and sorbitan monooleate polyoxyethylene (Tween 80); and bile salts or cholic acid derivatives as described herein, in particular sodium deoxycholate or taurodeoxycholate. Thus, in one aspect of the invention 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.

In one embodiment of the present invention, the vaccine comprises a vesicular adjuvant formulation comprising cholesterol, a saponin and an LPS derivative. In this regard the adjuvant formulation suitably comprises a unilamellar vesicle comprising cholesterol having a lipid bilayer suitably comprising dioleoyl phosphatidyl choline wherein the saponin and LPS derivative are associated with or included in the lipid bilayer. In one aspect, these adjuvant formulations comprise QS21 as saponin and 3 D-MPL as the LPS derivative, wherein the ratio of QS2 lxolesterol 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.

Suitably, the vaccines of the invention are used in combination with aluminum and are suitably adsorbed or partially adsorbed on aluminum adjuvants. Suitably the adjuvant is an aluminum salt, which may be in combination with 3D MPL, such as aluminum phosphate and 3D MPL. Aluminum hydroxide, optionally in combination with 3D MPL is also suitable.

In another aspect of the present invention the vaccine comprises combining HPV VLPs with an aluminum salt or an aluminum + 3D MPL salt. Aluminum hydroxide is suitable as aluminum salt.

The vaccine may also comprise aluminum or an aluminum compound as a stabilizer.

In another aspect the adjuvant may be a combination of an oil-in-water emulsion adjuvant and 3D MPL. In one aspect the oil-in-water emulsion comprises a metabolizable 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 (e.g., see W09961052 A2), topical, subcutaneous, mucosal (typically intravaginal), intravenous, intramuscular, intranasal, sublingual, intradermal and suppository.

Optionally the vaccine may also be formulated or co-administered with other HPV antigens or non-HPV antigens. Appropriately these non-HPV antigens may provide protection against other diseases such as sexually transmitted diseases such as herpes simplex virus, EBV, chlamydia and HIV. We particularly prefer that the vaccine comprises gD or a truncated HSV thereof. Thus the vaccine provides protection against both HPV and HSV.

The dosage of vaccine components will vary with the individual's condition, sex, age and weight, route of administration, and HPV of the vaccine. The amount can also be varied with the number of VLP types. Suitably, the release is of an amount of vaccine suitable to generate a protective immune response. Suitably, each vaccine dose comprises 1-100 μg of each VLP, in an aspect of 5-80 μg, in another aspect 5-30 μg of each VLP, in an additional 5-20 μg of each VLP, yet in an additional aspect 5 μg, 6 μg, 10 μg, 15 μg or

For all vaccines of the invention, in one aspect the vaccine is used for vaccination of adolescent girls 10 to 15 years of age, such as 10 to 13 years. However, older girls over 15 and adult women may also be vaccinated. The vaccine can also be given to women following an abnormal pap smear or after surgery following the removal of an HPV lesion or for those who are seronegative and DNA negative for HPV cancers.

In one aspect the vaccine of the invention is used to vaccinate men.

In one aspect, the vaccine is released on a dose 2 regimen, for example on a 0, 1 month or 0, 6 month regimen respectively. The vaccination regimen may incorporate a booster injection after 5 to 10 years, such as 10 years.

In one aspect, the invention relates to a triple dose vaccine, wherein (for example) a first vaccine and a second vaccine are released, the first and second vaccine being different, followed by a third vaccine containing one or more or more. all HPV elements of the first or second vaccines. In one aspect the first and second vaccines are completely different from the HPV L1 components.

For example, a first vaccine may comprise or consist of HPV 16 and HPV 18 L1 protein. The second vaccine may consist of HPV 31 and HPV 45 L1 protein. A third vaccine may comprise L1 protein of all 4 types of HPV, HPV 16, 18, 31 and 45.

In another aspect, the invention relates to a triple dose vaccine, wherein (for example) a first HPV vaccine and a second HPV vaccine are released, the first and second vaccine being different, followed by a third vaccine, the third vaccine containing none of the HPV ll components of the first or second vaccines.

In one aspect, the vaccine is a liquid vaccine formulation, although the vaccine may be lyophilized and reconstituted prior to administration.

The teaching of all references in this application, including patent applications and granted patents, are hereby incorporated by reference in their entirety.

The vaccines of the invention comprise certain HPV components as shown above. In a further aspect of the invention, the vaccine essentially consists of, or consists of, said components.

The term 'vaccine' as used in the present invention refers to a composition comprising an immunogenic component capable of eliciting an immune response in an individual, such as a human, optionally when appropriately formulated or adjuvanted. A vaccine suitably evokes a protective immune response against incident infection or persistent infection or cytological abnormality such as ASCUS, CIN1, CIN2 CIN3 or cervical cancer caused by one or more HPV types.

The present invention is now described with reference to the following examples which serve to illustrate the invention.

Example 1

Precise details of the experiment are provided in Harper et al, the Lancet. November 13, 2004; 364 (9447): 1757-65. In summary, healthy women between the ages of 15 and 25 were immunized with a mixture of HPV 16 and HPV 18 ll VLPs. The women enrolled were: 1) HPV-16 and HPV-18 seronegative; 2) negative for high-risk HPV infection of the cervix (detected by HPV PCR); 3) women who had 6 or fewer sexual partners in their lifetime and 4) who had normal PAP smears.

The mixture comprised, per 0.5 ml dose, 20 μg of HPV-16 VLP Ll, 20 μg of HPV-18 VLP Ll and was adjuvanted with 500 μg of aluminum hydroxide and 50 μg of 3D MPL. The placebo group was injected with 500 μg aluminum hydroxide alone.

The efficacy of the vaccine (V.E.) against certain types of HPV cancer has been evaluated, in which the V.E. is the% improvement in protection against vaccine infection or disease compared to a placebo group.

Cross-protection was assessed by detecting the presence of specific nucleic acid for various oncogenic types in vaccines and control group. Detection was performed using techniques as described in and references W003014402, particularly for non-specific HPV DNA amplification and subsequent detection of DNA types using a LiPA system as described in WO 99/14377 and Kleter et al. , [Journal of Clinical Microbiology (1999), 37 (8): 2508-2517], the total contents of which are specifically incorporated herein by reference.

Any suitable method, however, can be used for detecting HPV DNA in a sample, such as type-specific PCR using primers specific for each type of HPV of interest. Suitable primers are known to skilled persons or can be readily constructed as sequences of oncogenic HPV types are known.

In detail, the methods section of the Lancet document is reproduced here for completeness:

The main objective of this study was to evaluate the efficacy of the vaccine in preventing infection with HPV-16, HPV-18 or both (HP V-16/18) between 6 and 18 months in participants who were initially shown to be HPV-seronegative. 16/18 by ELISA and HPV-16/18 DNA negative by PCR. Secondary objectives included: evaluation of vaccine efficacy in preventing persistent HPV-16/18 infection and evaluation of vaccine efficacy in prevention of cytologically confirmed low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL) and histologically confirmed LSEL (CIN 1), HSIL squamous cell cancer (CIN 2 or 3) or adenocarcinoma associated with HPV-16/18 infection between 6 and 18 months and 6 and 27 months. Prevention of atypical squamous cells of undetermined significance cytology (ASCUS) associated with HPV-16/18 infection was added post-hoc to the outcome analyzes.

We will also do an exploratory analysis of the CIN 1 and 2 histopathological endpoints associated with HPV-16/18 DNA detected by PCR in lesional tissue. Other objectives included vaccine evaluation for immunogenicity, safety and tolerability.

Researchers in North America (Canada and the USA) and Brazil recruited women for this effective study through advertisements or prior participation in a cross-sectional HPV epidemiology study that took place between July and December, 2000.

For each of the 32 study sites, an institutional review framework approved the protocol, forms of permit and amendments. The women signed separate written permits for study participation and colposcopy. For those under 18, permission and consent from the participant's parents were required.

There were two study phases: an initial phase for vaccination and follow-up that was completed at month 18; and an extension blind follow-up phase that was completed in month 27.

Women eligible for the early stage (0 to 18 months) included healthy women 15 to 25 years of age with no more than six sexual partners, no history of an abnormal Pap test or ablative or excisional cervical treatment. of the uterus and no continuous treatment for external condylomata; and were cytologically negative, seronegative for HPV-16 and HPV-18 antibodies by ELISA and HPV-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 prior to study entry.

Women who completed the initial phase of the most recent study and who did not undergo ablative or excisional cervical therapy or hysterectomy after enrollment were eligible to participate in the study extension phase (months 18-27).

Procedures

Each dose of the divalent HPV-16/18 virus equivalent particle vaccine (GlaxoSmithKline Biologicals, Rixensart, Belgium) contained 20 μg of HPV-16 ll virus equivalent particle and 20 μg of HPV ll virus equivalent particle. -18. Each virus-equivalent particle type was produced on a Spodoptera frugiperda Sf-9 and Trichoplusia ni Hi-5 cell substrate with AS04 adjuvant containing 500 μg aluminum hydroxide and 50 μg 3-deacylated monophosphoryl A lipid (MPL, Corixa, Montana, USA) supplied in a single dose vial. The placebo contained 500 μg aluminum hydroxide per dose and was identical in appearance to the HPV-16/18 vaccine. Each study participant received a 0 to 5 ml dose of vaccine or placebo at 0 months, 1 month and 6 months.

Health care providers obtained cervical specimens with a cervical brush and spatula (washed at PreservCyt, Cytyc Corporation, Boxborough, MA, USA) by HPV DNA cytology and testing at evaluation and at 6, 12, and 18 months. At months 0 and 6 and thereafter every 3 months, women with self-obtained cervicovaginal specimens with two sequential strands (placed on PreservCyt) for HPV DNA testing. [DM Harper, WW Noll, DR Belloni and BF. Cole, Randomized clinical trial of PCR-determined human papillomavirus detection methods: self-sampling versus clinician-directed — biological agreement and women's preferences. Am J Obstet Gynecol 186 (2002), p. 365373] A central laboratory (Quest Diagnostics, Teterboro, NJ, USA) reported cytology results (ThinPrep, Cytyc Corporation) using the Bethesda 1991 grading system.

Protocol guidelines recommended colposcopy following two reports of ASCUS or one report of atypical glandular cells of undetermined significance, LSIL or HSIL, squamous cell carcinoma, in situ adenocarcinoma, or adenocarcinoma. These guidelines also recommended biopsy for any suspected lesions.

The central histology laboratory made an initial diagnosis from formalin fixed tissue specimens for clinical control. A chart of three pathologists prepared a subsequent consensual diagnosis for HPV-16 and HPV-18 associated lesions with the CIN system. This consensual diagnosis also included a review of the sections adopted during the microdissection period for PCR detection of lesional HPV DNA.

HPV DNA isolated from cytology specimen (MagNaPuro Total Nucleic Acid system, Roche Diagnostics, Almere, The Netherlands) and cervical biopsy specimen (K proteinase extraction) was amplified from an aliquot of purified total DNA with primers. SPF 10 broad-spectrum amplifiers that amplify a 65 base pair region of the LL gene [B Kleter, LJ van Doom, Jter Schegget et al., Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of human anogenital papillomaviruses. Am J Pathol 153 (1998), p. 1731-1739: L. van Doom, W Quint, B. Kleter et al., Genotyping of human papillomavirus in cervical cytology specimens by the PGMY Iine blot assay and the SPF (IO) Iine probe assay. J Clin Microbiol 40 (2002), p. 979-983 and WG Quint, G Scholte, LT van Doom, B Kleter, PH Smits and J. Lindeman, Comparative analysis of human papillomavirus infections in cervical scrapes and biopsy specimens by general SPF (IO) PCR and HPV genotyping. J Pathol 194 (2001), p. 51-58]. Amplification products were detected by a DNA enzyme immunoassay. An Iine probe assay (LiPA Kit HPVINNO LiPA HPV genotyping assay, version 1 of the SPF-10 system, Innogenetics, Gent, Belgium, manufactured by Labo Bio-medical Products, Rijswijk, The 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). [Β Kleter, L. van Doom, L. Schrauwen et al., Development and clinical evaluation of a highly sensitive PCR-reverse hybridization Iine probe assay for detection and identification of anogenital human papillomavirus. J Clin Microbiol 37 (1999), p. 2508-2517]. Any specimen that was positive by DNA enzyme immunoassay was assayed by HPV-16 and HPV-18 type-specific PCR. HPV-16 type-specific PCR primers amplified a 92 base pair segment of the E6 / E7 gene and HPV18 type-specific PCR primers amplified a 126 base pair segment of the L1 gene. [MF Baay, WG Quint, J Koudstaal et al., 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), p. 745-747]

We defined incident HPV-16/18 cervical infection as at least one HPV-16 or HPV-18 positive PCR result during the experiment and persistent HPV-16/18 infection as at least two HPV-DNA PCR assays. positive for the same viral genotype separated by at least 6 months. [H Richardson, G Kelsall, P. Tellier et al., The Natural History of Type-Specific Human Papillomavirus Infections in Female University Students. Cancer Epidemiol Biomarkers Prev 12 (2003), p. 485-490 and AB Moscicki, JH Ellenberg, S Farhat and J. Xu, Persistence of human papillomavirus infection in HTV-infected and -infected adolescent girls: risk factors and differences, by philogenetic type. J Infect Dis 190 (2004), p. 37-45]. HPV-DNA test results were hidden from investigators during the study and cytological and histological diagnoses were revealed for clinical control purposes only. Analyzes included HPV-16/18 DNA results for cervical specimens and combined and self-obtained cervical cervicovaginal specimens.

We collected serum from study participants at months 0,1,6, 7, 12 and 18 for immunogenicity assessment. Serological testing for HPV-16 and HPV-18 virus equivalent particle antibodies was performed by ELISA. Recombinant HPV-16 or HPV-18 virus equivalent particles were used as coating antigens for antibody detection (see web appendix http://image.thelancet.com/extras/04artl 0103_webappendix.pdf). Seropositivity was defined as a titer greater than or equal to the cut-off titer assay set at 8 HPV-16 ELISA units / ml and 7 HPV-18 ELISA units / ml. Typical natural titers were determined by the use of blood samples obtained from women in the previous epidemiology study that were found to be seropositive for HPV-16 or HPV-18 by ELISA.

Women reported symptoms experienced during the first 7 days after vaccination on daily cards with a three-degree symptom intensity scale. Additionally, they reported to the personal study by interview of all adverse events within the first 30 days after vaccination. Information on serious adverse events and pregnancy was collected throughout the study.

Statistical methods

Assuming a cumulative 6% incidence rate of both HPV-16 and HPV-18 infection over 12 months, we estimate that 500 women per treatment group would provide 80% power to assess a lower limit of 95% IC. of vaccine efficacy above zero. We assume an 80% retention rate for 18 months. Interim analyzes for efficacy, safety and immunogenicity were made for future study planning purposes only; The 0'Brien and Fleming methods were used to adjust the α value for the final analysis after the interim analyzes occurred (total α = 0.05; bilateral test). [PC O'Brien and TR. Fleming, A multiple testing procedure for clinical trials. Biometrics 35 (1979), p. 549-556].

Block randomization stratified according to validated algorithms was centralized with an internet randomization system. The stratification was according to age (15-17, 18-21 and 22-25 years) and region (North America and Brazil). Each vaccine dose was assigned a randomly chosen number based on information from the specific participant who entered the personal study computerized randomization system. The location of treatment remains hidden from researchers and women participating in a long-term follow-up study.

The intention-to-treat and protocol-based groups are shown in the figure, where the reasons for excluding analyzes are listed in order; Women who met more than one exclusion criterion were counted only once according to the higher classification criterion. We refer to the series of participants associated in the intention-to-treat analyzes and according to the protocol as groups, although the information used to restrict patient inclusion in according to the protocol was known only after follow-up.

We performed both protocol-based and intention-to-treat analysis for efficacy. Vaccine efficacy calculation in the month 18 analysis according to the protocol was based on the proportion of participants with HPV-16/18 infection in the vaccinated versus placebo groups. Vaccine efficacy was defined as 1 minus the ratio between these two proportions; 95% of CIs measured the accuracy of estimated efficacy. The ρ values were calculated with the bilateral Fisher exact test. Corresponding rates were expressed as case numbers with the result divided by the number of participants at risk. The month 18 group according to the protocol included registered women who received three scheduled doses of vaccine and agreed with the protocol as described in the figure.

Vaccine efficacy calculation in the intention-to-treat month 27 analyzes and according to the protocol was based on the Cox proportional hazard model using the time of occurrence of HPV-16/18 infection cases in the vaccinated versus placebos groups. This allowed the tracking of Tempo-persona results data in each group. Vaccine efficacy was calculated using 1 minus the risk ratio and ρ values calculated using the Iog rank test. Corresponding rates were expressed as the number of cases divided by Total Tempo-persona. All enrolled women who received at least one dose of vaccine or placebo were negative for high-risk HPV-DNA at month 0 and had any data available for outcome measurement included in the intention-to-treat group. The month-27 group according to the protocol included effect results from the month-18 group according to the protocol and results that occurred during the extension phase (18 months to 27 months).

Calculation of ρ values for safety analysis was performed using Fisher's exact test comparisons. The safety analysis group included all registered women who received at least one dose of vaccine or placebo and complied with specified minimum protocol requirements (see figure below): <table> table see original document page 35 </column> < / row> <table> Immunogenicity was assessed in a subgroup of the group according to the safety protocol, which included women with serology results at months 0, 7, and 18 who received all three doses of vaccine or placebo from the study. according to the program, condoning the blood sampling program and did not become positive for HPV-16/18 DNA during the experiment. Seropositivity rates between vaccine and placebo groups were compared with Fisher's exact test (p <0-001 considered significant). Significant mean titers were compared with the ANOVA and Kruskal-Wallis tests.

Block randomization and statistical analyzes were performed with SAS version 8.2 (SAS Institute, Cary, North Carolina).

Initial analysis and results

The results of the initial cross-protection analysis are presented in patent application W02004 / 056389, the total contents of which are incorporated herein by reference.

An initial analysis was performed on an "ITT" (Intention To Treat group, representing all individuals who received at least one dose of vaccine). These data are shown in Table A.

The results presented in the Tables BeC refer to the "ATP" group (according to the Protocol) for those patients who met all the criteria of the experiment. Table B is a central point analysis with data taken from all Tempo-persona patients in which at least 50% of the group were 18 months after their first vaccination. Table C gives the final result, all data being from patients 18 months after the 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.

As shown by the data presented in Table A, immunization with a mixture of HPV16 and HPV18 VLPs provided precursor cross protection against other HPV types. At this point, sample sizes are too small to account for rigorous statistical analysis, however the data show a positive trend and suggest that immunization with HPV 16 and HPV 18 VLPs will be effective against infection with other HPV types.

This has been confirmed as the advanced study.

Table B demonstrates that HPV 16 and HPV 18 provide statistically significant cross-protection against the high-risk cancer group types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.

Table C shows that except for related HPV-18 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 high risk types 12 (non-HPV-16/18) evaluated.

Additional analysis was performed on specific cross-protection against specific types.

The efficacy of the vaccine was evaluated against infections and diseases related to high risk cancer types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, HPV-16 related types. phylogenetics (the groups of; 31, 35 and 58; 31, 33, 35, 52 and 58) and phylogenetic HPV-18-related types (45 and 59).

An analysis was performed on an "ATP" group (According to Protocol) for those patients who met all the criteria of the experiment. In the ATP group all patients received 3 doses of vaccine at 0, 1 and 6 months and were seronegative at 6 months. Results

As shown by the data presented in Table 1, immunization with a mixture of HPV16 and HPV18 VLPs provided statistically significant cross-protection against incident HPV types 31, 52, and 45 infection compared to control. Statistically significant cross-protection against incident infection was also observed against the group of all HPV 16 related types (HPV-31, 33, 35, 52, and 58) and the group of all high-risk types excluding 16 and 18 (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68).

Statistically significant cross-protection against persistent infection was also observed against types 31 and 52 and was also observed against the group of all HPV 16-related types (see Table 2).

Statistically significant cross-protection was observed against cytological abnormalities associated with HPV 52 and was also observed against cytological abnormalities associated with the HPV 16-related group of all types (HPV-31, 33, 35, 52, and 58) and the all high-risk types excluding 16 and 18 (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Table A

<table> table see original document page 39 </column> </row> <table>

Samples were collected from patients 9, 12, 15, and 18 and tested for HPV infection by the types specified above. Table B

<table> table see original document page 40 </column> </row> <table> Table C

<table> table see original document page 41 </column> </row> <table>

Samples were collected within 18 months of the patients and tested for HPV infection by the above specified types. Table 1

Efficacy against Related Incident Infections 16/18 *

<table> table see original document page 42 </column> </row> <table> Table 2

<table> table see original document page 43 </column> </row> <table> TABLE 3

<table> table see original document page 44 </column> </row> <table> Example 2

10 groups of C57B1 / 6 mice were used, containing 24 mice per group.

Mice were vaccinated according to the information below in a dose 2 schedule on day 0 and day 28 using intramuscular administration (1 mouse group was either NaCl-initiated or reinforced, not listed below).

Vaccination was performed using different HPV virus equivalent particle combinations.

4 different VLPs were used: HPV 16 Ll Only VLPs, HPV 18 Ll Only VLPs, HPV 31 Ll Only VLPs, and HPV 45 Ll Only VLPs.

VLPs were manufactured and purified essentially in accordance with the disclosure of WO2003077942A2, incorporated herein by reference in its entirety.

In more detail, HPV 16 VLPs were purified using the sequential steps of: anion exchange chromatography (Dimethylaminoethyl - DMAE), anion exchange chromatography (trimethylaminoethyl - TMAE), hydroxyapatite chromatography, filtration and another anion, this period using a Diethylaminoethyl (DEAE) 5 step followed by a final filtration. HPV 31 VLPs were fabricated using the same sequence of steps.

HPV 18 VLPs were purified using the sequential steps of: anion exchange chromatography (Dimethylaminoethyl - DMAE), anion exchange chromatography (trimethylaminoethyl - TMAE), hydroxyapatite chromatography, filtration and an octyl sepharose column (interaction chromatography). hydrophobic), followed by a final filtration. HPV 45 VLPs were fabricated using the same sequence of steps. <table> table see original document page 46 </column> </row> <table> <table> table see original document page 47 </column> </row> <table>

Each antigen was used at a dose of 2 μ§. HPV 16, 18 and 45 VLPS were adsorbed

The adjuvant used was named AS04D, at 1/10 of the human dose, which is 5 μ§ of 3D MPL and 50 μl of aluminum hydroxide in total for each vaccine.

HPV virus equivalent particles were combined with aluminum hydroxide and 3D MPL as disclosed in W000 / 23105.

For example, in the tetravalent vaccine 16, 18, 31, 45, 2 μg of each of the HPV 16 VLPs were adsorbed on 5 μg of aluminum hydroxide. For HPV 18 and HPV 45 the same was done. For HPV 31 VLPS the 2 μg VLP was adsorbed on 2.5 μg aluminum hydroxide.

Separately 5 μg 3D-MPL was adsorbed on 17.5 μg aluminum hydroxide.

3D-MPL and VLPs were then mixed and additional aluminum hydroxide added to the result at 50 μg per vaccine dose.

Vaccination Program:

First Composition Second Composition

16/18 AS04D VLPs 16/18 AS04D VLPs

16/18/31/45 AS04D VLPs 16/18/31/45 AS04D VLPs

NaCl VLPs 31/45 AS04D

Readings

Antibody titers were determined using classical ELISA techniques well known in the art on day 14 after the initial vaccination and the second vaccination (after I and after II respectively).

Intracellular dyeing (ICS, Roederer et al. 2004 Clin. Immunol. 110: 199) was performed at 14 days post II to assess the MIC responses.

Results are given in Figures 1 - 18 below. In these post I and post II figures data are post-dose vaccine I and II measurements respectively. The bar given as "post I reinforcement" reflects the group in which

NaCl was given first, followed by a bivalent or tetravalent vaccine dose on day 28.

Figures 1-9 illustrate antibody responses generated by different vaccination regimens. Figures 10-18 illustrate the MIC responses generated by different vaccination regimens. Conclusions

1- A heterologous booster comprised of HPV 31 and 45 VLPs ll is effective in homologous enhancement of HPV 16 and 18 VLP ll primer induced VLP responses.

2- A heterologous booster comprised of HPV 31 and 45 VLPs ll is effective in heterologous enhancement of KQjV 31 and 45 VLP ll responses induced by the HPV 16 and 18 VLP ll primer. SEQUENCE LISTING

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G1u Cys Ile Being Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly

145 150 155 160

Cys Lys Pro Ile Gly Glu His Trp Gly Lys Gly Pro Cys Thr

165 170 175

Asn Val Val Wing Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn

180 185 190

Thr Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Met Wing

195 200 205

Asp Phe Thr Thr Read Gln Wing Asn Lys Ser Glu Val Pro Read Asp Ile

210 215 220

Cys Thr Be Ile Cys Lys Tyr Pro Aap Tyr Ile Lys Met Val Ser Glu

225 230 235 240

Pro Tyr Gly Asp Being Read Phe Phe Tyr Lèu Arg Arg Glu Gln «et Phe

245 250 255

Val Arg His Leu Phe Asn Arg Wing Gly Wing Val Gly Glu Asn Val Pro

260 265 270

Asp Asp Leu Tyr Ile Lys Gly Ser Gly Be Thr Wing Asn Leu Wing Ser

27S 280 285

Be Asn Tyr Phe Pro Thr Be Gly Be Met Val Thr Be Asp Wing

290 295 300

Gln Ile Phe Asn Lys Pro Tyr Trp Read Gln Arg Wing Glti Gly His Asn

305 310 315 320

Asn Gly Ile Cys Trp Gly Asn Gln Read Phe Val Val Val Val Asp Thr

325 330 335

Thr Arg Be Thr Asn Met Be Read Cys Wing Wing Ile Be Thr Be Glu

340 345 350

Thr Thr Tyr Lys Asn Thr Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu

355 360 365

Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ila Thr Leu Thr

370 375 380

Wing Asp Val Met Thr Tyr Ile His Be Met Asn Be Thr Ile Leu Glu

385 390 395 400

Asp Trp Asn Phe Gly Leu Gln Pro Pro Gly Gly Thr Leu Glu Asp

405 410 415

Thr Tyr Arg Phe Val Thr Be Gln Wing Ile Wing Cys Gln Lys His Thr

420 425 430

Pro Pro Wing Pro Lys Glu Asp Pro Read Lys Lys Tyr Thr Phe Trp Glu

435 440 445

Val Asn Leu Lys Glu Lys Phe Ser Wing Asp Leu Asp Gln Phe Pro Leu

450 455 460

Gly Arg Lys Phe Leu Leu Gln

465 470

<210> 2 <211> 472 <212> PRT

<213> PARTIAL SEQUENCE OF HUMAN PAPILOMAVIRUS hpv 18 <400> 2

Met Wing Read Trp Arg Pro Be Asp Asn Thr Val Tyr Leti Pro Pro

15 10 15

Ser Val Wing Arg Val Val Asn Thr Asp Asp Tyr Val Thr Arg Thr

20 25 30

Ile Phe Tyr His Wing Gly Be Ser Arg Read Leu Thr Val Gly Asn Pro

35 40 45

Tyr Phe Arg Val Pro Wing Gly Gly Gly Asly Lys Gln Asp Ile Pro Lya

SO 55 60

Val Ser Wing Tyr Gln Tyr Arg Val Phe Arg Goes Gln Leu Pro ABp Pro 65 70 75 80

Asn Lys Phe Gly Leu Pro Asp Asn Ser Ile Tyr Asn Pro Glu Thr Gln

85 90 95

Arg Leu Val Trp Cys Wing Val Gly Val Glu Ile Gly Arg Gly Gln Pro

100 105 110

Read Gly Val Gly Read Be Gly Hls Pro Phe Tyr Asn Lys Read Asp Asp

115 120 125

Thr Glu Be Be His Wing Ward Thr Be Asn Val Be Glu Asp Val Arg

130 135 140

Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys lie Leu Gly 145 150 155 160

Cy & Wing Pro Ile Wing Gly Glu His Tirp Wing Lys Gly Thr Wing Cys Lys

165 170 175

Be Arg Pro Read Be Gln Gly Asp Cys Pro Be Read Glu Leu Lys Asn

180 185 190

Thr Val Leu Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Met Wing

195 200 205

Asp Phe Be Thr Read Gln Asp Thr Lys Cys Glu Val Pro Read Asp Ile

210 215 220

Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Read Gln Met Ser Wing Asp 225 230 235 240

Pro Tyr Gly Asp Being Met Phe Phe Cys Leu Arg Arg Glu Gln Leu Phe

245 250 255

Arg Wing His Phe Trp Asn Arg Wing Gly Thr Met Gly Asp Thr Go Pro

260 265 270

Pro Be Read Tyr Ile Lys Gly Thr Gly Met Arg Wing Be Pro Gly Be

275 280 285

Cys Val Tyr Ser Pro Ser Pro Gly Ser Ile Val Thr Ser Asp Ser 290 Gln Leu 305

Asn gly

Thr arg

Pro gly

Glu Glu 370 Thr Wing 385

Glu Asp

Asp Thr

Wing wing

Asn Val 450 Leu Gly 465

<210> 3 <211> 1416 <212> DNA

<213> PARTIAL SEQUENCE OF HUMAN PAPILOMAVIRUS hpv 16

<400> 3

atgtctcttt ggctgcctag tgaggccact gtcfcacttgc ctcctgtccc agtatctaag 60 gttgtaagca cggatgaata tgttgcacgc acaaacatat attatcatgc aggaacatcc 120 agactacttg cagttggaca tccctatttt cctattaaaa aacctaacaa taacaaaata 180 ttagttccta aagtatcagg attacaatac agggtattta oaatacattt acctgacccc 240 aataagtttg gttttcctga cacctcattt tataatccag atacacagcg gctggtttgg 300 gcctgtgtag gtgttgaggt aggtcgtggt cagccattag gtgtgggcat tagtggccat 360 cctttattaa ataaattgga tgacacagaa aatgctagtg cttatgcagc aaatgcaggt 420 gtggataata gagaatgtat atctatggat tacaaacaaa cacaattgtg tttaattggt 480 tgcaaaccac ctatagggga acactggggc aaaggatccc catgtaccaa tgttgcagta 540 aatccaggtg attgtccacc attagagtta ataaacacag ttattcagga tggtgatatg 600 gttgatactg gctttggtgc tatggacttt actacattac aggctaacaa aagtgaagtt 660 ccactggata tttgtacate tatttgcaaa tatccagatt atattaaaat ggtgtcagaa 720 ccatatggcg acagcttatt tttttattta cgaagggaac aaatgtttgt tagacattta 780 tttaataggg ctggtgctgt tggtgaaaat gtaccagacg atttatacat 840 taaaggctct

29S

Phe Asn Lys Pro Tyr 310

will Cys Trp His Asn 325

Ser Thr Asn Leu Thr 340

Gln Tyr Asp Wing Thr 355

Tyr Asp Read Gln Phe 37S

Asp Val Met Ser Tyr 390

Trp Asn Phe Gly Val 405

Tyr Arg Phe Val Gln 420

Pro Wing Glu Asn Lys 435

Asp Leu Lys Glu Lys 455

Arg Lys Phe Leu Val 470

Trp Read His Lys 315

Gln Leu Phe Val 330

Ile Cys Ala Ser 345

Lys Phe Lys Gln 360

Ile Phe Gln Leu

Ile His Ser Met 395

Pro Pro Pro Pro 410

Ser Vai Ala Ile 425

ASp Pro Tyr Asp 440

Phe Ser Leu Asp Gln

300

Glrt Wing Gly His Asn 320

Thr Val Val Asp Thr 335

Thr Gln Ser Pro Val 350

Tyr Ser Arg His VaI 365

Cys Thr Ile Thr Leu 380

Asn Ser Ser Ile Leu 400

Thr Thr Be Leu Val 4X5

Thr Cys Gln Lys Asp 430

Lys Leu Lys Phe Trp 445

Leu Asp Gln Tyr Pro 460 gggtctactg caaatttagc cagttcaaat tattttcctá cacctagtgg ttctatggtt 900 acctctgatg cccaaatatt caataaacct tattggttac aacgagcaca gggccacaat 960 aatggcattt gttggggtaa ccaactattt gttactgttg ttgatactac acgcagtaca 1020 aatatgtcat tatgtgctgc catatctact tcagaaacta catataaaaa tactaacttt 1080 aaggagtacc tacgacatgg ggaggaatat gatttacagt ttatttttca actgtgcaaa 1140 ataaccttaa ctgcagacgt tatgacacac atacattcta tgaattccac tattttggag 1200 gactggaatt ttggtctaca acctccccca gtaggcacac tagaagatac ttagaggttt 1260 gtaacatccc aggcaattgc ttgtcaaaaa catacacctc cagcacctaa agaagatccc 1320 cttaaaaaat acactttttgggaagtaaat ttaaaggagatttt8080

<210> 4 <211> 1419 <212> DNA

<213> PARTIAL SEQUENCE OF HUMAN PAPILOMAVIRUS hpv 18 <400> 4

atggctttgt ggcggcctag tgacaatacc gtatatcttc cacctecttc Cgtggcaaga 60 gttgtaaata ccgatgatta tgtgactcgc acaagcatat tttatcatgc tggcagctct 120 agattattaa ctgttggtaa tccatatttt agggttcctg caggtggtgg caataagcag 180 gatattccta aggtttctgc ataccaatat agagtattta gggtgcagtt acctgaccca 240 aataaafcttg gtttacctga taatagtatt tataatcctg aaacacaacg tttagtgtgg 300 gagtggaaat tggcegtggt cagcctttag gcctgtgttg gtgttggcct tagtgggcat 360 ccattttata ataaattaga tgacactgaa agttcccatg ccgccacgtc taatgtttct 420 gaggacgtta gggacaatgt gtctgtagat tataagcaga cacagttatg tattttgggc 480 tgtgcccctg ccattgggga acactgggct aaaggcactg cttgtaaatc gcgtccttta 540 tcacagggcg attgcccccc ttbagaactt aaaaacacag ttttggaaga tggtgatatg 600 gtagatactg gatatggtgc catggacttt agtacattgc aagatactaa atgtgaggta 660 ccattggata tttgtcagtc tatttgtaaa tatcctgatt atttacaaat gtctgcagat 720 ccttatgggg attccatgtt tttttgctta cggcgtgagc agctttttgc taggcatttt 780 tggaataggg caggtactat gggtgacact gtgcctccat ccttatatat taaaggcaca 840 ggtatgcgtg cttcacctg g cagctgtgtg tattctccct ctccaagtgg ctctattgtt 900 acctctgact cccagttgtt taataaacca tattggttac ataaggcaca gggtcataac 960 aatggtgttt gctggcataa tcaattattt gttactgtgg tagataccac tcgcagtacc 1020 aatttaacaa tatgtgcttc tacacagtct cctgtacctg ggcaatatga tgctaccaaa 1080 tttaagcagc atageagaca tgttgaggaa tatgatttgc agtttatttt tcagttgtgt 1140 actactactt taactgcaga tgttacgtcc Catattcata gtatgaatag cagtatttta 1200 gaggattgga actttggtgt tccccccccg ccaactacta gtttggtgga taçatatcgt 1260 tttgtacaat ctgttgctat tacctgtcaa aaggatgctg caccggctga aaataaggat 1320 ccctatgata agttaaagtt ttggaatgtg gatttaaagg aaaagttttc tttagactta 1380 gatcaatatc cccttggacg taaatttttg gttcagtaa 1419

<210> 5 <211> 1419 <212> DNA

<213> Sequence PARTIAL Ll of human papillomavirus HPV 31 <400> 5 atgtctctgt ggcggcctag cgaggctact gtctacttac cacctgtece agtgtctaaa 60 gttgtaagca cggatgaata tgtaacacga accaacatat attatcacgc aggcagtgct 120 aggccgctta cagtaggcca tccatattat tccataccta aatctgacaa tcctaaaaaa 180 atagttgtac caaaggtgtc aggattacaa tatagggtat ttagggttcg tttaccagat 240 ccaaacaaat ttggatttcc tgatacatct ttttataatc ctgaaactca 300 acgcttagtt tgggcctgtg ttggtttaga ggtaggtcgc gggcagccat taggtgtagg tattagtggt 360 catccattat taaataaatt tgatgacact gaaaactcta atagatatgc cggtggtcct 420 ggcactgata atagggaatg tatatcaatg gattataaac aaacacaact gtgtttactt 480 ggttgcaaac cacctattgg agagcattgg ggtaaaggta gtccttgtag taacaatgct 540 attacecctg gtgattgtcc tccactagaa ttaaaaaatt cagttataca agatggggat 600 atggctgata caggctttgg agçtatggat tttactgctt tacaagacac taaaagtaat 660 gttcctttgg acatttgtaa ttctatttgt aaatatccag attatcttaa aatggttgce 720 gagccatatg gcgatacatt atttttttat ttacgcaggg aacaaatgtt tgtaaggcac 780 ttetttaata gatcag GCAC ggttggtgaa tcggtcccta ctgacttata tattaaaggc 840 tccggttcaa cagctacttt agctaacagt acatactttc ctacacctag cggctccatg 900 gttacttcag atgcacaaat tttcaataaa ccatattgga tgcaacgtgc tcagggacac 960 aataatggta tttgttgggg caatcagtta tttgttactg tggtagacac cacacgtagt 1020 accaatatgt ctgtttgtgc tgcaattgca aacagtgata ctacatttaa aagtagtaat atttaagaca tggtgaggaa tttaaagagt 1080 tttgatttac aatttatatt tcagttatgc 1140 aaaataacat tatctgcaga cataatgaca tatattcaca gtatgaatcc tgctattttg 1200 gaagattgga attttggatt gaccacacct ccctcaggtt ctttggagga tacetatagg 1260 tttgtcacct cacaggccat tacatgtcaa aaaactgccc cccaaaagcc caaggaagat 1320 ccatttaaag attatgtatt ttgggaggtt aatttaaaag aaaagttgtgtagtagtagtagtagtagggg

<210> 6

<211> 1428

<212> DNA

<213> HPV45 HUMAN LAPTOP PARTIAL SEQUENCE ll

<400> 6

atggctttgt ggcggcctag cgacagtacg gtatatcttc caccaccttc tgtggccaga 60 gttgtcaaca ctgatgatta tgtgtcccgc acaagcatat tttaccatgc aggcagttcc 120 cgattattaa ctgtaggcaa tccacatttt agggttgtac Ctagtggtgc aggtaataaa 180 caggctgttc ctaaggtatc cgcatatcag tatagggtgt ttagagtagc tttacccgat 240 cctaataaat ttggattacc tgattctact atatataatc ctgaaacaca acgtttggtt 300 tgggcatgtg taggtatgga aattggtcgt gggcagcctt caggtattgg cctaagtggc 360 catccatttt ataataaatt ggatgataca gaaagtgctc atgcggctac ggctgttgtt 420 acgcaggatg ttagggataa tgtgtcagtt gattataagc aaacacagct gtgtatttta 480 ggttgtgtac ctgctattgg tgagcactgg gccaagggca cactttgtaa acctgcacaa 540 tcgcagcctg gtgactgtcc tcctttggaa cttaaaaaca ccattattga ggatggtgat 600 atggtggata caggttatgg ggcaacggat tttagtacat cgcaggatac aaagtgcgag 660 gttccatcag acatttgtca atccatctgt gatccctatg gggattctat gtttttttgc ttttggaata gggcaggtgt tatgggtgac actagcgcta atatgcgtga aacccctggc tctattacta cttctgattc tcaattattt ggccataaca atggtacttg ttggcataat cgcagtacta atttaacatt atgtgcc tct cctactaagt ttaagcacta tagtagacat cagttgcgca ctattacttt aactgcagag agtatattgg aaaattggaa ttttggtgta acataccgtt ttgtgcaatc agttgctgtt aagcaggatc catatgataa attagatgatg cctact

aaatatceag attatttgca aatgtctgct 720 ctacgccgtg aacaactgtt tgcaagacat 780 acagtaccta cagacctata tattaaaggc 840 agetgtgtgt attccccttc tcccagtggc 900 aataagccat attggttaca taaggcccag 960 cagttgtttg ttactgtagt ggacactacc 1020 acacaaaatc ctgtgccagg tacatatgat 1080 gtggaggaat atgatttaca gtttattttt 1140 gttatgtcat atatccatag tatgaatagt 1200 cctccaccac ctactacaag tttagtggat 1260 acctgtcaaa aggatactac acctccagaa 1320 tggactgttg acctaaagga aaaattttcc 1380 aagtttttag ttcagtaa 1428

Claims (30)

1. Use of a human papillomavirus L1 protein or immunogenic fragment thereof from a first HPV type, characterized in that it is in the preparation of a medicament for enhancing an immune response previously evoked by a human papillomavirus L1 protein or immunogenic fragment of same from a different HPV type. Use according to claim 1, characterized in that the second type is phylogenetically related to the first type. Use according to claim 1 or 2 of an HPV 31 or HPV 52 L1 protein or immunogenic fragment thereof, characterized in that it is in the preparation of a medicament for enhancing an immune response to an HPV 16 vaccine comprising an L1 protein or fragment thereof. Use according to claim 3 of an HPV 31 protein or immunogenic fragment thereof, wherein it is in the preparation of a medicament for enhancing an immune response to an HPV 16 vaccine comprising an L1 protein or fragment thereof. same. Use according to claim 3 of an HPV 52 protein or immunogenic fragment thereof, characterized in that it is in the preparation of a medicament for enhancing an immune response to an HPV 16 vaccine comprising an L1 protein or fragment thereof. same. Use according to claim 1 or 2 of an HPV 16 L1 protein or immunogenic fragment thereof, wherein it is in the preparation of a medicament for enhancing an immune response to an HPV 31 or HPV 52 vaccine comprising an L1 protein or fragment thereof. Use according to claim 6 of an HPV 16 L1 protein or immunogenic fragment thereof, characterized in that it is in the preparation of a medicament for enhancing an immune response to an HPV 31 vaccine comprising an L1 protein or fragment. of the same. Use according to claim 6 of an HPV 16 ll protein or immunogenic fragment thereof, wherein it is in the preparation of a medicament for enhancing an immune response to an HPV 52 vaccine comprising a ll protein or fragment of the same. Use according to claim 1 or 2 of an HPV 45 L1 Protein or immunogenic fragment thereof, wherein it is in the preparation of a medicament for enhancing an immune response to an HPV 18 vaccine comprising a L1 protein. or fragment thereof. Use according to claim 1 or 2 of an HPV 18 L1 protein or immunogenic fragment thereof, wherein it is in the preparation of a medicament for enhancing an immune response to an HPV 45 vaccine comprising an L1 protein. or fragment thereof. Vaccination program for the protection against HPV infection and / or disease, characterized in that it comprises the release of a first HPV vaccine comprising an L1 protein or immunogenic fragment thereof of at least HPV 16 and HPV 18 and a second HPV vaccine that does not comprise the HPV 16 and HPV 18 Ll components of the first vaccine and the second vaccine comprises an ll protein or immunogenic fragment thereof of at least one other type of oncogenic HPV, wherein the first and second Vaccines can be released in any order and the release is separated by an appropriate interval. Method for the prevention of HPV infection and / or disease, characterized in that it comprises the release of a first HPV vaccine comprising an L1 protein or immunogenic fragment thereof of at least HPV 16 and HPV 18 and a second HPV vaccine that does not comprise HPV 16 and HPV 18 L1 components of the first vaccine and that the second vaccine comprises an L1 protein or immunogenic fragment thereof from at least one other type of oncogenic HPV, wherein the first and second vaccines may be released in any order and the release is separated by an appropriate time interval. Method or program according to claim 11 or 12, characterized in that the first vaccine comprises protein L1 or immunogenic fragment thereof of HPV 16, HPV 18 and optionally HPV 33. Method or program according to claim 13, characterized in that the first vaccine further comprises L1 protein or immunogenic fragment thereof of HPV 58. Method or program according to any one of claims 11 to 13, characterized in that the second vaccine comprises an HPV-31 and / or HPV 45 and / or HPV 52 protein or immunogenic fragment thereof. The method or program of claim 15, wherein the second vaccine comprises an L1 protein or immunogenic fragment thereof of HPV 31 and HPV 45. Method or program according to claim 16, characterized in that the second vaccine comprises an L1 protein or immunogenic fragment thereof of HPV 31, HPV 45 and HPV 52. A method or program according to any one of claims 11 to 17, characterized in that the first and second vaccines have no identical L1 protein or protein fragments in common. Method or program according to any one of claims 11 to 18, characterized in that the HPV 16 or HPV 18 component in the first vaccine protects against HPV infection and / or disease caused by at least one type of HPV in the second vaccine. 20. A vaccine composition, characterized in that it comprises a combination of an HPV 31 ll protein or immunogenic fragment thereof and an HPV 45 protein or immunogenic fragment thereof, the vaccine not containing HPV 16 protein ll or HPV 18 or immunogenic fragments thereof. Vaccine composition according to claim 20, characterized in that it includes an HPV 52 L1 protein or immunogenic fragment thereof. Kit, characterized in that it comprises a first and second vaccine composition as defined in claim 11. Kit according to claim 22, characterized in that the first vaccine component comprises the HPV 16 and HPV 18 proteins, or immunogenic fragments thereof, and the second vaccine component comprises the HPV 31 and HPV proteins. 45, or immunogenic fragments thereof. Use, method, program, vaccine or kit according to any one of the preceding claims, characterized in that the HPV L1 protein is in the form of a virus equivalent particle. Use, method, program, vaccine or kit according to any one of the preceding claims, characterized in that the first or second vaccine or both comprises an adjuvant. Use, method, program, vaccine or kit according to claim 25, characterized in that the adjuvant comprises 3D-MPL. Use, method, program, vaccine or kit according to claim 25, characterized in that the adjuvant comprises an aluminum salt. Use, method, program, vaccine or kit according to claim 26 or 27, characterized in that the adjuvant comprises an aluminum salt and 3D MPL. Use, method, program, vaccine or kit according to claim 26, characterized in that the adjuvant comprises an oil-in-water emulsion adjuvant and 3D-MPL. Use, method, program, vaccine or kit according to claim 29, characterized in that the oil-in-water emulsion comprises a metabolizable oil, a sterol and an emulsifying agent.