JP2015514696A - Vaccination method against human papillomavirus - Google Patents

Abstract

The present disclosure includes an immunogenic composition comprising HPV VLPs derived from one or more HPV types in combination with an adjuvant comprising a TLR agonist for use in a method for the prevention of HPV infection or disease in an individual. In the method, a first dose of an immunogenic composition comprising HPV VLP and a TLR agonist is administered, followed by an HPV VLP derived from one or more HPV types, but with a TLR. A second dose of the immunogenic composition containing no agonist is administered. [Selection figure] None

Description

BACKGROUND This disclosure relates to the field of human vaccines. More specifically, the present disclosure relates to pharmaceutical and immunogenic compositions for the prevention or treatment of human papillomavirus (HPV) infection or disease, and methods for vaccination against HPV infection or disease. Is.

  Papillomavirus is a small and highly species-specific DNA tumor virus. Human papillomavirus is a DNA virus that infects basal epithelial (skin or mucosa) cells. Over 100 distinct human papillomavirus (HPV) genotypes have been described. HPV is generally specific to the squamous epithelium of the skin (e.g. HPV-1 and -2) or specific to the mucosal surface (e.g. HPV-6 and -11) and usually for months Or cause a benign tumor (warts) that lasts for several years.

  Persistent infection with oncogenic human papillomavirus (HPV) is a necessary cause of cervical cancer, which is the second leading cause of cancer death in women worldwide. "High risk" genotypes, such as genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 73, can lead to cervical cancer There is also an international consensus that it is associated with other mucosal anogenital and head and neck cancers. Globally, HPV-16 and HPV-18 are the major carcinogenic forms and cumulatively account for over 70-80% of all invasive cervical cancer cases.

  Infections with other genotypes called “low risk” include benign or low-grade cervical tissue changes, as well as cervical, vagina, vulva and anus in women, and penis, scrotum or Can cause genital warts (cuspid condyloma), a tumor that occurs in the anus. They also cause pediatric and adult vocal cord epithelial tumors (juvenile respiratory papillomatosis or recurrent respiratory papillomatosis) that require surgical intervention.

  Two prophylactic HPV vaccines have recently been approved in many countries. Both utilize HPV-16 and -18 cervical precancerous lesions and virus-like particles (VLPs) consisting of individual HPV-type recombinant L1 capsid proteins to prevent cancer. CervarixTM (GlaxoSmithKline Biologicals) contains HPV-16 and -18 VLPs produced using the baculovirus expression vector system in the Trichoplusia ni insect cell substrate, and the immunostimulator 3- Formulated with O-desacyl-4'-monophosphoryl lipid A (3D MPL, also known as MPL) and aluminum hydroxide salt. Gardasil ™ (Merck) contains HPV-16 and -18 VLPs produced in the yeast Saccharomyces cerevisiae and is formulated with amorphous aluminum hydroxyphosphate sulfate. In addition, Gardasil ™ also contains VLPs derived from the non-carcinogenic types HPV-6 and -11 that are responsible for 75-90% of genital warts. For both vaccines, specific protection against infection with carcinogenic types HPV-16 and HPV-18 and related precancerous lesions has been demonstrated in randomized clinical trials.

  The list of oncogenic HPV types that cause cervical cancer includes at least HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 found in cervical cancer. 66, 68 and 73 (Mahdavi et al., 2005; Quint et al., 2006).

  Existing vaccines can provide specific protection to varying degrees against infection and / or disease by some of these HPV types. For example, Cervarix ™ provides a cross-protective effect against HPV types 33, 31, 45 and 51. HPV-16 / 18 and these four types cause about 85% of cervical cancers; furthermore, HPV-33 infection has a particularly high risk of progression to cervical lesions, and HPV-45 is associated with adenocarcinoma It accounts for a large proportion (Wheeler et al., 2012). However, it is potentially beneficial to provide the high degree of protection achieved by Cervarix ™ against cervical cancer, and also to provide some protection against infections and diseases caused by other HPV types Will. Providing a high degree of protection against cervical cancer and, in addition, providing an improved protection against genital warts caused by HPV-6 and HPV-11 over that provided by existing vaccines Would be potentially beneficial.

  It has now been found that certain benefits are obtained by administering one or more doses of HPV vaccine with adjuvant MPL in a vaccination scheme using another HPV vaccine that does not contain an MPL adjuvant. For example, the immune response against a particular HPV type (eg, HVP 18) present in a vaccine can be increased compared to a vaccination scheme using only an aluminum adjuvant. This is especially seen when the MPL-containing vaccine is first administered, but is not exclusive. Alternatively, or in addition, a cross-reactive immune response against a specific HPV type that is not present in the MPL adjuvanted vaccine but present in the aluminum adjuvanted vaccine is administered with the MPL-containing vaccine first followed by the addition of aluminum adjuvant. Administration of the vaccine can be equivalent or increased compared to vaccination using only the aluminum adjuvanted vaccine.

Overview The present disclosure relates to the use of TLR agonist-containing HPV vaccines to enhance vaccination against HPV. The present disclosure further relates to the use of various HPV vaccines, including TLR agonist-containing vaccines, in a particular order in a vaccination scheme. Specifically, this disclosure relates to improving response to certain HPV types through the use of TLR agonist-containing HPV vaccines in vaccination schemes using non-TLR agonist-containing HPV vaccines. The present disclosure further provides a priming vaccine that induces a cross-reactive immune response against one or more HPV types that are not present in the priming vaccine, followed by a cross-reactive response that is not present in the priming vaccine and is caused by the priming vaccine. It relates to a vaccination scheme using a boosting vaccine containing one or more induced HPV types. The immune response to the non-existing HPV type is enhanced by booster vaccines to a level that is at least equal to or higher than the immune response induced only by the equivalent number of dose booster vaccines. The use of different priming and booster vaccines also allows the use of different vaccines in one vaccination schedule.

In one embodiment, the present invention comprises an HPV VLP derived from one or more HPV types in combination with an adjuvant comprising a TLR agonist for use in a method for the prevention of HPV infection or disease in an individual. A first immunogenic composition is provided, the method comprising:
(i) administering to the individual at least one dose of the first immunogenic composition;
(ii) administering to said individual at least one dose of said second immunogenic composition comprising HPV VLPs derived from one or more HPV types and no TLR agonist;
Wherein the first immunogenic composition is present in the second immunogenic composition and is not present in the first immunogenic composition, or a type-specific immune response against HPV type or It increases at least one of the cross-reactive immune responses.

In a further aspect, the invention provides an HPV VLP derived from at least one HPV type for use in a method for the prevention of HPV infection or disease in an individual comprising an aluminum salt but no TLR4 agonist An immunogenic composition comprising in combination with a method comprising:
(i) administering to the individual at least one dose of a first immunogenic composition comprising HPV VLPs derived from one or more HPV types in combination with an adjuvant comprising a TLR agonist; and
(ii) administering to said individual at least one dose of a second immunogenic composition that is an immunogenic composition comprising HPV VLP in combination with an aluminum salt rather than a TLR4 agonist;
Wherein the first immunogenic composition is present in the second immunogenic composition and is not present in the first immunogenic composition, or a type-specific immune response against HPV type or It increases at least one of the cross-reactive immune responses.

In another aspect, the invention provides a method for the prevention of HPV infection or disease in an individual comprising:
(i) administering to the individual at least one dose of a first immunogenic composition comprising HPV VLPs derived from one or more HPV types in combination with an adjuvant comprising a TLR agonist; and
(ii) administering to said individual at least one dose of said second immunogenic composition comprising HPV VLPs derived from one or more HPV types and no TLR agonist;
The first immunogenic composition comprises a type-specific immune response or crossover against a type that is present in the second immunogenic composition and not present in the first immunogenic composition. Provided is the above method, which increases at least one of the reactive immune responses.

In another aspect, the present invention provides the following:
(i) a first immunogenic composition comprising VLPs derived from at least one HPV type in combination with an adjuvant comprising a TLR agonist; and
(ii) providing a kit comprising a second immunogenic composition comprising a VLP derived from at least one HPV type and not comprising a TLR agonist;

  In another aspect, the present invention provides a method of inducing antibodies against HPV in a human comprising administering to the human the first and second immunogenic compositions described herein.

  In another aspect, the present invention provides a method of inducing neutralizing antibodies against HPV in a human comprising administering to the human the first and second immunogenic compositions described herein. To do. It is also possible to induce cross-neutralizing antibodies by such a method.

  In another aspect, the invention provides a method for inducing cellular immunity against HPV in a human comprising administering to the human the first and second immunogenic compositions described herein. To do.

  In another aspect, the invention induces neutralizing antibodies and cellular immunity against HPV in a human comprising administering to the human the first and second immunogenic compositions described herein. Provide a way to do it. It is also possible to induce cross-neutralizing antibodies by such a method.

  In a further aspect, the present disclosure provides a first comprising HPV VLPs derived from one or more HPV types in combination with an adjuvant comprising a TLR agonist for use in a method for enhancing prevention of HPV infection or disease. With respect to an immunogenic composition, wherein the method comprises one or more doses of the immunogenic composition comprising HPV VLPs derived from one or more HPV types but no TLR agonist 1 or Administration of a multi-dose second immunogenic composition to an already administered individual.

BRIEF DESCRIPTION OF THE FIGURES FIGS . 1-20 and 22-33 are measured by ELISA and mock virus neutralization assay, respectively, in mice after immunization with various vaccination schemes using Cervarix ™ and Gardasil ™ 2 shows the total and neutralizing antibody response in mice. These are the results of three independent experiments. The data of Example 1 was grouped as FIGS. 1-16, the data of Example 2 as FIGS. 17-20, and the data of Example 3 as FIGS. 22-33. FIG. 21 shows the results of the protection assay forming part of Example 2, and FIGS. 34 to 38 show the results of the protection assay forming part of Example 3.

  Further details are as follows.

FIG. 1 shows the total anti-HPV 16 L1 VLP antibody response. FIG. 2 outlines the statistical analysis for the total anti-HPV-16 response. FIG. 3 shows the neutralizing anti-HPV-16 L1 VLP antibody response. FIG. 4 outlines the statistical analysis for neutralizing anti-HPV 16 response. FIG. 5 shows the total anti-HPV 18 L1 VLP antibody response. FIG. 6 outlines the statistical analysis for the total anti-HPV-18 response. FIG. 7 shows the neutralizing anti-HPV-18 L1 VLP antibody response. FIG. 8 outlines the statistical analysis for neutralizing anti-HPV 18 response. FIG. 9 shows the total anti-HPV-6 L1 VLP antibody response. FIG. 10 shows an overview of the statistical analysis for total anti-HPV6 antibody response. FIG. 11 shows the neutralizing anti-HPV-6 L1 VLP antibody response. FIG. 12 outlines the statistical analysis for neutralizing anti-HPV6 antibody response. FIG. 13 shows the total anti-HPV-11 L1 VLP antibody response. FIG. 14 outlines the statistical analysis for total anti-HPV11 antibody response. FIG. 15 shows the neutralizing anti-HPV-11 L1 VLP antibody response. FIG. 16 shows an overview of statistical analysis for neutralizing anti-HPV11 antibody responses. FIG. 17 shows the total anti-HPV-18 antibody response (Example 2). FIG. 18 shows the neutralizing anti-HPV-18 antibody response (Example 2). FIG. 19 shows the total anti-HPV-11 antibody response (Example 2). FIG. 20 shows the neutralizing anti-HPV-11 antibody response (Example 2). FIG. 21 shows the comparative protection rate and bioluminescence signal at 1 month after II in mice after the intravaginal challenge experiment of Example 2. FIG. 22 shows total anti-HPV-18 L1 VLP antibody with 1M PIII (Example 3). FIG. 23 shows total anti-HPV-18 L1 VLP antibody (Example 3) with 6M PIII. FIG. 24 shows the neutralizing anti-HPV-18 L1 VLP antibody with 1M PIII (Example 3). FIG. 25 shows neutralizing anti-HPV-18 L1 VLP antibody with 6M PIII (Example 3). FIG. 26 shows total anti-HPV-6 L1 VLP antibody (Example 3) with 1M PIII. FIG. 27 shows total anti-HPV-6 L1 VLP antibody with 6M PIII (Example 3). FIG. 28 shows neutralizing anti-HPV-6 L1 VLP antibody (Example 3) with 1M PIII. FIG. 29 shows neutralizing anti-HPV-6 L1 VLP antibody (Example 3) with 6M PIII. FIG. 30 shows total anti-HPV-11 L1 VLP antibody (Example 3) with 1M PIII. FIG. 31 shows total anti-HPV-11 L1 VLP antibody (Example 3) with 6M PIII. FIG. 32 shows neutralizing anti-HPV-11 L1 VLP antibody (Example 3) with 1M PIII. FIG. 33 shows a neutralizing anti-HPV-11 L1 VLP antibody (Example 3) with 6M PIII. FIG. 34 shows the comparative protection rate and bioluminescence signal (radiance, Ph / Sec / cm ² ) (Example 3) at 6M after III. FIG. 35 shows the comparative protection rate after 1M and the bioluminescence signal (radiance, Ph / Sec / cm ² ) (Example 3). FIG. 36 shows the comparative protection rate and bioluminescence signal (radiance, Ph / Sec / cm ² ) (Example 3) at 6M after III. FIG. 37 shows the comparative protection rate and bioluminescence signal (radiance, Ph / Sec / cm ² ) at 1M after III (Example 3). FIG. 38 shows the comparative protection rate and bioluminescence signal (radiance, Ph / Sec / cm ² ) at 6M after III (Example 3).

DETAILED DESCRIPTION The present invention relates to non-HPV types that are present in vaccines, particularly HPV types that are at high risk for cervical cancer or low-risk HPV types that cause genital warts. This is the first description of the use of a TLR agonist-containing HPV vaccine in an individual who is also administered a TLR agonist-containing HPV vaccine. The present invention further describes the use of TLR agonist-containing HPV vaccines to elicit a cross-reactive immune response against HPV type administered in the form of a second non-TLR agonist-containing vaccine. More specifically, the present invention describes a method for the prevention of HPV-related diseases or infections by administering different priming vaccines and booster vaccines, wherein the priming vaccine comprises the priming vaccine It induces an immune response against the HPV type that is not present in the vaccine but is present in the booster vaccine. The present invention relates to one vaccine schedule without reducing the immune response against HPV types not present in one of the vaccines, and more importantly, while improving the immune response against a particular HPV type, It offers the possibility of using a vaccine instead of another.

  In certain embodiments, the first immunogenic composition comprises HPV 16 and / or HPV 18 VLP. In certain embodiments, the first immunogenic composition comprises only HPV 16 and HPV 18 VLPs and no other HPV VLPs.

  In certain embodiments, the first immunogenic composition increases a type-specific immune response against HPV 16 or HPV 18, or both HPV 16 and HPV 18.

  An increase in type-specific immune response is an immune response to a particular HPV type when only a second dose of a second immunogenic composition (i.e., a composition not using TLR as an adjuvant) is administered. There may be an increase in immune response when compared.

  In certain embodiments, the first immunogenic composition elicits a cross-reactive immune response against one or more high-risk or low-risk HPV types present in the second immunogenic composition.

  The so-called “high risk” HPV types responsible for cervical cancer are genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 73 It will be appreciated that as more HPV types are discovered, they can be added to this list over time. So-called `` low risk '' mucosal HPV types are at low risk of causing cancer (e.g. HPV 6 and 11 causing genital warts), types associated with common warts (e.g. HPV 2 and 3) , As well as HPV 76 associated with benign skin warts. In one embodiment, the low risk HPV type present in the composition used in the present invention is HPV 6 or HPV 11 or HPV 6 and HPV 11.

  In certain embodiments, the first immunogenic composition has a cross-reactive immune response to a type that is present in the second immunogenic composition and not present in the first immunogenic composition, Increased compared to the immune response to that type when only an equivalent number of doses of the second immunogenic composition is administered.

  The immune response elicited against a particular HPV type is described in an appropriate assay for a specific antibody against that HPV type, such as the Examples herein, or Harper et al 2004, Dessy et al 2008 or Pastrana et al 2004. As measured by ELISA and / or pseudo-neutralization assays.

  In certain embodiments, the second immunogenic composition comprises HPV 6, HPV 11, HPV 16 and HPV 18 VLP, with or without additional HPV VLPs. Such additional HPV types include additional high-risk oncogenic HPV types (e.g., one or more of HPV 31, HPV 33, HPV 45, HPV 52, and HPV 58). May exist in various combinations. In certain embodiments, HPV 6, 11, 16, 18, 31, 33, 45, 52 and 58 VLPs are present in the second immunogenic composition in the form of a 9-valent HPV vaccine.

  As used herein, a priming composition is an immunogenic composition that is administered prior to a booster composition.

  Similarly, a booster composition is an immunogenic composition that is administered after a priming composition.

  The priming and booster compositions described herein are immunogenic compositions, i.e., they can elicit specific immune responses against pathogens such as human papillomavirus (e.g., in an experimental setting). A composition suitable for administration to a human or animal subject. Accordingly, an immunogenic composition comprises one or more antigens (eg, viral antigenic subunits, eg, polypeptides thereof) or antigenic epitopes. An immunogenic composition can also include one or more additional components (eg, excipients, carriers, and / or adjuvants) that can elicit or enhance an immune response. In certain cases, the immunogenic composition is administered to elicit an immune response that protects the subject from symptoms or symptoms induced by the pathogen. In some cases, a symptom or disease caused by a pathogen is prevented (or treated) by inhibiting replication of the pathogen (e.g., human papillomavirus) after exposure of the subject to the pathogen, e.g., Reduced or improved). For example, in the context of the present disclosure, immunogenicity intended for administration to a subject or population of subjects intended to elicit a protective or palliative immune response against human papillomavirus A particular embodiment of the composition is a vaccine composition or vaccine.

  The term “vaccine” refers to a composition comprising an immunogenic component capable of eliciting an immune response in an individual, such as a human, optionally containing an adjuvant. A vaccine for HPV is suitably a protective immune response against accidental or persistent infections caused by one or more HPV types, or cytological abnormalities such as ASCUS, CIN1, CIN2, CIN3, or cervical cancer To trigger.

  The dosage of the immunogenic composition described herein can be a human dose. The term “human dose” means a dose having a volume suitable for human use. A human dose includes an amount of antigen suitable for eliciting an immune response in humans. Generally, the human dose volume is a liquid with a volume of 0.3-1.5 mL. In certain embodiments, the human dose is 0.5 mL. In further embodiments, the human dose is greater than 0.5 mL, for example 0.6, 0.7, 0.8, 0.9 or 1 mL. In a further embodiment, the human dose is between 1 mL and 1.5 mL.

  The immune response elicited by one HPV type against another HPV type is a cross-reactive immune response. The presence or absence of a cross-reactive immune response as described herein is detected and measured by any suitable assay for measuring specific antibodies against related HPV types, particularly related HPV type VLPs. be able to. Methods for screening antibodies are well known in the art. By using an ELISA, such as the ELISA described in the Examples herein, the cross-reactivity of antibodies can be assessed. A suitable ELISA is also described in Harper et al., 2004 (see webappendix). The cross-reactive response can be cross-neutralization, and the antibody can be neutralized and cross-linked using an appropriate assay such as, for example, a pseudovirus neutralization assay as described in the Examples herein. Can be tested for neutralizing properties. Suitable pseudovirus neutralization assays are described in Dessy et al., 2008 and Pastrana et al., 2004.

  The first and second immunogenic compositions described herein typically comprise at least one pharmaceutically acceptable diluent or carrier and optionally (second immunogenicity). With respect to the composition) an adjuvant is also included.

  An “adjuvant” is a substance that enhances the generation of an immune response in a non-specific manner. Common adjuvants include suspensions of minerals to which the antigen is adsorbed (alum, aluminum hydroxide, aluminum phosphate); emulsions such as water-in-oil and oil-in-water emulsions (and variations thereof such as multiphase emulsions) And reversible emulsions), liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids (e.g., CpG oligonucleotides), liposomes, Toll-like receptor agonists (specifically, TLR2, TLR4, TLR7 / 8 and TLR9 agonists), as well as various combinations of such components.

  In certain embodiments, a VLP in either or both of the first or second immunogenic compositions is used in combination with aluminum, and the VLP is used with an aluminum adjuvant, such as aluminum hydroxide or amorphous aluminum. It can be adsorbed or partially adsorbed on hydroxyphosphate sulfate.

  In certain embodiments, the TLR agonist in the first immunogenic composition is a non-toxic derivative of lipid A, such as monophosphoryl lipid A, or more specifically 3-O-desacyl-4′-mono. Phosphoryl lipid A (3D-MPL), or QS21. In some embodiments, MPL is used in combination with aluminum hydroxide.

  In certain embodiments, the second immunogenic composition comprises an aluminum salt, such as amorphous aluminum hydroxyphosphate sulfate.

  If the VLP is adsorbed to an aluminum-containing adjuvant, the VLP can be adsorbed to the aluminum adjuvant prior to mixing the VLP to make the final vaccine product.

  Thus, in some embodiments, the priming composition includes an aluminum salt. VLPs may be adsorbed on aluminum salts or partially adsorbed. In certain embodiments, the adjuvant is aluminum hydroxide and 3D MPL. Compositions comprising such adjuvants according to the present disclosure can be prepared as described, for example, in WO 00/23105, which is incorporated herein by reference.

  In certain embodiments, the second immunogenic composition comprises an aluminum salt. VLPs can be adsorbed on aluminum salts or partially adsorbed. In certain embodiments, the aluminum salt is amorphous aluminum hydroxyphosphate sulfate.

  In certain embodiments, the first immunogenic composition comprises aluminum hydroxide and 3D MPL, and the second immunogenic composition comprises amorphous aluminum hydroxyphosphate sulfate.

  In certain embodiments, the TLR agonist for use with the HPV antigen in the first immunogenic composition described herein is a non-toxic bacterial lipopolysaccharide derivative. One example of a suitable non-toxic derivative of lipid A is monophosphoryl lipid A, or more specifically 3-deacylated monophosphoryl lipid A (3D-MPL), as already described. 3D-MPL is sold under the name MPL by GlaxoSmithKline Biologicals N.A. and shall be referred to as MPL or 3D-MPL throughout this specification. See, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL primarily promotes CD4 + T cell responses with an IFN-γ (Th1) phenotype. 3D-MPL can be manufactured according to the method disclosed in GB2220211 A. Chemically, 3D-MPL is a mixture of 3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. In the composition of the present invention, small particle 3D-MPL can be used. Small particle 3D-MPL has a particle size such that it can be sterile filtered through a 0.22 μm filter. Such preparations are described in WO94 / 21292.

In other embodiments, the lipopolysaccharide can be a β (1-6) glucosamine disaccharide as described in US Pat. No. 6,005,099 and European Patent No. 0 729 473 B1. Those skilled in the art will be able to readily produce various lipopolysaccharides (eg, 3D-MPL) based on the teachings of these references. In addition to the aforementioned immunostimulants (whose structure is similar to that of LPS or MPL or 3D-MPL), acylated monosaccharide and disaccharide derivatives that are sub-portions to the above structure of MPL Is also a suitable adjuvant. In other embodiments, the adjuvant is a synthetic derivative of lipid A, some of which are TLR-4 agonists and by way of example, but not limited to:
OM174 (2-deoxy-6-o- [2-deoxy-2-[(R) -3-dodecanoyloxytetra-decanoylamino] -4-o-phosphono- □ -D-glucopyranosyl] -2- [ (R) -3-Hydroxytetradecanoylamino] -β-D-glucopyranosyl dihydrogen phosphate), (WO 95/14026)
OM 294 DP (3S, 9 R) -3-[(R) -Dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9 (R)-[(R) -3-hydroxytetradeca Noylamino] decane-1,10-diol, 1,10-bis (dihydrogen phosphate) (WO 99/64301 and WO 00/0462)
OM 197 MP-Ac DP (3S-, 9R) -3- □ (R) -Dodecanoyloxytetradecanoylamino] -4-oxo-5-aza-9-[(R) -3-hydroxytetradecanoyl Amino] decane-1,10-diol, 1-dihydrogen phosphate 10- (6-aminohexanoate) (WO 01/46127)
Is mentioned.

  Other TLR4 ligands that may be used are alkyl glucosaminide phosphates (AGP), such as AGP disclosed in WO 98/50399 or US Pat. No. 6,303,347 (the process for the preparation of AGP is also disclosed). Suitably a pharmaceutically acceptable salt of AGP as disclosed in RC527 or RC529 or US Pat. No. 6,764,840. Some AGPs are TLR4 agonists and some are TLR4 antagonists. Both are considered useful as adjuvants.

  Other suitable TLR-4 ligands (Sabroe et al., JI 2003 p1630-5) that can trigger a signaling response via TLR-4 are, for example, lipopolysaccharides and derivatives thereof, or fragments thereof from Gram-negative bacteria In particular non-toxic derivatives of LPS (eg 3D-MPL). Other suitable TLR agonists are: heat shock protein (HSP) 10, 60, 65, 70, 75 or 90; surfactant protein A, hyaluronan oligosaccharide, heparan sulfate fragment, fibronectin fragment, fibrinogen peptide and b-defensin- 2, as well as muramyl dipeptide (MDP). In certain embodiments, the TLR agonist is HSP 60, 70 or 90. Other suitable TLR-4 ligands are those described in WO 2003/011223 and WO2003 / 099195, for example, disclosed on pages 4-5 of WO2003 / 011223 or pages 3-4 of WO2003 / 099195. Compounds I, II and III, and especially those compounds disclosed in WO2003 / 011223 as ER803022, ER803058, ER803732, ER804053, ER804057, ER804058, ER804059, ER804442, ER804680, and ER804764. For example, one suitable TLR-4 ligand is ER804057.

  In one embodiment of the invention, a TLR agonist is used that is capable of causing a signaling response through TLR-1. Suitably, TLR agonists that can trigger a signaling response via TLR-1 are: triacylated lipopeptide (LP); phenol-soluble modulin; LP of Mycobacterium tuberculosis S- (2,3-bis (palmitoyloxy)-(2-RS) -propyl) -N-palmitoyl- (R) -Cys- (S) -Ser- mimics the acetylated amino terminus of bacterial lipoproteins Selected from (S) -Lys (4) -OH trihydrochloride (Pam3Cys) LP and OspA LP from Lyme disease Borrelia burgdorfei. In an alternative embodiment, a TLR agonist is used that is capable of causing a signaling response through TLR-2. Suitably, TLR agonists that can trigger a signaling response through TLR-2 are derived from lipoproteins, peptidoglycans, M tuberculosis, Lyme disease B burgdorferi or T pallidum. Bacterial lipopeptides; peptidoglycans derived from species including Staphylococcus aureus; lipoteichoic acid, mannuronic acid, Neisseria porins, bacterial cilia, Yersina virulence factor, CMV One or more of virions, measles hemagglutinin, and zymosan from yeast. In an alternative embodiment, a TLR agonist is used that is capable of causing a signaling response through TLR-3. Suitably, TLR agonists that can trigger a signaling response through TLR-3 are double-stranded RNA (dsRNA), or polyinosine polycytidylic acid (Poly IC), a molecular nucleic acid pattern associated with viral infection . In an alternative embodiment, a TLR agonist is used that is capable of causing a signaling response through TLR-5. Suitably, the TLR agonist capable of causing a signaling response through TLR-5 is bacterial flagellin. In an alternative embodiment, a TLR agonist is used that is capable of causing a signaling response through TLR-6. Suitably, TLR agonists capable of triggering a signaling response through TLR-6 are mycobacterial lipoproteins, diacylated LPs, and phenol soluble modulins. Additional TLR6 agonists are described in WO 2003/043572. In an alternative embodiment, a TLR agonist is used that is capable of causing a signaling response through TLR-7. Suitably, a TLR agonist capable of causing a signaling response via TLR-7 is a single stranded RNA (ssRNA), loxoribine, a guanosine analog at positions N7 and C8, or an imidazoquinoline compound, or a derivative thereof It is. In certain embodiments, the TLR agonist is imiquimod. Further TLR7 agonists are described in WO 2002/085905.

  The amount of 3D-MPL used in a single dose can suitably enhance the immune response to the antigen in humans. Specifically, an appropriate amount of 3D MPL is determined by comparing the immunological ability of the composition compared to a composition without adjuvant or compared to a composition using another amount of 3D MPL as an adjuvant. An amount that improves and at the same time is acceptable from the reactivity profile. The amount of 3D-MPL in each human dose of vaccine is, for example, 1 to 200 μg per dose, or 10 to 100 μg, or 20 to 80 μg, such as 25 μg, or 40 to 60 μg per dose, such as 50 μg. sell.

  The immunogenic compositions described herein can also include aluminum or an aluminum compound as a stabilizer.

  In certain embodiments, a single dose of a first immunogenic composition is administered, followed by one or more doses of a second immunogenic composition, such as 1 or 2 or 3 doses. A second immunogenic composition is administered.

  In another embodiment, two doses of a first immunogenic composition are administered, followed by one or more doses of a second immunogenic composition, e.g., one or two doses of a first immunogenic composition. Two immunogenic compositions are administered.

  In certain embodiments, a single dose of the first immunogenic composition is administered followed by a two dose of the second immunogenic composition, or a two dose of the first immunogenic composition. A single dose of the second immunogenic composition is administered after administering the immunogenic composition.

  In certain embodiments, no more than two doses of the first immunogenic composition are administered.

  For the kits described herein, the frequency of administration of each composition may be as described for the use or method.

  Accordingly, the methods and uses and kits described herein provide for a single dose of a first immunogenic composition, or a single dose of a second immunogenic composition, or a single dose. Both the first immunogenic composition and the second immunogenic composition can be used.

  In certain embodiments, the first and second immunogenic compositions comprise HPV VLP in an amount of 20 μg or more per dose. Each dose may contain, for example, 30 μg of each VLP, or 40 μg of each VLP, or 60 μg of each VLP. Various VLPs may be present in the same or different amounts. The first and second immunogenic compositions may contain different amounts of the same HPV VLP.

  In certain embodiments, the first immunogenic composition comprises HPV 16 and HPV 18 VLP in an amount of 20 μg per dose.

  In certain embodiments, the second immunogenic composition comprises HPV 6, HPV 11, HPV 16 and HPV 18 VLP in amounts of 20 μg, 40 μg, 40 μg and 20 μg, respectively, per dose.

  Administration of the immunogenic composition can be any schedule for vaccination administered twice or three or more times, for example, 0, 1 month schedule, 0, 2 month schedule for 2 dose vaccines 0, 3 month schedule, 0, 4 month schedule, 0, 5 month schedule or 0, 6 month schedule; 0, 1, 6 month schedule, 0, 2, 6 month schedule for 3 doses vaccination, 0 , 3, 6 months schedule, 0, 4, 6 months schedule. Thus, the second dose may be administered, for example, 1 month or 2 months or 3 months or 4 months or 5 months or 6 months or up to 12 months or up to 24 months after the first dose. Similarly, the third dose may be administered 1 month or 2 months or 3 months or 4 months or 5 months or 6 months or up to 12 months or up to 24 months after the second dose.

  HPV VLPs and methods for producing VLPs are well known in the art. VLPs are typically composed of viral HPV L1 protein and may also include L2 protein. Regarding VLP, refer to, for example, WO9420137, US5985610, W09611272, US6599508B1, US6361778B1, EP595935.

  In any of the embodiments described herein, the HPV VLP can comprise the HPV L1 protein or an immunogenic fragment thereof, with or without another protein or peptide, such as an L2 protein or peptide.

  In certain embodiments, the VLP in the first immunogenic composition comprises an HPV L1 protein or immunogenic fragment thereof (e.g., present by reference) into which one or more epitopes of L2 are inserted. And the like described in WO 2010/149752 incorporated in the specification). In certain embodiments, the first immunogenic composition comprises such HPV L1 VLPs into which one or more epitopes of L2 have been inserted, along with HPV L1 only VLPs (HPV L1 only VLPs), For example, a combination of HPV 16 and HPV 18 L1 single VLPs and HPV L1 VLPs into which one or more epitopes of L2 are inserted within L1.

  In another embodiment, the VLP in the first immunogenic composition is a L1-only VLP that is a VLP comprising L1 or an immunogenic fragment thereof and not L2.

  In certain embodiments, the VLP in the second immunogenic composition is a L1-only VLP comprising L1 or an immunogenic fragment thereof and not L2.

  In certain embodiments, the VLP in the first immunogenic composition comprises truncated L1.

  In certain embodiments, the VLP in the second immunogenic composition comprises full length L1.

  Suitable immunogenic fragments of HPV L1 include truncation, deletion, substitution, or insertion variants of L1. Such immunogenic fragments may be capable of generating an immune response, which may distinguish L1 proteins, such as L1 in the form of viral particles or VLPs, from the HPV type from which the L1 protein is derived. it can.

  Immunogenic L1 fragments that can be used include truncated L1 proteins. In certain embodiments, truncation removes the nuclear localization signal and optionally also removes the DNA binding pattern of the L1 C-terminal region. In another embodiment, the truncation is a C-terminal truncation. In a further embodiment, C-terminal truncation removes less than 50 amino acids, such as less than 40 amino acids. If L1 is derived from HPV 16, then in another embodiment, C-terminal truncation removes 34 amino acids from the carboxy terminus of HPV 16 L1. If L1 is derived from HPV 18, then in a further embodiment, C-terminal truncation removes 35 amino acids from the carboxy terminus of HPV 18 L1. Thus, truncated L1 protein is cleaved on the C-terminal side compared to wild-type L1, eg, nuclear localization signal by removing less than 50 or less than 40 amino acids from the C-terminal end of the protein And, in some cases, the DNA binding pattern. Specific examples of such truncated proteins for L1 from HPV 16 and 18 are set forth below as SEQ ID NOs: 1 and 2. Truncated L1 proteins are also described in US 6,060,324, US 6,361,778, and US 6,599,508, which are incorporated herein by reference.

である。 In certain embodiments, the HPV 16 L1 amino acid sequence has the following sequence: (SEQ ID NO: 1)

It is.

  HPV 16 L1 sequences may be those disclosed in WO94 / 05792 or US 6,649,167, for example, those that are appropriately truncated. Appropriate truncation products have been cleaved at positions equivalent to the positions previously indicated as assessed by sequence comparison and using the criteria disclosed herein.

である。 In one embodiment, the HPV 18 L1 amino acid sequence has the following sequence: (SEQ ID NO: 2)

It is.

  Alternative HPV 18 L1 sequences are disclosed in WO96 / 29413, which can be appropriately truncated. Appropriate truncation products have been cleaved at positions equivalent to those indicated above, as assessed by sequence comparison and utilizing the criteria disclosed herein.

  In certain embodiments, the HPV VLP of the first immunogenic composition is an L1 only VLP comprising truncated L1 and the HPV VLP of the second immunogenic composition is an L1 alone comprising full length L1. Type VLP.

  VLPs can be made using the baculovirus system in any suitable cell substrate such as yeast cells or bacterial cells or insect cells, for example in insect cells such as cells obtained from Trichoplusia ni. Also, techniques for the preparation of VLPs are well known in the art (eg, WO9913056, US 6416945B1, US 6261765B1 and US6245568, and references thereof, the entire contents of which are incorporated herein by reference).

  In certain embodiments, HPV VLPs in the first immunogenic composition are expressed in insect cells.

  In certain embodiments, HPV VLPs in the second immunogenic composition are expressed in yeast.

  VLPs can be made by disassembly and reassembly techniques. 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, from insect cells to obtain a uniform preparation of VLPs. Degradation and reconstitution of purified recombinant L1 HPV 11 VLP is described. WO99 / 13056 and US6245568 also describe a disassembly / reconstitution process for making HPV VLPs.

  In some embodiments, HPV VLPs are made as described in WO99 / 13056 or US6245568.

  Alternatively, VLP expresses an L1 protein or immunogenic fragment, which is extracted from its production system or cell substrate, while the protein is mainly in the form of L1 monomer or pentamer (capsomer). Can be purified, and then VLP can be formed from the purified protein. In certain embodiments, the extraction and / or purification steps are performed in the presence of a reducing agent such as β-mercaptoethanol (BME) to prevent VLP formation. In some embodiments, this process includes spontaneously forming a VLP by removing a reducing agent such as BME.

  VLP formation can be assessed by standard techniques such as, for example, electron microscopy and dynamic laser light scattering.

  In some cases, the immunogenic composition can be formulated or co-administered with other non-HPV antigens. Suitably, these non-HPV antigens can provide protection against sexually transmitted diseases such as other diseases such as herpes simplex virus (HSV). For example, the vaccine may contain gD from HSV or a truncated product thereof. In this way, the vaccine provides protection against both HPV and HSV.

  In certain embodiments, the immunogenic composition is provided as a liquid vaccine formulation, but the composition can be lyophilized and reconstituted prior to administration.

  The immunogenic compositions described herein can be of various routes including oral, topical, subcutaneous, mucosal (typically intravaginal), intravenous, intramuscular, intranasal, sublingual, intradermal and the like. It can be administered either by way of or via a suppository. Intramuscular and intradermal delivery is preferred.

  The dose of VLP may vary depending on the individual's condition, sex, age and weight, route of vaccine administration and HPV. The amount can also vary depending on the number of VLP types. Suitably the delivery will be a delivery of an amount of VLP suitable to elicit an immunological protective response. Suitably each vaccine dose is 1-100 μg of each VLP, suitably at least 5 μg, or at least 10 μg, such as 5-50 μg of each VLP, most suitably 10-50 μg of each VLP, such as 5 μg, Contains 6 μg, 10 μg, 15 μg, 20 μg, 40 μg or 50 μg.

  The immunogenic compositions described herein can be tested using standard techniques, eg, in standard preclinical models, to confirm that the vaccine is immunogenic. .

  All methods and uses and kits described herein may be for use with adolescent girls aged 9 years or older, such as 10-15 years, such as 10-13 years. However, it is also possible to vaccinate girls and adult women older than 15 years. Similarly, vaccines can be administered to younger age groups, such as 2-12 years old. Vaccines are also available to women after abnormal cervical cancer test results, women following surgery following removal of lesions caused by HPV, or women who are serologically negative and DNA negative for HPV cancer types. It can also be administered.

  In certain embodiments, the methods and uses and kits described herein include the following age groups: 9-25 years, 10-25 years, 9-19 years, 10-19 years, 9-14 years, For use with women who fall into one or more of 10-14 years, 15-19 years, 20-25 years, under 14 years, under 19 years, under 25 years.

  The methods and uses and kits described herein can be used for men and boys.

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

References

Three-dose immunogenicity test in mice
BALB / c mice (23 mice per group) received intramuscular injections on days 0, 21 and 120 for all groups. All doses were 1/10 of the human dose of antigen. Two control groups included Cervarix ™ (HPV-16 / 18 L1 VLP 2/2 μg + AS04) or Gardasil ™ (HPV-16 / 18/6/11 L1 VLP 4/2/2/4 μg + Merck aluminum hydroxyphos Three injections of fate sulfate (MAA ^* )) vaccine were given. Four other additional groups include Cervarix ™ on day 0, Gardasil ™ on days 21 and 120; Cervarix ™ on days 0 and 21, then Gardasil ™ on day 120 Gardasil ™ was injected on day 0, Cervarix ™ on days 21 and 120, or Gardasil ™ on days 0 and 21, and Cervarix ™ on day 120.

  Blood was collected at day 42 (D21 PII) and day 162 (D42 PIII) and analyzed for total antibody titer (ELISA) against HPV-16 / 18/6 and 11 L1 VLPs. Neutralizing antibody titers (PBNA) against HPV-16 / 18/6 and 11 were also measured on day 162. Based on previous experiments and using ANOVA-one-way analysis, a sample size of 23 mice was required to detect a 2-fold difference between 6 groups with 91% power.

^* MAA = Merck aluminum hydroxyphosphate sulfate The following 6 groups of mice were used.

Adjuvant formulation (1/10 of human dose)

Results The humoral response to HPV-16, 18, 6 and 11 L1 VLPs after injection of various immunization schemes was monitored by total and neutralizing antibody responses (see the method described at the end of Example 1). I want to be)

1.HPV-16 L1 VLP response
1.1 Total antibody response HPV16 (after ELISA, II and III)
A comparison of total antibody responses after immunization with different vaccination schemes (ELISA—see Materials and Methods section below) is shown in FIG. A summary of the statistical analysis comparing all groups to the Cervarix ™ or Gardasil ™ control group is shown in FIG. Note that the syringe-like portion in the figure corresponds to the time of injection.

1.2 Neutralization response HPV16 (PBNA, D42 PIII)
A comparison of neutralizing antibody responses after immunization with various vaccination schemes (mock neutralization assay—see Materials and Methods section below) was performed at III after D42 and is shown in FIG. A summary of the statistical analysis comparing all groups to the Cervarix ™ or Gardasil ™ control group is shown in FIG.

2. HPV-18 L1 VLP response
2.1 Total antibody response HPV18 (after ELISA, II and III)
A comparison of total antibody response (ELISA) after immunization with different vaccination schemes is shown in FIG. A summary of the statistical analysis comparing all groups to the Cervarix ™ or Gardasil ™ control group is shown in FIG.

2.2 Neutralization response HPV18 (PBNA, D42 PIII)
A comparison of the neutralizing antibody response after immunization with different vaccination schemes (pseudoneutralization assay) was performed at III after D42 and is shown in FIG. A summary of the statistical analysis comparing all groups to the Cervarix ™ or Gardasil ™ control group is shown in FIG.

3. HPV-6 L1 VLP response
3.1 Total antibody response HPV6 (after ELISA, II and III)
A comparison of total antibody response (ELISA) after immunization with different vaccination schemes is shown in FIG. A summary of the statistical analysis comparing all groups to the Cervarix ™ or Gardasil ™ control group is shown in FIG.

3.2 Neutralization response HPV6 (PBNA, D42 PIII)
A comparison of neutralizing antibody responses after immunization with different vaccination schemes (pseudoneutralization assay) was performed after III and shown in FIG. A summary of the statistical analysis comparing all groups to the Cervarix ™ or Gardasil ™ control group is shown in FIG.

4. HPV-11 L1 VLP response
4.1 Total antibody response HPV11 (after ELISA, II and III)
A comparison of total antibody response (ELISA) after immunization by various vaccination schemes is shown in FIG. A summary of the statistical analysis comparing all groups to the Cervarix ™ or Gardasil ™ control group is shown in FIG.

4.2 Neutralization response HPV11 (PBNA, D42 PIII)
A comparison of neutralizing antibody responses after immunization with different vaccination schemes (pseudoneutralization assay) was performed at III after D42 and is shown in FIG. A summary of the statistical analysis comparing all groups to the Cervarix ™ or Gardasil ™ control group is shown in FIG.

CONCLUSIONS: Positive effect of single dose CervarixTM priming on total and neutralizing anti-HPV-6 and 11 responses after III compared to GardasilTM priming (3.5-32 fold, p <0.0001) → CGG> GCC and GGC
-Positive effect of double dose Cervarix (TM) priming on total anti-HPV-6 and 11 responses only after III compared to Gardasil (TM) priming (3.1-5.8 fold, p <0.0001) → CCG> GCC and GGC
Positive effect (1.9-2.6) of CervarixTM priming (1 or 2 doses) on total and neutralizing anti-HPV-16 responses at day 42 post III compared to GardasilTM priming Times, p ≦ 0.0006) → CCG to CGG ≧ GGG, GCC and GGC
Positive effect (1.7-4.2 fold, p ≦ 0.0066) of double-dose CervarixTM priming on total anti-HPV-18 response 42 days after III compared to GardasilTM priming → CCG ≧ GGG, GCC and GGC
Comparison of Cervarix (TM) priming and Gardasil (TM) priming reproduces Cervarix (TM) priming on ELISA antibody responses to all HPV L1 VLPs (including 6 and 11) and PBNA responses to HPV-16, 6 and 11 Sexual positive effects were shown.

  A single priming with Cervarix (TM) is sufficient to induce an antibody response similar to Cervarix (TM) for HPV-16, but to ensure a titer similar to Cervarix (TM) for HPV-18 Has also been shown to require priming with at least two doses of Cervarix ™.

  In conclusion, the above immunogenicity data show that at least 1x (HPV-16, 6 and 11) or 2 x (HPV-16, 6 and 11) or 2x (compared to a complete vaccination schedule with CervarixTM or GardasilTM). The added value of priming with Cervarix ™ of HPV-18) was demonstrated.

Table: Ranking based on total and neutralizing antibody responses of vaccination schemes for HPV 6 and HPV 11

  The added value of priming with a single dose of Cervarix (TM) is a higher total anti-HPV18 response in a double dose scheme at 1/50 HD compared to a double dose Gardasil (TM) And maintained by showing a similar total anti-HPV11 response. See Example 2.

Materials and methods
Anti-HPV 16/18/6/11 L1 VLP ELISA
Quantification of anti-HPV-16 / 18/6/11 L1 VLP antibody was performed by ELISA using HPV-16, HPV-18, HPV-6 and HPV-11 truncated L1 VLP as a coating. Antigen was diluted to a final concentration of 1, 2 or 5 μg / mL in PBS and then adsorbed overnight at 4 ° C. to the wells of a 96-well microtiter plate (Maxisorp Immuno-plate, Nunc, Denmark). The plates were then incubated for 1 hour at 37 ° C. with PBS containing 0.1% Tween 20 + 1% BSA (saturation buffer). Serum diluted with a saturation buffer was added to the HPV L1 coated plate and incubated at 37 ° C. for 1 hour 30 minutes. The plate was washed 4 times with PBS 0.1% Tween 20, and then biotin-conjugated anti-mouse Ig (Dako, UK) diluted with a saturated buffer was added to each well and incubated at 37 ° C. for 1 hour 30 minutes. After the washing step, streptavidin-horseradish peroxidase (Dako, UK) diluted in saturation buffer was added for an additional 30 minutes at 37 ° C. The plates were washed as above and then incubated for 20 minutes at room temperature with a solution of 0.04% o-phenylenediamine (Sigma) 0.03% H ₂ O ₂ in 0.1% Tween 20, 0.05M citrate buffer pH 4.5. The reaction was stopped with 2N H ₂ SO ₄ and then read at 492/620 nm. ELISA titers were calculated from reference values by SoftMaxPro (using a 4-variable equation) and expressed in EU / mL.

Pseudoneutralization assay (PBNA)
Pseudovirus (PsV) was generated by transfection of 293TT cells (human fetal kidney cell line + SV40 T antigen) with a plasmid expressing both L1 and L2 and a reporter plasmid p2CMVSEAP (SEAP = secreted alkaline phosphatase). Briefly, 20 million 293TT cells were seeded 16 hours prior to transfection. Example: For production of HPV16 pseudovirus, the cells were transfected with 27 μg each of pYSEAP, p16L1h, and p16L2h (Lipofectamine 2000 / Invitrogen) and then harvested 40-48 after transfection. The extracted pseudovirion particles were then further purified using Optiprep (Sigma). The preparations are examined for purity with 10% SDS-Tris-glycine gel (Bio-Rad), titrated to 293TT cells and tested for infectivity by SEAP detection (Chemiluminescence, BD Clontech) and then pooled. And frozen at −80 ° C. until use.

  To assay the neutralization titer of serum samples, 293TT target cells were pre-seeded at 30000 hours at 30,000 cells / well in 96 well flat bottom plates. The pseudovirus preparation was diluted appropriately to obtain alkaline phosphatase (SEAP) for 30-70 relative light output (RLU) output reading. The diluted pseudovirus stock was placed in a 96-well plate, combined with the diluted serum, and then left on ice for 1 hour. The pseudovirus / antibody mixture was then transferred onto previously seeded cells and incubated for 72 hours. At the end of the incubation, the supernatant was collected and clarified at 1500 × g for 5 minutes. The SEAP content of the clarified supernatant was measured using the Great ESCAPE SEAP Chemiluminescence Kit (BD Clontech) as per manufacturer's instructions. Twenty minutes after adding the substrate, the samples were analyzed using a MLX Microplate Luminometer (Dynex Technologies) set to Glow-Endpoint in white Microlite 1 (Dynex) or Optiplate-96 (Perkin-Elmer) opaque 96-well plates. Read at 0.20 sec / well.

  Serum neutralizing titer is defined as the reciprocal of the highest dilution factor that reduces SEAP activity by at least 50% compared to serum free controls. Positive neutralization in HPV-16, HPV-18, HPV-6 and HPV-11 assays when serum neutralizes at a dilution factor at least 4 times higher than the titer observed in the BPV1 neutralization assay (negative control) Considered.

Statistical analysis group means were compared using one-way analysis of variance (ANOVA 1). The analysis was performed based on log10 conversion data for the purpose of normalization. When a significant difference was detected between the group mean values (p value <0.05), pairwise comparison between the mean values was performed at a significance level of 0.05 (Tukey-HSD comparison judgment method).

UL / LL = Upper / lower limit of 95% confidence interval (CI).

Two-dose immunization test in mice (including challenge test)
This preclinical experiment was undertaken to compare the specific protection induced against HPV-18 and 11 after vaccination with CC, CG, GG or GC schemes. In this experiment, the vaccine was used at a dose of 1/50 of the human dose.

Part I-Immunogenicity test
BALB / c mice (10 mice per group) were given two doses of CervarixTM 1/50 HD, two doses of GardasilTM 1/50 HD on days 0 and 21. A single dose of Cervarix ™ 1/50 HD followed by a single dose of Gardasil ™ 1/50 HD, or a single dose of Gardasil ™ 1/50 HD followed by a single dose Intramuscular injections were given using an amount of Cervarix ™ 1/50 HD.

  Blood was collected on day 28 after II and analyzed by ELISA for total antibody titers against HPV-18 and 11 L1 VLPs after vaccination with CC, GG, CG or GC. Neutralizing antibody titers against HPV-18 and 11 were also measured 28 days after II (by PBNA).

  To assess the specific and cross-protection induced by the various immunization schemes described above, mice were challenged with PsV-18 and 11 one month after II.

group

Adjuvant formulation (1/50 HD)

^* MAA = Merck aluminum hydroxyphosphate sulfate
Results The humoral response to HPV-18 and 11 L1 VLP after injection of various immunization schemes was monitored by total (ELISA) antibody response and neutralization (PBNA) antibody response.

1. HPV-18 L1 VLP response
1.1 Total antibody response HPV-18 (ELISA, D28 PII)
A comparison of total antibody response (ELISA) 28 days after PII immunization with different vaccination schemes is shown in FIG.
-CC to CG (2.3 to 4.8 times, p ≦ 0.0613) ≧ GG to GC

1.2 Neutralizing antibody response HPV-18 (PBNA, D28 PII)
A comparison of the neutralizing antibody response after immunization by various vaccination schemes (pseudoneutralization assay-see Example 1 Materials and Methods section) is shown in FIG.
-CC ~ CG ~ GG ~ GC

2. HPV-11 L1 VLP response
2.1 Total antibody response HPV-11 (ELISA, D28 PII)
A comparison of total antibody response (ELISA) after immunization by various vaccination schemes is shown in FIG.
-CG to GG (1.8 to 3.5 times, p = 0.0038 to 0.2924) ≥ GC (5.1 times, p = 0.0001)> CC

2.2 Neutralizing antibody response HPV-11 (PBNA, D28 PII)
A comparison of neutralizing antibody responses (pseudoneutralization assay NCI) after immunization with various vaccination schemes is shown in FIG.
-GG (5.6 to 11.5 times, p ≦ 0.0001)> GC to CG (58 to 120 times, p <0.0001)> CC
-No positive response was observed with CC 1/50 HD (cut-off value).

Conclusion
Similar total anti-VLP18 titers were observed for CC and CG, which were higher than the GG and GC schemes.

  For GG and CG, a statistically significant higher total anti-VLP11 response was observed compared to GC and CC.

  Similar specific neutralizing antibody titers against HPV-18 were observed for all vaccination schemes.

  For CG, statistically significant neutralizing antibody titers against HPV-11 were observed, which were low compared to GG but higher than the CC scheme.

Part II-Intravaginal challenge and defense
Specific protection induced after CC, GG, CG or GC vaccination schemes, after challenge with luciferase PsV-18 and 11 (see Materials and Methods section below) of vaccinated mice 1 month after II evaluated.

1. PsV-18 Challenge
Given the unexpected protection (60%) observed in the NaCl group (ie non-vaccine control), no conclusions could be made about the level of protection after challenge with PsV-18 (thus data not shown).

2. PsV-11 Challenge
A comparison of protection against PsV-11 induced after CC, GG, CG or GC vaccination is shown in FIG.

Note: Due to variations in intravaginal challenge, up to 20% protection (full or partial protection) in the NaCl group is tolerated.

-Complete protection (100%) rate was observed for GG, CG and GC-No protection from CC vaccination was observed-No protection was observed when neutralizing antibody response was not measured
Conclusion
Compared to GG, 100% protection against PsV-11 was observed in these two schemes despite the low neutralization response to HPV-11 in the CG and GC vaccination schemes. Furthermore, in the CC vaccination, the absence of neutralizing antibodies against HPV-11 was observed and at the same time no protection against this isotype was observed, suggesting a correlation between the presence of neutralizing antibodies and the protection rate.

Result highlights:
・ Total antibody (ELISA)
○ HPV-18 : CC ~ CG≥GG ~ GC
○ HPV-11 : GG ~ CG ≧ GC> CC
・Neutralizing antibody (PBNA: HPV-6 / 11)
○ HPV-18 : CC ~ CG ~ GG ~ GC
○ HPV-11 : GG> GC to CG> CC
・Efficacy (intravaginal challenge mouse model)
○ HPV-18 : Uncertain data due to unexpected protection in NaCl group ○ HPV-11 : GG - CG - GC> CC vaccination (100% full protection or 0%)
Conclusion
Priming with a single dose of Cervarix (TM) followed by a single dose of Gardasil (TM) is a total anti-HPV-18 response similar to a double dose of Cervarix (TM) (CC), And similar anti-HPV-11 responses were induced compared to two doses of Gardasil ™ (GG). Furthermore, 100% protection against PsV-11 was observed in CG vaccination, as in GG. These observations confirmed the potential added value for starting a vaccination scheme using Cervarix ™ based on ELISA titers and protection rates.

Materials and methods
Two weeks after In Vivo challenge immunization, mice were synchronized with the hormonal cycle by subcutaneous injection of 3 mg / 100 μL of depoprovera. Four days later, mice were pre-treated in their vagina with 50 μL of Conceptrol (a CMC-based spermicide containing 4% nonoxynol-9 used to destroy the epithelium of the vaginal tract). did. The mice were challenged intravaginally 6 hours later with 30 μL luciferase-pseudovirion diluted in 1.5% low viscosity carboxymethylcellulose. The pseudovirion is composed of HPV L1 and L2 surface proteins with an encapsulated reporter plasmid that expresses the luciferase protein. PsV infection was monitored by measuring luciferase expression in the genital tract 2 days after challenge. Anesthetized mice were injected intravaginally with 20 μL luciferin (15 mg / mL) and after 5 minutes exposed and imaged for 2 minutes using a Xenogen IVIS Spectrum in vivo imaging device (Caliper LifeSciences).

  Protection was defined when the mouse showed a signal below the mean + 3 SD of the signal obtained in NaCl vaccinated mice challenged with PsV-18 expressing SEAP (negative control).

Mice were considered fully protected when the bioluminescent signal obtained after challenge had a cut-off value of less than 939 ph / sec / cm ² . This value was determined by a statistician using a bioluminescent signal measured in an unrelated thoracic zone (# 10 experiment). Mice were considered partially protected if the measured bioluminescence signal was above the cut-off value of 939 ph / sec / cm ² but below the lower limit of CI95 observed in the negative NaCl control group.

Comparative short-term and long-term protection induced by Cervarix ™ and Gardasil ™ vaccines in a three-dose vaccination scheme (Day 0, Day 45, Day 120, 1/50 of the human dose)
These preclinical experiments were undertaken to compare the specific and cross-protection induced against HPV-18 / 6 and 11 after vaccination with the CCC, GGG, CGG, CCG, GCC and GGC schemes. This was assessed at 1 and 6 months after III to mimic short and long term protection. The clinical 0 / M2 / M6 vaccination scheme was mimicked by utilizing the vaccination scheme D0 / 45/120.

BALB / c mice (20 mice per group) were given intramuscular injections on days 0, 45 and 120. Two groups include Cervarix ™ 1/50 HD (HPV-16 / 18 L1 VLP 0.4 / 0.4 μg + AS04) vaccine or Gardasil ™ 1/50 HD (HPV-16 / 18/6/11 L1 VLP 0.8 /0.4/0.4/0.8 μg + MAA ^* ) The vaccine was injected three times. Four other additional groups include Cervarix ™ 1/50 HD on day 0 and Gardasil ™ 1/50 HD on days 45 and 120; Cervarix ™ 1/50 on days 0 and 45 50 HD, then Gardasil ™ 1/50 HD on day 120; Gardasil ™ 1/50 HD on day 0, then Cervarix ™ 1/50 HD on days 45 and 120, or 0 and 45 days The eyes were injected with Gardasil ™ 1/50 HD followed by Cervarix ™ 1/50 HD on day 120.

  Blood was collected at 1 month post III (20100801) or 6 months post III (20100810) and immediately before challenge to analyze total antibody titers (ELISA) against HPV-18 / 6 and 11 L1 VLPs.

Neutralizing antibody titers (PBNA) against HPV-18 / 6 and 11 were also measured 1 or 6 months after III.

  To assess the specific protection induced by these various immunization schemes, mice were challenged with PsV-18 / 6 or 11 at 1 or 6 months after the third dose.

group

Adjuvant formulation (1/50 human dose)

^* MAA = Merck aluminum hydroxyphosphate sulfate
Results Humoral responses to HPV-18, 6 and 11 L1 VLPs after injection of various immunization schemes were determined by total (ELISA) and neutralizing (PBNA) antibody responses at 1 or 6 months after vaccination. Monitored.

1.1. Humoral response
1.1.1. HPV-18 L1 VLP response
1.1.1.1. Total antibody response HPV-18 (ELISA, 1 or 6M PIII)
A comparison of total antibody response (ELISA) at 1M and 6M PIII after immunization with various vaccination schemes is shown in FIGS.

The outline of statistical analysis is as follows.

1.1.1.2. Neutralizing antibody response HPV-18 (ELISA, 1 or 6M PIII)
A comparison of neutralizing antibody response (ELISA) at 1M and 6M PIII after immunization with various vaccination schemes is shown in FIGS.

The outline of statistical analysis is as follows.

1.1.2. HPV-6 L1 VLP response
1.1.2.1. Total antibody response HPV-6 (ELISA, 1 or 6M PIII)
A comparison of total antibody response (ELISA) at 1M and 6M PIII after immunization with various vaccination schemes is shown in FIGS. 26 and 27, respectively.

The outline of statistical analysis is as follows.

1.1.2.2. Neutralizing antibody response HPV-6 (ELISA, 1 or 6M PIII)
A comparison of neutralizing antibody response (ELISA) at 1M and 6M PIII after immunization with various vaccination schemes is shown in FIGS. 28 and 29, respectively.

The outline of statistical analysis is as follows.

1.1.3. HPV-11 L1 VLP response
1.1.3.1. Total antibody response HPV-11 (ELISA, 1 or 6M PIII)
A comparison of total antibody response (ELISA) at 1M and 6M PIII after immunization with various vaccination schemes is shown in FIGS.

The outline of statistical analysis is as follows.

1.1.3.2. Neutralizing antibody response HPV-11 (ELISA, 1 or 6M PIII)
A comparison of neutralizing antibody response (ELISA) at 1M and 6M PIII after immunization with various vaccination schemes is shown in FIGS. 32 and 33, respectively.

The outline of statistical analysis is as follows.

1.1.4. Conclusion At 1 and 6 months after vaccination, similar (<2 fold, p = 0.0001 to 1.000) total anti-HPV18 responses were observed for all vaccination schemes tested. The positive effect of CervarixTM boost compared to classic GardasilTM three doses and the positive effect of 2X CervarixTM priming on total anti-HPV18 response compared to GardasilTM priming It was not confirmed in the experiment.

  In this experiment, the reproducible added value of 1X Cervarix ™ priming to total and neutralizing anti-HPV-6 and 11 responses at 1 and 6 months after III compared to Gardasil ™ priming Was not shown.

  See overall conclusion.

1.2. Intravaginal Challenge and Protection Specific protection induced after various vaccination schemes was evaluated after challenge of vaccinated mice with luciferase PsV-18 / 6 or 11 one or six months after III.

1.2.1. PsV-18 Challenge A comparison of the protection rate with 6M PIII after immunization by various vaccination schemes is shown in FIG.

  Unexpected findings of protection observed for the NaCl group one month after vaccination (80%), and no conclusions could be made about the short-term protection level after challenge with PsV-18 (data shown )

^* Due to variability in intravaginal challenge, up to 20% protection (full or partial protection) in the NaCl group is tolerated.

• 100% protection was observed in all vaccination schemes except CGG (80%).

1.2.2. PsV-6 Challenge Comparison of protection rates with 1M and 6M PIII after immunization by various vaccination schemes is shown in FIGS. 35 and 36, respectively.

^* Due to variability in intravaginal challenge, up to 20% protection (full or partial protection) in the NaCl group is tolerated.

In the first month after III, 100% protection was observed for GGG, CGG, CCG, GCC and GGC, in contrast to CCC vaccination with no protection against PsV-6 → GGG ~ CGG ~ CCG ~ GCC ~ GGC> CCC

• Six months after III, 100% protection was observed for GGG, CGG, CCG, GCC and GGC, in contrast to CCC vaccination with no protection against PsV-6 → GGG ~ CGG ~ CCG ~ GCC ~ GGC> CCC
1.2.3. PsV-11 Challenge Comparison of protection rates with 1M and 6M PIII after immunization by various vaccination schemes is shown in FIGS. 37 and 38, respectively.

1 month after III 100% protection was observed for GGG and CGG. Good protection rate was observed for CCG, GCC and GGC, with a tendency to better quality of protection with GCC. Protection (20%) was observed for CCC.

• Six months after III, 100% protection was observed for GGG, CGG, CCG, GCC and GGC, in contrast to CCC vaccination with no protection against PsV-11 → GGG to CGG ~ CCG ~ GCC ~ GGC> CCC
Conclusion The obtained data show good sustained protection against PsV-18, 6 and 11 up to 6 months after vaccination, confirming the potential benefits of Cervarix ™ / Gardasil ™ mix The The intravaginal challenge mouse model shows similar conclusions for humans for specific protection.

Correlation of protection rate and total / neutralizing antibody level By examining the correlation between total and neutralizing antibody level and protection rate, the minimum amount of antibody required to induce protection can be assessed. The data is summarized in the table below.

1 month after III data

Data for 6 months after III

• Lack of protection against PsV-6 and PsV-11 in CCC appears to correlate with low levels of total anti-HPV6 / 11 response and lack of neutralizing antibodies to HPV-6 and 11.

Results of Example 3-General highlights:
Immunogenic total antibodies (ELISA)
○ No effect of Cervarix (Trademark) on HPV-18 ELISA antibody response at 1 and 6 months after vaccination → GGG-GCC-GGC
○ Negative impact of Cervarix ™ on HPV-6 ELISA: Cervarix was unable to enhance the existing HPV-6 response 1 month after III → GGG> GCC to GGC
○ Negative effects of Cervarix (trademark) 2X on HPV-6 ELISA at 6 months after III were observed, but similar responses were observed in GGG and GGC → GGG-GGC> GCC
○ Negative effects of Cervarix (TM) 2X on HPV-11 ELISA at 1 and 6 months after III were observed, but similar responses were observed for GGG and GGC → GGG-GGC> GCC
・ Neutralizing antibody (PBNA)
A similar neutralizing antibody response to HPV-18 was observed for all vaccination schemes at 1 month of PIII, and a lower response was observed only for GGG compared to CCC at 6 months after vaccination.
O Neutralizing antibody responses to HPV-6 and 11 were similar when Cervarix ™ priming followed by two doses of Gardasil ™ was compared to classic Gardasil ™ three doses.

Effectiveness (intravaginal challenge mouse model)
• HPV-18: All six vaccination schemes showed high sustained protection until 6 months after III.

Overall conclusion of Example 3
The added value of priming with Cervarix ™ compared to a three-dose vaccination scheme with Cervarix ™ or Gardasil ™ was not confirmed in this experiment. This can be linked to a vaccination schedule corresponding to D0 / 45/120 compared to previous data observed in the classical D0 / 21/120 scheme. Of note, CCC did not respond as clinically (see Einstein et al. 2009) compared to GGG.

  Vaccination with one or two doses of CervarixTM in a three-dose vaccination scheme is 100% against PsV-6 and 11, similar to the classic GardasilTM three-dose scheme. Indicates full defense. In addition, high (80-100%) protection against PsV-18 was observed with the normal Cervarix ™ and Gardasil ™ three-dose schemes, but such protection was also observed in the group injected with the combined vaccines. These data confirm the potential benefits of the Cervarix ™ / Gardasil ™ mix.

  No protection against PsV-6 and PsV-11 was observed after vaccination with 3 doses of Cervarix ™, which correlates with clinical data and is specific for PsV-6 / 18 and 11 Shows the validity of the intravaginal challenge mouse model in the context of mechanical and cross-reactive responses.

Overall conclusions of Examples 1, 2 and 3
Immunogenic serological data show the added value of priming with Cervarix ™ at least 1X (total and neutralizing HPV-16 / 18 response) compared to a three-dose vaccination scheme with Gardasil ™ )It has been shown. Total and neutralizing antibody responses to HPV-11 were also measured using the classic Gardasil ™ 3 when priming with a single dose of Cervarix ™ followed by two doses of Gardasil ™. It was higher compared to the single dose. The added value of priming with Cervarix (TM) (1 or 2 doses) compared to 3 doses of Gardasil (TM) was observed for total anti-HPV6 response, but for neutralizing antibodies Was not.

  Priming with a single dose (HPV-6 and 11) or two doses (HPV-16) of CervarixTM compared to the classic three doses of CervarixTM is HPV-16 / 6 And induce higher total and neutralizing responses to 11.

  These data were observed in a three-dose scheme with 1/10 HD but were not confirmed with 1/50 HD. When this vaccine dilution was tested in the D0-45-120 scheme, the data obtained did not show a higher anti-VLP18 response in CCC compared to GGG as usual. Given the fact that a higher anti-VLP18 response is maintained in Cervarix (TM) in a two-dose scheme with 1/50 HD, the D0-45-120 vaccination schedule is optimal for this evaluation Is unthinkable.

  The added value of priming with a single dose of CervarixTM is higher in the total resistance in a double dose scheme with 1/50 HD compared to a double dose GardasilTM. Maintained by showing HPV18 response and similar total anti-HPV11 response.

Efficacy Efficacy data after vaccination with 3 doses (CCC, GGG, CCG, CGG, GCC or GGC) or 2 doses (CC, GG, CG or GC) with 1/50 HD for each vaccine Obtained.

  Specific protection against PsV-18 was shown up to 6 months after III in all three dose vaccination schemes. In addition, 100% protection against PsV-6 and PsV-11 was demonstrated not only in the classic three-dose Gardasil ™, but also in GCC, GGC, CGG and CCG, and this protection persisted until 6 months after vaccination . As expected, no cross-protection against PsV-6 and PsV-11 was observed after vaccination with 3 doses of Cervarix ™.

  Surprisingly, 100% protection against PsV-11 was achieved even after vaccination with 1/50 HD of CG, but neutralizing antibody response was despite high levels of ELISA titer induction. I was not able to admit. Similar to the three-dose vaccination scheme, no cross protection against PsV-11 was observed with CC.

  These data show the possibility of mixing Cervarix ™ / Gardasil ™ vaccines while maintaining a high level of protection against a particular type (HPV-18 / 6/11). CG, CCG and CGG immunization schemes can be excellent candidates because they simultaneously achieve protection against high-risk HPV types and genital warts.

Claims (34)

A first immunogenic composition comprising HPV VLPs derived from one or more HPV types in combination with an adjuvant containing a TLR agonist for use in a method for the prevention of HPV infection or disease in an individual. The method is as follows:
(i) administering to the individual at least one dose of the first immunogenic composition;
(ii) administering to said individual at least one dose of said second immunogenic composition comprising HPV VLPs derived from one or more HPV types and no TLR agonist;
Wherein the first immunogenic composition is present in the second immunogenic composition and is not present in the first immunogenic composition, or a type-specific immune response against HPV type or The first immunogenic composition described above that increases at least one of the cross-reactive immune responses. An immunogenic composition comprising HPV VLPs derived from at least one HPV type in combination with an adjuvant containing an aluminum salt but no TLR4 agonist for use in a method for the prevention of HPV infection or disease in an individual And the method is as follows:
(i) administering to the individual at least one dose of a first immunogenic composition comprising HPV VLPs derived from one or more HPV types in combination with an adjuvant comprising a TLR agonist; and
(ii) administering to said individual an at least one dose of a second immunogenic composition that is an immunogenic composition that does not include a TLR4 agonist and that includes HPV VLP;
Wherein the first immunogenic composition is present in the second immunogenic composition and is not present in the first immunogenic composition, or a type-specific immune response against HPV type or The immunogenic composition described above, which increases at least one of the cross-reactive immune responses. A method for the prevention of HPV infection or disease in an individual comprising:
(i) administering to the individual at least one dose of a first immunogenic composition comprising HPV VLPs derived from one or more HPV types in combination with an adjuvant comprising a TLR agonist; and
(ii) administering to said individual at least one dose of said second immunogenic composition comprising HPV VLPs derived from one or more HPV types and no TLR agonist;
The first immunogenic composition comprises a type-specific immune response or crossover against a type that is present in the second immunogenic composition and not present in the first immunogenic composition. The method above, wherein the method increases at least one of the reactive immune responses.   4. Use or method according to any one of claims 1 to 3, wherein the first immunogenic composition comprises HPV 16 or HPV 18 VLP, or HPV 16 and HPV 18 VLP.   5. Use or method according to claim 4, wherein the first immunogenic composition increases the type specific immune response against HPV 16 or HPV 18 or both HPV 16 and HPV 18.   The first immunogenic composition augments the immune response compared to a type-specific immune response to the HPV type when only the second number of doses of the second immunogenic composition are administered Use or method according to any one of claims 1-5.   The first immunogenic composition elicits a cross-reactive immune response against one or more high-risk or low-risk HPV types present in the second immunogenic composition. The use or method according to any one of.   The first immunogenic composition of any of claims 1-7, wherein the first immunogenic composition elicits a cross-reactive immune response against one or more low risk HPV types present in the second immunogenic composition. Use or method according to paragraph 1.   9. The method of any one of claims 1-8, wherein the first immunogenic composition elicits a cross-reactive immune response against HPV 6, and the second immunogenic composition comprises HPV 6 VLP. Use or method.   The first immunogenic composition elicits a cross-reactive immune response against HPV 11, and the second immunogenic composition comprises HPV 11 VLP. Use or method.   A cross-reactive immune response against a type in which the first immunogenic composition is present in the second immunogenic composition and not present in the first immunogenic composition is obtained in an equivalent number of times. 11. Use or method according to any one of claims 1 to 10, wherein the use or method is increased compared to the immune response to the type when only a second dose of the second immunogenic composition is administered.   12. Use or method according to any one of claims 1-11, wherein said second immunogenic composition comprises HPV 6, 11, 16, and 18 VLP and optionally other types.   13. Use or method according to any one of claims 1 to 12, wherein the second immunogenic composition comprises HPV 6, 11, 16, 18, 31, 45, 52 and 58.   14. The use or method according to any one of claims 1 to 13, wherein the first immunogenic composition comprises a TLR4 agonist.   15. Use or method according to claim 14, wherein the TLR4 agonist is MPL.   16. Use or method according to claim 15, wherein the first immunogenic composition comprises MPL and an aluminum salt.   17. Use or method according to claim 16, wherein the aluminum salt is aluminum hydroxide.   18. Use or method according to any one of claims 1 to 17, wherein the second immunogenic composition comprises an aluminum salt.   19. Use or method according to claim 18, wherein the aluminum salt in the second immunogenic composition is aluminum hydroxyphosphate sulfate.   21. Any one of claims 1-19, wherein two doses of the first immunogenic composition are administered, followed by a single dose or multiple doses of the second immunogenic composition. The method or use according to paragraph 1.   21. Any one of claims 1-19, wherein a single dose of the first immunogenic composition is administered, followed by one or two or more doses of the second immunogenic composition. Use or method according to paragraph.   An immune response against one or more HPV types present in both the first and second immunogenic compositions is administered as three doses of the second immunogen. 22. Use or method according to claim 20 or 21, wherein the use or method is increased compared to the immune response against the HPV type when only the sex composition is administered.   An immune response against one or more HPV types that is administered in three doses and is present only in the second immunogenic composition is a three dose only of the second immunogenic composition 23. The use or method according to any one of claims 20 to 22, wherein the use or method is greater than the immune response to the HPV type when administered.   24. Use or method according to any one of claims 1 to 23, wherein the HPV VLP comprises L1 or an immunogenic fragment thereof.   25. Use or method according to any one of claims 1 to 24, wherein the HPV VLP is a L1-alone VLP. Less than:
(i) a first immunogenic composition comprising VLPs derived from at least one HPV type in combination with an adjuvant comprising a TLR agonist; and
(ii) A kit comprising a second immunogenic composition comprising VLPs derived from at least one HPV type and no TLR agonist.   27. The kit of claim 26, wherein the first and second immunogenic compositions comprise HPV 16 and HPV 18 VLP.   28. The kit according to claim 26 or 27, wherein the second immunogenic composition further comprises HPV 6 and / or HPV 11 VLP not present in the first immunogenic composition.   29. A kit according to any one of claims 26 to 28, wherein the second immunogenic composition comprises HPV 6, 11, 16, and 18 VLPs and optionally other types.   30. The kit of any one of claims 26 to 29, wherein the first immunogenic composition comprises a TLR4 agonist.   32. The kit of claim 30, wherein the TLR4 agonist is MPL.   32. The kit according to any one of claims 26 to 31, wherein the first immunogenic composition comprises an aluminum salt.   33. A kit according to any one of claims 26 to 32, wherein the second immunogenic composition comprises an aluminum salt.   34. The kit of claim 33, wherein the first immunogenic composition comprises aluminum hydroxide and the second immunogenic composition comprises aluminum hydroxyphosphate sulfate.

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