ES2394448T3 - Vaccine against HPV16 and HPV18 and at least one other type of HPV selected from HPV 31, 45 or 52 - Google Patents

Abstract

An immunogenic composition comprising the PLV or capsomers of HPV 16 and HPV 18 and at least one other type of HPV cancer, the other type of cancer being selected from the list consisting of the types of HPV 31, 45 and 52, in which the The dose of the PLV or capsomere of at least one other type of cancer is reduced relative to that of HPV16 or 18.

Description

Vaccine against HPV16 and HPV18 and at least one other type of HPV selected from HPV 31, 45 or 52.

The present application refers to vaccines against human papillomaviruses.

Background of the invention

5 Papillomaviruses are small DNA tumor viruses, which are highly species specific. So far, over 100 individual human papillomavirus (HPV) genotypes have been described. HPVs are generally specific to either the skin (for example, HPV-1 and -2) or mucosal surfaces (for example, HPV 6 and -11) and usually produce benign tumors (warts) that persist for several months or years . Such benign tumors may be worrisome to the individuals they concern but are not threatening to the

10 life, with a few exceptions.

Some HPVs are also associated with cancers, also known as oncogenic types of HPV. The strongest positive association between HPV and human cancer is that between HPV-16 and HPV-18 and cervical carcinoma. Cervical cancer is the most common malignancy in developing countries, with approximately 500,000 new cases occurring worldwide each year.

15 Other HPVs of particular interest with respect to cancer are types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82, 26, 53, and 66.

HPV virus-like particles (PLV) have been suggested as potential vaccines for HPV treatment. A bivalent vaccine that uses PLV has been shown to be effective in preventing infection with HPV types 16 and 18 in young women (Lancet, vol 364, publication 9447, November 2004, pages 1757-1765). He

WO 03/077942 discloses a vaccine composition composed of an equal proportion mixture of HPV 16, 18, 31 and 45.

There is still a need for a vaccine that protects against multiple types of HPV (for example> 2).

The present invention addresses this need.

Summary of the invention

The present invention relates to an immunogenic composition comprising HPV PLV 16 and 18 and at least one other type of HPV cancer, the other type of cancer is selected from the list consisting of types 31, 45 and 52 of HPV, in which the dose of PLV from at least one other type of cancer is reduced relative to that of HPV 16 or 18.

The invention further relates to an immunogenic composition as defined above in combination with an adjuvant and / or vehicle.

The invention further relates to a vaccine comprising an immunogenic composition as defined above with a pharmaceutically acceptable excipient.

The invention also relates to a method of prevention of infection and / or HPV disease comprising the administration to an individual in need thereof of a composition or vaccine as has been

35 defined above.

The invention further relates to a method for preparing an immunogenic composition as defined above comprising mixing the HPVs of HPV 16 and 18 with at least one other type of HPV cancer, the other type of cancer being selected from the list consisting of types 31, 45, and 52 of HPV, in which the dose of PLV of at least other types of cancer is reduced relative to that of HPV 16 or 18.

The above aspects of the invention can also employ HPV capsomeres instead of the PLV.

Detailed description

The inventors have surprisingly determined that HPVs of HPV 16 and 18 can provide cross protection against infection and / or disease by certain types of cancer other than HPV, ie HPV 31, HPV 45 and HPV 52. The data is provided in the examples in this specification.

45 Cross-protection can be considered as the protection provided by a vaccine that contains a type of HPV against infection (incident or persistent) and / or disease caused by a different type of HPV. Incident and persistent infection are defined as in Lancet's publication by Harper et al., Vol. 364, publication 9447, November 2004, pages 1757-1765. Cross protection can be assessed considering the efficacy of the vaccine (V. E.), in which V. E. is the% improvement in protection against vaccine infection compared to

50 a placebo group for a given type.

According to the above, HPV vaccines comprising HPV PLVs 16 and 18 can be formulated using a lower dose of other types of cancer PLVs (non-HPV 16/18) (31, 45 or 52) that of otherwise they would be required in the absence of HPV PLV 16 and 18, while still achieving the same protective response against incident and / or persistent HPV infection for that other type. It may be advantageous to reduce the dose of PLV from another type of cancer in a multivalent vaccine setting, without significant impact on the protection produced by those other types when the total amount of antigen can be limited; for example, through physical, chemical or regulatory restrictions. More doses of a vaccine are also allowed to be produced for a given amount of antigen and can potentially reduce the overall cost of the vaccine.

The dose of PLV in this specification is conveniently the amount of PLV, measured by weight.

In other words, in order to achieve the same protective response against incident and / or persistent infection, the dose of other PLVs of the cancerous type (not HPV 16 / HPV 18) that need to be used in combination with the HPVs of HPV 16 and / or HPV 18 they can be reduced compared to the level that is required in the absence of such types of PLV 16 and / or 18.

Thus, the invention relates to an immunogenic composition comprising the PLVs of HPV 16 and 18 and at least one other type of HPV cancer, the other type of cancer being selected from the list consisting of types 31, 45 and 52 of HPV, in which the PLV dose of at least other types of cancer is reduced relative to that of HPV 16 or 18.

In an alternative aspect the invention relates to an immunogenic composition comprising the PLV of HPV 16 and 18 and at least one other type of HPV cancer, the other type of cancer being selected from the list consisting of types 31, 45 and 52 HPV, in which the PLV dose of at least other types of cancer is reduced relative to those required otherwise, in the absence of HPV PLV 16 and 18, which generates the same protection against infection by HPV incident and / or persistent by that type another type of cancer.

In one aspect the dose of the other PLV of the cancerous type (not HPV 16, 18) is sufficient to provide protection against incident and / or persistent infection by that type, and in one aspect of the invention the protection of the invention against at least infection. incident.

In one aspect of the invention the composition of the invention is suitable for protection from infection and / or disease in human subjects.

An appropriate dose can be determined by, for example, tests on humans such as those described in the examples herein.

Protection against incident and / or persistent infection by a given type of HPV, such as HPV 16, 18, 31, 45 or 52, protection is conveniently in 50% of a population vaccinated against such infection, and in one aspect of the invention it is 60% protection, in an additional aspect 70% protection, in an additional aspect 80% protection, in an additional aspect 90% protection, in an additional aspect 95% protection, in an additional 96% protection aspect, an additional 97% protection aspect, an additional 98% protection aspect, an additional 99% protection aspect and still an additional 100% protection aspect.

This protection is conveniently evaluated over a period of at least 6 months, such as for a period of at least 9 months, at least 1 year, at least 18 months, conveniently over a period of 2 years or more than 2 years.

In one aspect of the invention, protection is seen against infection or disease caused by HPV 16 and / or HPV 18, and in one aspect infection and / or disease by both HPV 16 and HPV 18.

Infection prevention can be assessed by analysis of HPV species present in vaccinated individuals, for example by PCR analysis and / or hybridization techniques such as those described in WO 03014402 and WO 9914377, incorporated herein as reference.

When the immunogenic composition of the invention comprises both the HPV 16 and HPV PLV then the non-HPV 16/48 cancer type PLV is type 31, or type 45, or type 52, or a combination thereof. In one aspect the immunogenic composition of the invention comprises HPV PLVs 16, 18, 31 and 45. In one aspect the immunogenic composition of the invention comprises HPV PLVs 16, 18, 31 and 52. In one aspect the immunogenic composition of the invention comprises the HPV PLV 16, 18, 45 and 52. In one aspect the immunogenic composition of the invention comprises the HPV PLV 16, 18, 31, 52 and 45. When there are 2 or more other PLVs of the cancerous type (for example, 31, 45, 31 and 52, 45 and 52), then at least one of these other types of cancer is present at a dose that is reduced to that of HPV 16 or HPV 18.

In one aspect the dose of HPV 31 is reduced relative to that of HPV 16.

In one aspect the dose of HPV 52 is reduced relative to that of HPV 16.

In one aspect the dose of HPV 45 is reduced in relation to that of HPV 18.

Conveniently the immunogenic compositions as defined above provide protection against incident infection and / or persistent infection and / or disease caused by HPV 16, HPV 18 and one or more of the other types of HPV (31, 45 or 52) present in the groups listed above, such as incident infection.

When an immunogenic composition of the invention comprises the HPV 16 PLV but not the HPV 18 5 PLV then conveniently the non-HPV 16/18 PLV cancer type is type 31 and / or type 52.

When an immunogenic composition of the invention comprises HPV PLV 18 but not HPV 16 PLV then conveniently the type of HPV 16/18 PLV cancer is type 45.

In one aspect of the invention the composition comprises the HPVs of HPV 16 and 18 in combination with any or the PLVs of both HPV 31 and HPV 45.

In one aspect of the invention the composition comprises at least the HPV PLV 16 in combination with the HPV 31 PLV.

The composition of the invention may comprise, in addition to the PLV at a reduced dose, other HPV PLV at any suitable dose. For example, the composition of the invention may comprise "high risk" cancers such as one or more of HPV 33, 35, 39, 51, 56, 58, 59, 66, or 68.

In one aspect the composition may comprise so-called "genital warts" types such as HPV 6 and / or 11, or also called "skin types" such as HPV 5 and / or 8. In one aspect additional PLVs are present at the same dose or higher than HPV 16 and / or HPV 18.

In one aspect of the invention the composition comprises the HPV of HPV 39 and HPV 51 and the dose of at least one of these is reduced relative to HPV 16 and / or HPV 18.

In one aspect the amount of any additional PLV is selected so as to provide some degree of protection against infection or disease against the additional type (s).

Certain compositions of the invention, individualized below, comprise the following doses of PLV:

Composition number

HPV PLV 16 (μg) HPV PLV 18 (μg) HPV 31 PLV (μg) HPV PLV 16 (μg)

one

twenty twenty 10 10

2

twenty 30 10 10

3

twenty 30 twenty twenty

4

30 twenty 10 10

5

30 twenty twenty twenty

Conveniently there is no significant or biologically relevant interference between HPV PLVs in the

Composition of the invention, so that the combined PLV vaccine of the invention is capable of offering effective protection against infection by each type of VHP PLV represented in the vaccine. Conveniently the immune response against a type of PLV given in the combination is at least 50% of the immune response of the same type of PLV when measured individually, preferably 100% or substantially 100%. For responses of the components of VHP 16 and VHP 18, the combined vaccine of the invention preferably

30 stimulates an immune response that is at least 50% of that provided by a VHP 16 / VHP PLV vaccine

18. Conveniently the immune response generated by the vaccine of the invention is at a level at which the protective effect of each type of PLV is still seen. The immune response can be conveniently measured, for example, by antibody responses using conventional techniques such as ELISA, and by clinical trials as described herein.

In one aspect the composition of the invention does not comprise a heat shock protein or fragment thereof.

In one aspect the composition of the invention does not comprise a V2P L2 protein or peptide. In another aspect the composition of the invention comprises a VHP L2 protein or peptide.

VHP PLVs and procedures for the production of PLVs are well known in the art. The PLV

40 are typically constructed from the virus's L1 and L2 structural proteins, see for example, WO 9420137, WO 9629413 and WO 9405792. Any VHP PLV can be used in the present invention such as LV or L1 + PLV L2

The formation of PLV can be evaluated by conventional techniques such as, for example, electron microscopy and dynamic laser light scattering.

In one aspect of the invention the PLV is a PLV L1 only.

The PLV may comprise the full length L1 protein.

In one aspect of the invention the L1 protein used to form the PLV is a truncated L1 protein. Truncated VHP L1 proteins are described in, for example, US 6361778, incorporated herein by reference. In a further aspect the truncation is a terminal C truncation. In a further aspect, C-terminal truncation eliminates less than 50 amino acids, conveniently and preferably less than 40 amino acids. Conveniently the L1 sequence of VHP 16 begins at the second methionine codon,

10 for example as shown in the sequence below, or similar positions in the other types of VHP. When the PLV is a PLV of VHP 16 then in one aspect the C terminal truncation removes 34 amino acids from the L1 sequence of VHP 16. When the PLV is a PLV of VHP 18 then in one aspect the C terminal truncation eliminates 35 amino acids from the L1 sequence of VHP 18.

In one aspect the sequence of VHP 16 is the following sequence:

The VHP sequence 16 may also be that described in WO 9405792 or US 6649167, for example, conveniently truncated. Suitable truncations are truncated in an equivalent position in the manner shown above, as determined by sequence comparison.

20 In one aspect the sequence of VHP 18 is the following sequence:

An alternative VHP sequence 18 is described in WO 9629413, which may be conveniently truncated. Suitable truncations are truncated at a position equivalent to that shown above, as determined by sequence comparison.

Other sequences of VHP 16 and VHP 18 are known in the art and may be suitable for use in the present invention.

Suitable truncation of HPV 31, VHP 45 and VHP 52 can also be performed, suitably removing the equivalent C terminal portions of the L1 protein to those described above as determined by sequence alignment.

Truncated L1 proteins are described in, for example, WO 9611272 and US 6066324, which are incorporated herein by reference.

In one aspect the truncated L1 proteins are conveniently derived from the functional L1 protein, capable of

5 inducing an immune response (if necessary, when conveniently accompanied by adjuvant), said immune response being able to recognize a PLV consisting of the full-length L1 protein and / or the type of VHP from which the protein is derived L1.

The PLVs of the invention may also comprise other types of functional protein derivatives, including mutants of the full-length or truncated VHP L1 proteins such as suppression mutants,

10 replacement, or insertion. The L1 protein or derivative may also be a fusion protein, such as the fusion of the L1 protein with L2 or an early protein. The L1 protein of the functional protein derivative is capable of forming a PLV, and the formation of PLV can be determined by conventional techniques such as, for example, electron microscopy and dynamic laser light scattering.

PLVs can also be prepared on any suitable cell substrate such as yeast cells or cells

15, for example, baculovuirus cells, and techniques for the preparation of PLVs are well known in the art, such as WO 9913056 and US 6245568, and references therein, the contents of which are incorporated herein as reference.

In one aspect, PLVs are prepared by decoupling and regrouping techniques, which can provide the most stable and / or homogeneous papillomavirus PLVs. For example, McCarthy et al., 1988

20 “Quantitative Disassembly ad Reassembly of Human Papillomavirus Type 11 Viruslike Particles in Vitro” J. Virology 72 (1): 33-41, describes the decoupling and regrouping of purified VHP 11 L1 PLVs from insect cells in order to obtain a homogeneous preparation of the PLV. WO 9913056 and US 6245568 also describe decoupling and regrouping procedures for preparing VHP PLVs.

In one aspect the VHP PLVs of the invention are prepared as described in WO 9913056 and US 25 6245568.

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

It has long been known that enterobacterial lipopolysaccharide (LPS) is a potent stimulator of

30 immune system, although its use in adjuvants has been restricted by its toxic effects. A non-toxic derivative of LPS, lipid A monophosphoryl (MPL), produced by the elimination of the central carbohydrate group and the lower end glucosaline phosphate, has been described by Ribi et al (1986, Immunology and Immunopharmacology of bacterial endotoxins, Plenum Publ Corp., NY, p. 407-419) and has the following structure:

An additional detoxified version of MPL is produced by the removal of the acyl chain from position 3 of the central disaccharide structure, and is called lipid A 3-O-deacylated monophosphoryl (3D-MPL). It can be purified and prepared by the procedures shown in GB 2122204B, whose reference also discloses the preparation of diphosphoryl lipid A, and the 3-O-deacylated variants thereof.

In one aspect the 3D-MPL is in the form of an emulsion having an average particle size of less than 0.2 µm in diameter, and its manufacturing process is described in document 94/21292. Aqueous formulations comprising monophosphoryl lipid A and a surfactant have been described in WO 984367OA2.

The bacterial lipopolysaccharide derived from adjuvants to be formulated in the compositions of the present invention can be purified and processed from bacterial sources, or alternatively they can be synthetic. For example, purified monophosphoryl lipid A is described in Ribi et al., 1986 (above), and monophosphoryl lipid A or 3-O-deacylated diphosphoryl derived from Salmonella sp. It is described in GB 2220211 and US 4912094. Other purified and synthetic lipopolysaccharides have been described (Hilgers et al., 1986, Int. Arch. Allergy. Immunol., 79 (4): 392-6; Hilgers et al., 1987, Immunology, 60 (1): 141-6; and EP 0 549 074 B1). In one aspect the bacterial lipopolysaccharide adjuvant is 3D-MPL.

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

Saponins are described in: Lacaille - Dubois, M and Wagner H. (1996. A review of the biological and pharmacological activities of saponins. Phytomedicine vol. 2 pp. 363-386). Saponins are steroid glycosides or triterpenes widely distributed in the plant and marine animal kingdom. Saponins are observed to form colloidal solutions in water that foam after stirring, and that precipitate cholesterol. When the saponins are near the cell membranes they create pore-like structures in the membrane that causes the membrane to burst. Erythrocyte 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. The adjuvant and hemolytic activity of individual saponins has been studied extensively in the art (Lacaille-Dubois and Wagner, previously). For example, Quil A (derived from the bark of the South American tree Quillaza Saponaria Molina), and the fractions thereof, are described in US 5,057,540 and "Saponins as vaccine adyuvants", Kensil,

C. R., 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), comprising Quil A fractions are hemolytic and have been used in vaccine manufacturing (Morein, B., EP 0 109 942 B1; WO 96/11711; WO 96 / 33739). Hemolytic saponins QWS21 and QS17 (HPLC-purified fractions of Quil A) have been described as potent systemic adjuvants, and the process of their production is described in U.S. 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 other plant species such as Gypsophila and Saponaria (Bomford et al., Vaccine, 10 (9): 572-577, 1992).

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

In one aspect the adjuvant is a particularly potent adjuvant formulation that involves QS21 and 3D-MPL in an oil-in-water emulsion and is described in WO 95/17210.

In accordance with one embodiment of the present invention there is provided a composition accompanied by adjuvant with detoxified lipid A or a non-toxic derivative of lipid A, more preferably accompanied by adjuvant with a monophosphoryl lipid A or derivatives thereof.

In one aspect the composition additionally comprises a saponin, more preferably QS21.

In one aspect the adjuvant formulation additionally comprises an oil-in-water emulsion. The present invention also provides a process for producing a vaccine formulation comprising mixing a PLV of the present invention together with a pharmaceutically acceptable excipient, such as 3D-MPL.

Additional components that are preferably present in a composition accompanied by adjuvant according to the invention include non-toxic detergents such as octoxinols and polyoxyethylene esters as described herein, particularly t-hydroxyapatitaphenoxy polyethoxyethanol (Triton X-100) and polyoxyethylene monooleate sorbitan (Tween 80); and bile salts or derivatives of colic acid as described in

this specification, in particular sodium deoxycholate or sodium taurodeoxycholate. In one aspect the adjuvant formulation comprises 3D-MPL, Triton-100, Tween 80 and sodium deoxycholate, which can be combined with an HPV PLV providing a suitable vaccine.

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

In one aspect the composition of the invention is used in combination with aluminum, and is suitably absorbed or partially absorbed in aluminum adjuvant. Conveniently the adjuvant is an aluminum salt, which in one aspect is in combination with 3D MPL, such as aluminum phosphate and 3D MPL.

In another aspect the adjuvant is aluminum hydroxide, optionally in combination with 3D MPL. In other aspects the composition is the combination of the PLV with an aluminum salt or with an aluminum salt + 3D MPL. In one aspect of the invention the aluminum salt is aluminum hydroxide.

The composition of the invention may also comprise aluminum or an aluminum compound as a stabilizer.

The present invention generally relates to combinations of PLV. However, it is appreciated that the essential component of PLV is an L1 protein. The L1 proteins are associated to form pentamers (capsomeres) that are then tested (grouped) to form the PLV. As such the present invention relates to immunogenic compositions as described above comprising L1 protein, or capsomers comprising L1 protein, instead of PLV as described herein. Conveniently L1 proteins are capable of stimulating a protective immune response. Conveniently L1 proteins are conformationally correct.

For the avoidance of doubt, the invention thus also refers to the use of functional L1 derivatives as described above, such as L1 truncation, suppression, substitution or insertion mutants, and fusion proteins, conveniently those that are capable of causing an immune response capable of recognizing an HPV virus. Capsomeres comprising such proteins are also included in the present invention. Capsomeres as immunogenic agents are described in, for example, WO 0204007. WO 9901557 also describes compositions containing HPV capsomeres. L1 proteins, derivatives and capsomers can be used in the same manner as described for PLVs above.

Thus, the invention can also be seen to refer to an immunogenic composition comprising an L1 protein, or a functional derivative thereof, from HPV 16 and 18 and at least one other cancerous type of HPV, the other type of cancer being selected from the list consisting of HPV types 31, 45 and 52, in which the dose of L1 protein, or derivative thereof, of at least one other type of cancer is reduced relative to that of HPV 16 or 18.

Thus the invention can be seen to refer to an immunogenic composition comprising an HPV capsomer of HPV 16 and 18 and at least one other type of HPV cancer, the other type of cancer being selected from the list consisting of the types HPV 31, 45 and 52, in which the dose of the capsomer of at least one other type of cancer is reduced relative to that of HPV 16 or 18.

In one aspect the invention relates to an immunogenic composition as described above in combination with a pharmaceutically acceptable excipient. Suitable excipients are well known in the art and include for example buffers and water.

The compositions and vaccines of the invention can be provided and distributed by any of a variety of routes such as oral, topical, subcutaneous, mucosal (typically intravaginal), intravenous, intramuscular, intranasal, sublingual, intradermal and suppository.

In one aspect of the invention the composition or vaccine may be formulated or co-administered with an early HPV antigen, for example an antigen selected from the list consisting of HPV E1, E2, E3, E4, E5, E6, E7, and E8. In an alternative aspect vaccine may lack an early HPV antigen, for example an antigen selected from the list consisting of HPV E1, E2, E3, E4, E5, E6, E7 and E8.

Optionally the composition or vaccine can also be formulated or co-administered with non-HPV antigens. Conveniently these non-HPV antigens can provide protection against other diseases, most preferably sexually transmitted diseases such as herpes simplex virus, HBV, chlamydia and HIV. The inventors particularly prefer that the composition or vaccine comprises gD or a truncation thereof.

of VSH, conveniently a C terminal truncation of VSH-2 known as gD2t. In this way the composition or vaccine provides protection against both HPV and VSH.

The dosage of the vaccine composition or components will vary with the condition, sex, age and weight of the individual, the route of administration and HPV of the vaccine. The amount may also vary with the number of types of PLV. Conveniently the distribution is of an adequate amount of vaccine to generate an immunologically protective response. Conveniently each dose of vaccine comprises 1 - 100 µg of each PLV, in an aspect 5 - 80 µg, in an additional aspect 5 - 30 µg of each PLV, in an additional aspect 5 - 20 µg of each! G of each PLV with still additional aspects being specifically 5! g, 6! g, 10! g, 15! g or 20! g.

Doses suitable for use in humans typically include 20-40 µg of HPV 16 and HPV 18 PLV, with reduced doses of the other types of HPV cancer (31, 45, 52) as described in this specification. , conveniently at a level less than 20 µg per PLV, conveniently at a level that is capable of eliciting an immune protective response in at least some vaccinated individuals.

Other doses suitable for use in humans may comprise lower amounts of HPV 16 and / or 18, provided such doses are protective in humans as can be determined using assays indicated herein. For example, such doses may be appropriate when the PLVs of the invention are combined with strong adjuvants.

In one aspect the composition of the invention comprises 20 µg of HPV 16, 20 µg of HPV 18 and between 5-18 µg of each PLV of the other type of cancer (31, 45 or 52), for example 5-15 ! g, and in an additional aspect specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 µg of PLV of each type of non-HPV cancer 16/18.

In one aspect the composition of the invention comprises 10-15 µg of HPV 16, 10-15 µg of HPV 18 and between 5-9 µg of each PLV of the other type of cancer (31, 45 or 52), and in one aspect specifically 5, 6, 7, 8 or 9 µg of PLV of each type of non-HPV cancer 16/18.

In one aspect the weight ratio of PLV of HPV 16 to another PLV of cancerous type (31, 45 or 52) is in the range of 1: 0.9-1: 0.1 (HPV 16: other type), conveniently in the range of 1: 0.9-1: 0.3, conveniently 1: 0.8 1: 0.4.

In one aspect the weight ratio of HPV PLV 18 to another PLV of cancerous type (31, 45 or 52) is between 1: 0.9 1: 0.1, (HPV 18: other type), conveniently in the range from 1: 0.9-1: 0.32, conveniently 1: 0.8-1: 0.4.

In other words, a reduced dose is conveniently 10-90% of the dose of the HPV of HPV 16 or HPV 18, and in one aspect is 20-80% of the dose of the HPV of HPV 16 or HPV 18, in one additional aspect 30 - 70%, still in an additional aspect 30 - 60%, and specifically 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the HPV dose 16 or HPV18.

In one aspect the composition is conveniently used to prevent one or more of: HPV-16 and / or HPV-18 infection, persistent HPV-16 and / or HPV-18 infection and cervical neoplasm associated with HPV-16 and / or HPV - 18.

Conveniently the use of the immunogenic composition of the invention is used to prevent cervical neoplasia and / or incident infection and / or persistent infection associated with infection by other oncogenic types (not HPV 16, 18).

Conveniently the immunogenic composition of the invention is used in the active immunization of adults and adolescent women from the age of 10 years and older. For all the compositions and vaccines of the invention the composition or vaccine is conveniently used for the vaccination of adolescent girls aged between 10 and 15, preferably 10-13 years. The composition or vaccine can also be administered to older adult women after an abnormal PAP smear or after surgery that follows the removal of an HPV lesion, or that is seronegative and negative DNA for HPV cancer types. . 10 women

-

 55 years are another suitable target group. In another aspect the vaccine can be used in girls and women of all ages, from girls onwards, in an additional aspect it can be provided to boys or men. In a further aspect the vaccine can be used therapeutically in women who are seropositive for the HPV virus.

In one aspect the composition of the invention is used to prevent or treat cervical cancer, or CIN I, CIN II or CIN III pathological conditions caused by HPV infection.

In one aspect the invention the vaccine is distributed in a 2 or 3 dose regimen, for example a regime of 0, 1 month or a regimen of 0, 2 months, or a regimen of 0, 2, 6 months or a regimen 0 , 1 and 6 months respectively. Conveniently the vaccination regimen incorporates a booster injection after 3 to 10 years, or 5-10 years, such as preferably 3, 4, 5, 6, 7, 8, 9 or 10 years. In one aspect the composition or vaccine of the invention is a liquid vaccine formulation, although the vaccine can be lyophilized and reconstituted before administration. Topical formulations such as, for example, intravaginal creams can also be used.

The teachings of all references in the present application, which includes patent applications and granted patents, are incorporated herein in their entirety by reference.

The compositions and vaccines of the invention comprise certain components of HPV as set forth above. In a further aspect of the invention the vaccine consists essentially of, or is that they consist of said components.

The present invention is illustrated in this specification as a reference to the following non-limiting examples in relation to cross-protection by HPV 16 and HPV 18 PLVs, and showing the production of HPV PLVs.

Example 1

Healthy women between the ages of 15 and 25 were immunized with a mixture of PLV of L1 from HPV 16 and HPV

18. The women in the enlistment were: 1) seronegative for HPV-16 and HPV-18; 2) negative for high-risk HPV infection of the cervix (detected by HPV PCR); 3) they had 6 or less lifetime sexual partners and 4) they had normal PAP smears.

The mixture comprised, per 0.5 ml dose, 20 µg of PLV of L1 of HPV-16, 20 µg of PLV of L1 of HPV-18 and was accompanied by adjuvant with 500 µg of aluminum hydroxide and 50 ! g of 3D MPL. The placebo group was injected with 500 µg of aluminum hydroxide alone.

The efficacy of the vaccine (V. E.) against HPV types of high-risk cancer was evaluated, in which V. E. is the% improvement in protection against vaccine infection compared to a placebo group.

Cross protection was determined by detecting the presence of specific nucleic acid for various oncogenic types in vaccines and control group. Detection was carried out using techniques as described in WO 03014402, and references in this specification, particularly for non-specific extension of HPV DNA and subsequent detection of DNA types using a LiPA system as described in WO 99/14377, and in Kleter et al., [Journal of Clinical Microbiology (1999), 37 (8): 2508-2517], the total contents of which are incorporated herein by reference.

However, any suitable procedure can be used for the detection of HPV DNA in a sample, such as type-specific PCR that uses specific primers for each type of HPV of interest. Suitable primers are known to those skilled in the art, or can be easily constructed since the sequences of the oncogenic HPV types are known.

The efficacy of the vaccines was determined against infections for the 12 high-risk cancers, phylogenetically related types with HPV-16 (groups of, 31, 35, and 58; 31, 33, 35, 52 and 58) and the phylogenetically related types 30 HPV - 18 (45 and 59).

An initial analysis was carried out on an “ITT” (intention to treat cohort, which represents all individuals who received at least one dose of vaccine). These data are shown in table 1.

The results presented in tables 2 and 3 refer to the “ATP” group (according to the protocol) for those patients who meet all the criteria of the trial. Table 2 is a midpoint analysis with data taken from all patients at the time when at least 50% of the cohort was 18 months after their first vaccination. Table 3 provides the final results, all data being among subjects 18 months after the first vaccination (month 0). In the PTA group all patients received 3 doses of vaccine at months 0, 1 and 6 were seronegative at 6 months.

As demonstrated by the data presented in Table 1, immunization with a mixture of HPV 16 and HPV 18 provided obvious cross protection against other types of HPV. At this point the sample sizes are too small to provide a rigorous statistical analysis, however the data show a positive trend and suggest that immunization with the HPV of HPV 16 and HPV 18 will be effective against infection with other types of HPV.

This was confirmed as the study progressed.

The details of the protocol are further described in example 3.

Table 2 demonstrates that HPV 16 and HPV 18 provide statistically significant cross protection against the group of high-risk cancers 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68.

Table 3 demonstrates that, except for HPV-18-related types (which show a very low trend), there is statistically significant cross protection against the groups of: HPV 31, 35, 58; HPV 31, 33, 35, 52, 58; and the 12 high-risk types (non-HPV types - 16/18) evaluated.

Subsequent analysis of the test data has indicated that the HPV vaccine 16 and 18 used in examples 1

provides statistically significant cross protection against statistical infection caused by HPV 31 (vaccine efficacy 75.1%, p = 0.007). While the sample size does not yet allow statistically significant conclusions to be drawn on other types, data on other types such as 39, 45, 51 and 52 demonstrate a positive trend and suggest that immunization with HPV of HPV 16 and HPV 18 will be effective against infection with other types of HPV.

The data presented in Example 3 provide additional data obtained in the study sample, and focuses on the cross-protection provided against certain specific types.

Table 1

Types of HPV analyzed

Numbers of infected women (vaccine group) % of infected women (vaccine group) = A Numbers of infected women (placebo group) % of infected women (placebo group) = B % vaccine efficacy (1 (AB) x 100, adjusted for relative vaccine size and placebo group 95% confidence limits - lower limit 95% confidence limits - upper limit P

HPV 31, 35, 58

5 1.1 eleven 2.4 55.1  -29.1 84.4 0.127

HPV 31, 33, 35, 52, 58

17  3.8 24 5.4 30.3 -29.7 62.6 0.222

HPV 45, 59

3 0.7 6 1.3  50.6 -99.7 87.6 0.309

HPV 31,33,35,39, 45, 51, 52, 56, 58, 59, 66, 68

27 6.3 40 9.4 34.6 -6.5 59.9 0.086

10 Patient samples were taken at 9, 12, 15 and 18 months and tested for HPV infection by the other types specified above.

Table 2 - Efficacy of vaccine after three doses in the prevention of incident heterologous infections.

Table 2: Efficacy of vaccine against infection with phylogenetically related types with HPV - 16, types 15 phylogenetically related with HPV - 18, phylogenetically related types with HPV - 16 and / or HPV - 18 and all exclusive high risk exclusive types of HPV - 16 and HPV - 18 cohort (month 6 - 18)

Type of infection

Attack rate Vaccine efficacy

Vaccine

Placebo

N n AR

N n AR % Cla 95% P value

HPV -16 related

446 12 28 438 24 55 49.4 0.2 74.4 0.060

HPV-16 related *

423 29 6.9 423 46 6.9 37.0 1.6 59.6 0.052

HPV -18 related

442 9 2.0 449 16 3.6 42.9 -27.9 74.5 0.223

Related to HPV -16/18

433 21 4.9 438 41 9.4 48.2 13.8 68.9 0.012

Related to HPV -16/18

423 34 8.0 423 56 13.2 39.3 9.0 59.5 0.019

High risk **

383 53 13.8 386 88 22.8 39.6 17.7 55.7 0.001

N = number of subjects in the specified cohort n = number of subjects with HPV infection incident AR = attack rate = n / N 95% CI = 95% confidence interval

Lower limit = 1 - exp (log (arvg / arp) + 1.96 * square root of (1 / nv - 1 / Nv + 1 / np - 1 / Np))

Upper limit = 1 - exp (log (arv / arp) - 1.96 * square root of (1 / nv - 1 / Nv + 1 / np - 1 / Np)) when the number of cases in vaccine = 0 Lower limit * = 1 - exp (log (arvg * / arp *) + 1.96 * square root of (1 / (nv + 0.5) - 1 / (Nv + 0.5) + 1 / (np + 0, 5)

1 / (Np + 0.5)))

Upper limit * = 1 - exp (log (arv * / arp *) - 1.96 * square root of (1 / (nv + 0.5) - 1 / (Nv + 0.5) + 1 / (np + 0.5) -

1 / (Np + 0.5)))

with: arv = attack rate in vaccine recipients

arp = placebo receptor attack rate

nv = number of cases in vaccine recipients

Nv = number of cases and not cases in vaccine recipients

np = number of cases in placebo recipients

Np = number of vessels and no cases in placebo recipients

related to HPV - 16: types 35, 31, 58 phylogenetically related to HPV - 16 without considering others

HPV types

related to HPV - 16 *: types 35, 31, 58, 33, 52 phylogenetically related to HPV - 16 without considering other types of HPV related to HPV - 18: types 45, 59 related phylogenetically to HPV - 16 without considering other types

HPV

related to HPV - 16 and / or HPV - 18: types 35, 31, 58, 45, 59 phylogenetically related to HPV - 16 and / or HPV - 18 without considering other types of HPV related to HPV - 16 and / or HPV - 18 *: types 35, 31, 58, 33, 52, 45, 59 phylogenetically related to

HPV - 16 and / or HPV - 18 without considering other types of HPV * * = high-risk types exclusive to HPV - 16 and HPV - 18

(Cont.) Table 3

Type of HPV analyzed

Total number of subjects with information available per group Number of infected women (vaccine group) % of infected women (vaccine group) = A Number of infected women (placebo group) % of infected women (placebo group) = B % vaccine efficacy (1 - (AB) x 100 adjusted for relative vaccine size and placebo group 95% confidence limit - lower limit 95% confidence limit - upper limit P

HPV 31, 35, 58

412 eleven 2.7 26 6.3 57.9 15.9 78.9 0.012

HPV 31, 33, 35, 52, 58

403 28 6.9 48 12.2 43.0 11.0  63.5  0.015

HPV 45, 59

421  10 2.4 fifteen 3.6 33.5 -46.3 69.8 0.319

HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68

368 58 15.8 90 25.3 37.7 16.2  53.6  0.002

Samples were taken from patients at 18 months and tested for HPV infection by the other types specified above.

Example 2

5 The HPVs of HPV 16 and HPV 18 were produced as follows:

Example 1:

The combination of the HPVs of HPV 16 and HPV 18 is detailed in this specification. The L1 protein of other HPV genotypes can be easily produced by similar procedures, known in the art.

10 A Preparation of HPV PLV 16/18

HPV production of HPV 16 and HPV 18 was carried out using conventional protocols - for example, see WO 9913056, HPV 16/18 proteins are expressed in Trichoplusia ni (High Five ™) cells (at a density of approximately 350000 cells / ml) infected with recombinant baculovirus (MOI of 0.3) encoding the 16 or 18 HPV L1 gene of interest. Cells were collected approximately 72 hours after

15 the infection.

4.1 B Cell collection / antigen extraction

The antigen (L1-16/18) was extracted from Hi5 cells in a three-stage process of concentration, extraction, clarification. The concentration stage removes up to 90% of the culture medium, and was performed by tangential flow filtration. The extraction stage was performed with a hypoptonic buffer (20 mM Tris, pH 8.5). An equal volume

20 to the volume of the crop was used to perform the extraction. A contact time of at least one hour and a half was used in gentle agitation. Clarification was performed by tangential flow filtration.

C Purification

The purification procedure was carried out at room temperature. �-Mercaptoethanol (4% w / w) was added to the extract in order to dissociate the PLVs into capsomers, for both antigens, L1-16/18. Glycerol was added up to a concentration of 10% by weight just before the addition of �-mercaptoethanol.

All buffers used were filtered on 0.22 µm filters before storing at 2 ° C - 8 ° C. Before each purification, the gel matrices are sanitized and equilibrated with appropriate buffer before loading the sample.

Purification regimens are provided for separate purification of L1 from both HPV 16 and HPV 18. These schemes are broadly similar in meaning, and involve the steps of:

30 Anion exchange chromatography (Di methyl amino ethyl - DMAE), Anion exchange chromatography (Tri methyl amino ethyl - TMAE), Hydroxyapatite chromatography, Nanometric filtration (Planova), Ultrafiltration,

35 Hydrophobic interaction chromatography (using octyl sepharose) for HPV 18 or anion exchange chromatography (DEAE) for HPV 16; and sterile filtration.

4.1.1 Specifically:

4.1.2 C1 Purification of L1-18 antigen

40 4.1.2.1 DMAE anion exchange chromatography

The clarified extract (protein at a concentration of approximately 1g / ml, with the L1 protein at approximately 150 mg / ml) is applied to an anion exchange column (Di methyl amino ethyl). The elution is

performed with buffer (20 mM Tris / 200 mM NaCl / �-BME mercaptoethanol 4%) pH 7.9 ± 0.2. The antigen is eluted with approximately 5 column volumes and the elution profile is controlled at 280 nm.

4.1.2.2 TMAE anion exchange chromatography

The eluate from the first stage is diluted with 1 volume of 4% H2O / BME. The diluted eluate is then applied to a second anion exchange column (Trimethyl amino ethyl).

Elution is performed with buffer (20 mM Tris / 200 mM NaCl / 4% BME) pH 7.9 ± 0.2. The antigen is eluted with approximately 4 column volumes and the elution profile is controlled at 280 nm.

4.1.2.3 Hydroxyapatite chromatography The eluted from the TMAE stage is applied to a hydroxyapatite (HA) column. After sample application, the gel is eluted with approximately 2.5 volumes of buffer column

(100 mM NaH2PO4 / 30 mM NaCl / 4% BME), pH 6.0 ± 0.2.

4.1.2.4 Nanometric filtration (Planova)

The eluate of HA is diluted in order to reach the following conditions: buffer (25 mM NaH2PO4 / 10 mM NaCl / 4% BME), pH 7.5 ± 0.2.

It is then filtered successively on a 0.2 µm prefilter and on a 0.12 m2 15 N filter. Filtration is carried out at a constant pressure of 200 mbar (20 kPa) ± 20 (2 kPa) mbar.

4.1.2.5 Ultrafiltration

Ultrafiltration is performed with a tangential flow ultrafiltration system equipped with polyethersulfone membranes (Centramate module 0.1 m2, 100 kD).

The eluted from Planova is treated to reach the following conditions: (100 mM NaH2PO4 / 30 mM NaCl / 4% BME), pH 6.0 ± 0.2; It is then loaded into the system, concentrated five times and diafiltered with continuous injection of approximately 10 starting volumes of buffer (20 mM NaH2PO4 / 500 mM NaCl), pH 6.0 ± 0.2.

4.1.2.6 Hydrophobic interaction chromatography (Octil Sepharose)

The ultrafiltration permeate is applied to an octyl Sepharose column. This chromatography step is carried out in the negative mode with approximately 5 column volumes of buffer (20 mM Na3PO4 / 500 mM NaCl), pH 6.0 ± 0.2.

4.1.2.7 Sterile filtration The purified L1-18 antigen solution is sterilized by filtration on a 0.22 µm membrane.

4.1.4 C2 Purification of L1-16 antigen

4.1.4.1 DMAE ion exchange chromatography

The clarified extract is applied to an anion exchange column (Di methyl amino ethyl). Elution is performed with buffer (20 mM Tris / 180 mM NaCl / 4% BME) pH 7.9 ± 0.2. The antigen is eluted with approximately 4 column volumes and the elution profile is controlled at 280 nm.

4.1.4.2 TMAE anion exchange chromatography

The eluate from the first stage is diluted with 1 volume of 4% H2O / BME. The diluted eluate is then applied to a second anion exchange column (Trimethyl amino ethyl).

Elution is performed with buffer (20 mM Tris / 180 mM NaCl / 4% BME) pH 7.9 ± 0.2. The antigen is eluted with approximately 5 column volumes and the elution profile is controlled at 280 nm.

4.1.4.3 Hydroxyapatite (HA) chromatography The eluted from the TMAE stage is applied to an HA column. After application of the sample, the gel is eluted with approximately 3 volumes of buffer column

(100 mM NaH2PO4 / 30 mM NaCl / 4% BME), pH 6.0 ± 0.2.

4.1.4.4 Nanometric filtration (Planova)

The eluate of HA is diluted in order to reach the following conditions: buffer (25 mM NaH2PO4 / 10 mM NaCl / 4% BME), pH 7.5 ± 0.2.

It is then filtered successively on a 0.2 µm prefilter and on a 0.12 m2 15 N filter. Filtration is carried out at a constant pressure of 200 mbar (20 kPa) ± 20 (2 kPa) mbar.

4.1.4.5 Ultrafiltration

Ultrafiltration is performed with a tangential flow ultrafiltration system equipped with polyethersulfone membranes (Centramate module 0.1 m2, 100 kD).

The eluted from Planova is treated to reach the following conditions: (100 mM NaH2PO4 / 30 mM NaCl / 4% BME), pH 6.0 ± 0.2; It is then loaded into the system, concentrated five times and diafiltered with continuous injection of approximately 10 starting volumes of buffer (20 mM NaH2PO4 / 500 mM NaCl), pH 6.0 ± 0.2.

4.1.4.6 DEAE anion exchange chromatography

The ultrafiltration eluate is adjusted to the conductivity of the equlibrio buffer, (20 mM Na3PO4 / 250 mM NaCl), pH 6.0 ± 0.2 and is applied to an anion exchange column / Di ethyl amino ethyl).

Elution is performed with buffer (20 mM NaH2PO4 / 500 mM NaCl), pH 6.0 ± 0.2. The antigen is eluted with approximately 3 column volumes and the elution profile is controlled at 280 nm.

4.1.4.7 Sterile filtration The purified L1-16 antigen solution is sterilized by filtration on a 0.22 µm membrane. C3 Each type of PLV is adsorbed independently to produce a concentrated absorbed monovalent. Preparation of concentrated monovalent adsorbed PLV16:

60 µg of purified HPV of HPV16 are adsorbed in 150 µg of Al + 3 of Al (OH) 3, at a pH of 6.0 ± 0.2, for one hour at room temperature with gentle agitation. This concentrated monovalent adsorbed is stored at + 4 ° C. Adsorption is checked by centrifugation of the preparation and quantification of the PLVs in the supernatant.

Preparation of concentrated monovalent adsorbed PLV18: 60 µg of purified HPV of HPV18 are adsorbed in 150 µg of Al + 3 of Al (OH) 3, at a pH of 6.0 ± 0.2, for one hour at room temperature with gentle agitation. This concentrated monovalent adsorbed is stored at + 4 ° C. Adsorption is checked by centrifugation of the preparation and quantification of the PLVs in the supernatant.

D. Preparation of final vaccine

The concentrated monovalent adsorbed prepared by the above procedure can be combined to form a suspension containing 20 µg of each of the PLVs per dose. The final vaccine is stored at + 4 ° C.

The addition of PLVs from other types of cancer can be added as appropriate, at a suitable concentration according to the invention. Sequences of such types are known in the art and those skilled in the art can easily express PLVs comprising such proteins.

The combined adsorbed masses, or individual adsorbed masses, can also be mixed with adjuvants such as 3D-MPL.

Example 3

The precise details of the experiment carried out are provided in Harper et al., The Lancet 2004 Nov 13; 364 (9447): 1757-65.

In summary, healthy women between the ages of 15 and 25 were immunized with a mixture of HPV L1 PLV

18. The women in the enlistment were: 1) seronegative for HPV-16 and HPV-18; 2) negative for high-risk HPV infection of the cervix (detected by HPV PCR); 3) they had 6 or less lifetime sexual partners and 4) they had PAP smears.

The mixture comprised, per dose of 0.5 ml, 20 µg of PLV of L1 of HPV-16, 20 µg of PLV of L1 of HPV-18 and was accompanied by adjuvant with 500 g of aluminum hydroxide and 50! g of 3D MPL. The placebo group was injected with 500 µg of aluminum hydroxide alone.

HPV 16 PLVs consisted of a 471 amino acid HPV L1 protein, terminally truncated at C, with a 34 amino acid suppression. HPV 18 PLVs are constituted by a L1 HPV protein terminally truncated at C of 472 amino acids, with a 35 amino acid suppression.

The efficacy of the vaccine (V. E.) against certain cancerous HPV types was determined, in which V. E. is the% improvement in protection against infection by the vaccine compared to a placebo group.

Cross protection was determined by detecting the presence of specific nucleic acid for various oncogenic types in vaccines and control group. Detection was carried out using techniques as described in WO 03014402 and references therein, particularly for non-specific amplification of HPV DNA and subsequent detection of DNA types using a LiPA system as described in the document. WO 99/14377, and in Kleter et al., [Journal of Clinical Microbiology (1999), 37 (8): 2508-2517], whose total content is incorporated herein specifically by reference.

However, any suitable procedure can be used for the detection of HPV DNA in a sample, such as type-specific PCR using primers specific for each type of HPV in a sample. Suitable primers are known to those skilled in the art, or can be easily constructed as long as the sequences of the oncogenic HPV types are known.

In detail, the procedure section of the Lancet publication is reproduced in this specification as a reference, for full view:

Harper et al., The Lancet, November 13, 2004; 364 (947); 1757-65 - experimental details. The main objective of this study was to determine the efficacy of the vaccine in preventing infection with HPV - 16, HPV - 18, or both (HPV - 16/489, between months 6 and 18 in participants who were initially shown to be seronegative for HPV - 16/18 by ELISA and negative for HPV DNA 16/18 by PCR The secondary objectives included: evaluation of vaccine efficacy in the prevention of persistent infection with HPV - 16/18, and evaluation of efficacy of the vaccine in the prevention of cytologically confirmed low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and LSIL (CIN 1), HSIL (CIN 2 or 2) squamous cell cancer, histologically confirmed, adenocarcinoma associated with HPV - 16/18 infection between months 6 and 8, and months 6 and 27. The prevention of atypical squamous cell cytology undetermined significance (ASCUS) associated with HPV - 16/18 infection post-hoc was added to the analysis of the results.

The inventors did not perform an exploratory analysis of the histopathological CIN 1 and 2 endpoints associated with HPV-16/18 DNA detected by PCR in injured tissue. Other objectives included the determination of vaccine immunogenicity, safety, and tolerance.

Researchers in North America (Canada and the United States) and Brazil recruited women for this efficacy study through announcements or prior participation in the HPV cross-sectional epidemiology study that took place between July and December 2000.

For each of the 32 study sites, an institutional review panel approved the protocol, consensus forms, and amendments. The women signed separate written consents for participation and colposcopy. For those under 18, parental consent and participant consent were mandatory

There were two phases of the study: an initial phase for vaccination and the follow-up that ended in the 18th month; and a blind follow-up extension phase that ended on month 27.

Women who can be chosen for the initial phase (months 0-18) included healthy women aged 15-25 years, who had not had more than 6 sex partners, with no history of an abnormal Pap trial or treatment by ablation or excision of the cervix, and no ongoing treatment for external condylomata; and that they were cytologically negative, seronegative for HPV-16 and HPV-18 antibodies by ELISA, and negative for HPV DNA by PCR for 14 high-risk types of HPV (16, 18, 31, 33, 35, 39, 45 , 51, 52, 56, 58, 59, 66, and 68) no more than 90 days before the start of the study.

Women who completed the initial phase of the earliest of studies, and who did not have ablation therapy or excision of the cervix, or hysterectomy after enlistment, could be chosen to participate in the extension phase of the study (months 18 - 27 ).

Procedures

Each dose of particle vaccines that look like HPV-16/18 virus (GlaxoSmithKline Bilogicals, Rixensart, Belgium) contained 20 µg of particle that looks like HPV L1 virus - 16 and 20 µg of particle that looks like L1 virus de! g - 18. Each type of particle that looks like virus was produced on Spodoptera frigiperda Sf cell substrate

-

 9 and Trichoplusia ni Hi-5 with adjuvant AS04 containing 500 µg of aluminum hydroxide and 50 µg of 3- 3-deacylated monophosphoryl lipid (MPL, Corixa, Montana, United States) provided in a single dose vial. The placebo contained 500 µg of aluminum hydroxide per dose, and was identical in appearance to the! G - 16/18 vaccine. Each study participant received a dose of 0-5 ml of vaccine or placebo at 0 months, 1 month, and 6 months.

Healthcare Providers obtained samples of the cervix with a brush and spatula of the cervix (washed in PreservCyt, Cytyc Corporation, Boxborough, MA, United States) for HPV cytology and DNA testing at selection and 6, 12 months , and 18. In months 0 and 6, and subsequently every 3 months, cervicovaginal samples obtained from the women themselves with two samples with successive swabs (placed in PreservCyt) for HPV DNA assay. [DM Harper, WW Noll, DR Belloni and BF, Cole, Randomized clinical trial of PCR - determined human papillomavirus detection methdos: self - sampling versus clinician - directed - biologic concordance and women’s preferences. Am. F Obstet Gynecol 186 (2002), pp. 365-373]. A central laboratory (Quest Diagnostics, Teterboro, NJ, United States) reviewed cytological results (ThinPrep, Cytyc Corporation) using the 1991 Bethesda classification system.

The protocol guidelines recommended colposcopy after two reports of ASCUS, or a report of atypical glandular cells of undetermined significance, LSIL or HSIL, squamous cell carcinoma, adenocarcinoma in situ, or adenocarcinoma. These guidelines also recommended biopsy for any suspicious lesions.

The central histology laboratory made an initial diagnosis of tissue samples fixed by formalin for clinical treatment. A panel of three pathologists made a subsequent consensus diagnosis for lesions associated with HPV-16 and HPV-18 with the CIN system. This consensus diagnosis also included a review of the sections taken at the time of microsection for the PCR detection of HPV DNA from injury.

HPV DNA isolated from the cytological sample (MagNaPure Total Nucleic Acid system, Roche Diagnostics, Almere, The Netherlands) and from cervical biopsy samples (proteinase K extraction) was amplified from an aliquot of total DNA purified with the SPF10 broad spectrum primers that amplify a 65 base pair region of the L1 gene [B. Kleter, LJ van Doom, J ter Schegget et al., Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses. Am J Pathol 153 (1998), pp. 1731-1739: LJ van Doom, W Quint, B Kleter et al., Genotyping of human papillomavirus in liquid cytology cervical specimens by the PGMY line blot assay and the SPF (10) line probe assay. J Clinb Microbiol 40 (2002), pp. 979 983 and WG Quint, G Scholte, LJ van Doom, B Kletter, PH Smits and J. Lindeman, Comparative analysis of human papilloma virus infections in cervical scrapes and biopsy specimens by general SPF (10) PCR and HPV genotyping.

J. Pathol 194 (2001), pp. 51-58]. Amplification products were detected by a DNA enzyme immunoassay. A linear probe test LiPA kit, HPV INNO LiPA HPV genotype identification test, SPF-10 system, version 1, Inogenetics, 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). [B. Kleter, LJ van Doom, L Schauwen et al., Development and clinical evaluation of a highly sensitive PCR - reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus J. Clin Microbiol 37 (1999), pp. 2508-2517]. Any sample that was positive by DNA enzyme immunoassay was tested by HPV-16 and type-specific HPV-18 PCR. The HPV-16 type specific PCR primers amplified a 92 base pair segment of the E6 / E7 gene and HPV-18 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 first pairs for detection of human papillomavirus DNA by PCR in paraffin - embedded cervical carcinomas. J. Clin Microbiol 34 (1996), pp. 745-747]

The inventors defined invention of the cervix incident with HPV-16/18 with at least one positive PCR result for HPV-16 or HPV-18 during the trial, and persistent infection with HPV-16/18 as at least two HPV assays - Positive DNA for the same viral genotype separated in at least 6 months. [H Richardson, Kelsall, P Tellier et al., The natural history of type - specific human papillomavirus infections in female university students. Cancer Epidemiol Biomarkers Prev 12 (2003), pp. 485-490 and AB Moscicki, JH Ellenberg, S Farhat and J. Xu, Persitence of human papillomavirus infection in HIV - infected and uninfected adolescent girls: risk factors and differences, by phylogenetic type. J. Infect Dis 190 (2004), pp. 37-45] The results of HPV-DNA assays were hidden from the researchers during the study and cytological and histological diagnoses were revealed for clinical treatment purposes only. The analyzes included HPV-16/18 DNA results for cervical samples and combined cervical and cervicovaginal samples.

The inventors collected serum from the study participants at months 0, 1, 6, 7, 12, and 18 for immunogenicity determinations. The sexological test for antibodies to particles that look like HPV-16 and HPV-18 viruses was by ELISA. Particles that look like HPV-16 and HPV-18 viruses were used as

coating antigens for antibody detection (see the appendix of the website http://imaee.thelancet.com/extras/04art10103webappendix.pdf). Seropositiovity was defined as a titer greater than or equal to the cut-off titer of the assay set at 8 units / ml of ELISA for HPV-16 and 7 units / ml of ELISA for HPV-18. Typical natural titres were determined by 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.

The women recorded symptoms experienced during the first 7 days after vaccination on daily cards with a scale of three degrees of symptom intensity. Additionally, they reviewed the study staff by interviewing all adverse events in the first 30 days after vaccination. Information on serious adverse events and pregnancies was collected throughout the study.

Statistical procedures

Assuming a cumulative incidence rate of 60 infections of both the HPV-16 and HPV-18 type over 12 months, the inventors estimated that 500 women per treatment group would provide 80% power to assess a lower limit of 95% CI of vaccine efficacy above zero. The inventors assumed an 80% retention rate for 18 months. Provisional analyzes for efficacy, safety, and immunogenicity were made for future study planning purposes only; the O'Brien and Fleming procedure was used to adjust the value for the final analysis after the interim analyzes took place (p overall = 0.05; two-way trial). [PC O’Brien and TR Fleming, A multiple testing procedure for clinical trials. Biometrics 35 (1979), pp. 549-556]

The randomized stratified, block distribution according to validated algorithms was centralized with a random internet distribution system. Stratification was according to age (15-17, 18-21, and 22-25 years) and region (North America and Brazil). Each vaccine dose was attributed to a randomly chosen number based on the specific participant's information embedded in the computerized randomized distribution system by the study staff. The location of the treatment remains hidden for researchers and women participating in a long-term follow-up study.

The intention-to-treat and according-to-the-protocol cohorts are shown in the figure, in which the reasons for the exclusion of the analyzes are listed in order of category women who met more than one exclusion criterion were counted only according to the highest category criterion. The inventors refer to sets of participants introduced in the intention-to-treat and according-the-protocol analyzes as cohorts, although the information used to restrict the inclusion of subjects in the according-the-protocol was only known after follow-up. .

The inventors performed both according to the protocol and intention to treat for efficacy. The calculation of vaccine efficacy in the analysis according to - the - protocol was based on the proportion of participants with HPV - 16/18 infection in the groups vaccinated against placebo. Vaccine efficacy was defined as 1 minus the relationship between these two proportions; 95% CI measured the accuracy of efficacy estimates, p values were calculated with Fisher's exact two-way test. The corresponding rates were expressed as the number of cases with the result divided by the number of participants at risk. The cohort according to the 18-month protocol included enlisted women who received three scheduled doses of vaccine and complied with the protocol as described in the figure.

The calculation of vaccine efficacy in the intention-to-treat and according-to-the-27-month protocol analyzes was based on the Cox proportional model using the time of occurrence of cases with HPV infection 16/18 in the groups. vaccinated versus placebo. This allowed control for the accumulated person-time data. Vaccine efficacy was calculated using 1 minus the chance ratio and p values calculated using the category log test. The corresponding rates were expressed as the number of cases divided by the total time person. All enlisted women who received at least one dose of vaccine or placebo, were negative for HPV-high-risk DNA at month 0, and had any data available for the measurement of results were included in the intention-to-treat cohort. The cohort according to the 27-month protocol included the results of the cohort according to the 18-month protocol and the results that occurred during the extension phase (from 18 months to 27 months).

The calculation of p values for safety analyzes was performed using the Fisher's exact test comparisons. The cohort for safety analyzes included all enlisted women who received at least one dose of vaccine or placebo and met the minimum, specified protocol requirements (see figure below).

Immunogenicity was assessed in a subset of the safety cohort according to -the protocol that included women with serology results in months 0, 7, and 18, who received the three doses of vaccine or placebo from the study according to the program, met with the blood sampling program, and they did not become positive for HPV-16/18 DNA during the trial. The seropositivity rates between the vaccine and placebo groups were compared with Fisher's exact trial (p <0.001 judged significantly). The geometric mean of the titles was compared with ANOVA and Kruskal - Wallis essay.

Randomized block and statistical distribution analyzes were performed with SAS version 8.2 (SAS Institute, Cary, North Carolina).

Results

The results of the initial cross-protection analysis are presented in patent application WO 2004/056389, the total content of which is incorporated herein by reference.

5 Additional analysis

An analysis was carried out on an “ATP” group (according to protocol) for those patients who met all the criteria of the trial. In the ATP group, all patients received 3 doses of vaccine at months 0, 1 and 6 and were seronegative at 6 months.

As demonstrated by the data presented in Table 4, immunization with a mixture of HPV PLV 16

10 and HPV 18 provided statistically significant cross protection against infection caused by HPV types 31, 52 and 45 compared to the control.

The statistically significant cross-protection incident infection was also observed against the group of all HPV-related types 16 (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).

15 Statistically significant cross protection against persistent infection was also observed against types 31 and 52 (see table 5), and was also observed against the group of all HPV-related types 16 (see table 5).

Statistically significant cross protection was also observed against cytological abnormalities associated with HPV 52, see table 6. Statistically significant protection was also observed against the group of all

20 the types related to HPV 16 (HPV - 31, 33, 35, 52, and 58) and the group of all high-risk types, excluding 16 and 18 (31, 33, 35, 39, 45, 51, 52 , 56, 58, 59, 66, and 68).

Table 4

Efficacy against incident infections with types related to 16/18 *

* Uterus necks samples: ATP cohort

Table 5

Efficacy against persistent infections with types related to 16/18 *

* All samples; ATP cohort

Table 6 Efficacy against cytological abnormalities. With the types related to 16/18 *

5 * ATP cohort

In tables 4, 5 and 6

N = number of subjects in the specific cohort AR = attack rate = n (number of subjects with HPV or incident infection, persistent infection or cytological abnormality, as appropriate for the table) / N

10% vaccine efficacy is 1 - (A / B) x 100, adjusted for the relative size of the vaccine group and placebo, in which A =% of women in the vaccine group with incident infection, persistent infection or abnormality cytological, as appropriate for the table

15 B =% of women in the placebo group with incident infection, persistent infection or cytological abnormality, as appropriate for the table

Claims (18)

one.

 An immunogenic composition comprising the PLV or capsomers of HPV 16 and HPV 18 and at least one other type of HPV cancer, the other type of cancer being selected from the list consisting of types of HPV 31, 45 and 52, in the that the dose of the PLV or capsomer of at least one other type of cancer is reduced in relation to that of HPV 16 or 18.

2.

 A composition according to claim 1 wherein the other type of cancer is HPV 31.

3.

 A composition according to claim 1 wherein the other type of cancer is HPV 45.

Four.

 A composition according to claim 1 wherein the other type of cancer is HPV 52.

5.

 A composition according to claim 1 wherein the other types of cancers are HPV 31 and HPV 45.

6.

 A composition according to claim 1 wherein the other types of cancers are HPV 31 and HPV 52.

7.

 A composition according to claim 1 wherein the other types of cancers are HPV 52 and HPV 45.

8.

 A composition according to claim 1 wherein the other types of cancers are HPV 31, HPV 45 and HPV 52.

9.

 An immunogenic composition according to any one of the preferred claims wherein the composition comprises 10 µg, or more, of the HPVs of HPV 16 and / or HPV 18 or capsomer and between 2-9 µg of PLV or capsomer of the other type Of cancer.

10.

 An immunogenic composition according to any one of claims 1-8 wherein the composition comprises 20 µg, or more, of the HPVs of HPV 16 and / or HPV 18 or capsomeres and between 5-15 µg of PLV or capsomer of Another type of cancer.

eleven.

 An immunogenic composition according to claim 10 wherein the composition comprises 10 µg of PLV or capsomer of the other type of cancer.

12.

 An immunogenic composition according to any one of claims 1-11 in combination with an adjuvant.

13.

 A composition according to claim 12 wherein the adjuvant is an aluminum salt.

14.

 A composition according to claim 13 wherein the adjuvant is aluminum hydroxide.

fifteen.

 A composition according to claim 12 wherein the adjuvant is a derivative of lipid A.

16.

 A composition according to claim 15 wherein the adjuvant is 3D MPL.

17.

 A composition according to claim 16 wherein the adjuvant is 3D MPL and aluminum hydroxide.

18.

 A vaccine comprising an immunogenic composition according to any preceding claim with a pharmaceutically acceptable excipient.