CN1976718B

CN1976718B - Vaccine against HPV 16 and HPV 18 and at least another HPV type selected from HPV 31, 45 or 52

Info

Publication number: CN1976718B
Application number: CN2005800195155A
Authority: CN
Inventors: S·德布鲁斯; M·-T·马丁; R·J·斯蒂芬; J·斯蒂芬尼; M·A·C·维滕多夫
Original assignee: GlaxoSmithKline Biologicals SA
Current assignee: GlaxoSmithKline Biologicals SA
Priority date: 2004-06-16
Filing date: 2005-06-14
Publication date: 2012-10-10
Anticipated expiration: 2025-06-14
Also published as: NL300774I2; NL300774I1; NL300771I1; GB0413510D0; CY1113830T1; JP2012102132A; PT1758609E; SI1758609T1; NL300771I2; TW200612983A; ZA200610057B; DK1758609T3; CN1976718A; HUS1500061I1; LTC1758609I2; NL300773I2; KR20120123617A; HUS1500059I1; NL300773I1; KR101359943B1

Abstract

An immunogenic composition and methods for producing said composition, the composition comprising VLPs from HPV 16 and 18 and at least one other HPV cancer type, the other cancer type being selected from the list consisting of HPV types 31, 45 and 52, wherein the dose of the VLP of the at least one other cancer types is reduced relative to that of HPV 16 or 18.

Description

Anti-HPV16 and HPV18 and at least a other vaccine that is selected from HPV31,45 or 52 HPV type

The present invention relates to anti-human papillomavirus's vaccine

Background of invention

Human papillomavirus is little DNA oncovirus, has the height species specificity.Up to now, individual human papillomavirus (HPV) genotype on the books is above 100 kinds.HPV has specificity to skin (for example HPV-1 and-2) or mucomembranous surface (for example HPV-6 and-11) usually, generally can cause to continue several months or several years benign tumor (wart).This type benign tumor may cause ill worries, but except few exceptions, can not threaten to life.

Some HPVs also with related to cancer, be called as carcinogenic HPV type.The strongest sure contact is to be present in getting in touch between HPV-16 and HPV-18 and the cervical cancer between HPV and human cancer.In developing country, cervical cancer is modal malignant tumor, annual in the world newly-increased about 500,000 new cases.

There is special other HPV that gets in touch that type 31,33,35,39,45,51,52,56,58,59,68,73,82,26,53 and 66 is arranged with cancer.

The someone proposes, and HPV virus-like particle (VLP) might become the vaccine that is used to treat HPV.Confirm to use the bivalent vaccine of VLP can effectively prevent the young woman and infected HPV16 and 18.

Still need resist the vaccine of multiple (for example＞2) HPV type.

The present invention has satisfied this needs.

Summary of the invention

On the one hand; The present invention relates to immunogenic composition; Comprise from HPV16 and 18 with the VLP of at least a other HPV cancer types, other cancer types is selected from: HPV type 31,45 and 52, wherein the dosage of the VLP of at least a other cancer types is less than the dosage of HPV16 or 18.

On the other hand, the present invention relates to comprise the immunogenic composition from the VLP of HPV16 and at least a other HPV cancer types, other cancer types is selected from HPV type 31 and 52, and wherein the dosage of at least a other cancer types is less than the dosage of HPV16.

At related aspect, the present invention relates to comprise immunogenic composition from the VLP of HPV18 and HPV45, wherein the dosage of HPV45VLP is less than the dosage of HPV18.

The invention further relates to the immunogenic composition of as above definition and the combination of auxiliary agent and/or carrier.

The invention further relates to the immunogenic composition that comprises as above definition and the vaccine of pharmaceutically acceptable excipient.

The invention further relates to that prevention HPV infects and/or the method for disease, comprise compositions or vaccine that the individuality of administration needs as above defines.

The invention further relates to the method for the immunogenic composition that preparation as above defines; Comprise that mixing is from HPV16 and 18 and the VLP of at least a other HPV cancer types; Said other cancer types is selected from HPV type 31,45 and 52, and wherein the VLP dosage of at least a other cancer types is less than the dosage of HPV16 or 18.

The HPV capsomers more excellent than VLP also can be used in above-mentioned aspect of the present invention.

Specify

We have found surprisingly that it is the infection of HPV31, HPV45 and HPV52 and/or the cross-protection of disease that HPV16 and 18VLP can provide anti-some other HPV cancer types.In this paper embodiment, data are provided.

Cross-protection can be thought the protective effect that the vaccine by the HPV type that comprises infection that a kind of anti-different HPV types cause (accidental or continue) and/or disease provides.People such as the definition of chance and persistent infection such as Harper are at Lancet paper Vol 364, and issue 9447, and November 2004, the description among the pages 1757-1765.Cross-protection can be estimated through measuring efficacy of vaccines (V.E.), and wherein V.E. is for to compare with the placebo group of given type, and the % of the infection protective effect that vaccine provides increases.

Therefore; Comprise HPV16 and 18VLP the HPV vaccine can be lower than at other (non-HPV16/18) cancer types VLP (31, the 45 or 52) preparation that does not have required dosage under HPV16 and the 18VLP, this dosage still can obtain the chance of identical anti-those other types and/or the protective response that lasting HPV infects.When the antigen total amount possibly be restricted: for example physics, chemistry or adjusting restriction, the minimizing of other cancer types VLP dosage in the polyvalent vaccine scheme (scenario) under the protective effect that not appreciable impact is caused by those other types, possibly be favourable.For the antigen of specified rate, also allow to prepare the more vaccine of multiple dose, and possibly reduce total vaccine expense.

VLP dosage among this paper is for the total amount of suitable VLP, with gravimetry.

In other words; In order to obtain the identical protective response of anti-chance and/or persistent infection and/or disease; With do not exist such 16 and/or 18VLP under the level of needs compare, ' ' dosage of VLP can reduce other cancer types to unite (the non-HPV16/HPV18) of use needs with HPV16 and/or HPV18VLP.

Therefore; The present invention relates to comprise from HPV16 and 18 with the immunogenic composition of the VLP of at least a other HPV cancer types; Other cancer types is selected from HPV type 31,45 and 52, and the dosage of wherein at least a other cancer types VLP is less than the dosage of HPV16 or 18.

On the other hand; The present invention relates to comprise from HPV16 and 18 with the immunogenic composition of at least a other HPV cancer types; Other cancer types is selected from HPV type 31,45 and 52; The dosage of wherein at least a other cancer types VLP is less than at the dosage that does not have needs under HPV16 and the 18VLP, and this dosage can produce the chance of identical anti-this other type and/or the protective effect that lasting HPV infects.

In one aspect, the dosage of (non-HPV16,18) other cancer types VLP enough provides the chance of anti-the type and/or the protective effect of persistent infection, in one aspect of the invention, and anti-at least accidental infection of protective effect.

In one aspect of the invention, compositions of the present invention is suitable for protecting human patients to avoid infecting and/or disease.

Test to confirm appropriate dosage through human Test Example like those that in this paper embodiment, describe.

Anti-given HPV type for example HPV16,18,31,45 or 52 chance and/or the protective effect of persistent infection refers to, the protective effect that for example provides anti-the sort of type to infect to 50% the crowd of inoculation aptly in one aspect of the invention, provides 60% protective effect; On the other hand, 70% protective effect is provided, aspect further, 80% protective effect is provided; Further, 90% protective effect is provided, further, 95% protective effect is provided; Further, 96% protective effect is provided, further, 97% protective effect is provided; Further, 98% protective effect is provided, further; 99% protective effect is provided, and aspect also further, 100% protective effect is provided.

Aptly, this protective effect of inner evaluation in period at least 6 months, for example at least 9 months, at least 1 year, at least 18 months, at least 2 years or aptly above 2 years.

In one aspect of the invention, the anti-infection that causes by HPV16 and/or HPV18 or the protective effect of disease can be seen,, the anti-infection that causes by HPV16 and HPV18 and/or the protective effect of disease can be seen in another aspect of the present invention.

Can inoculate the HPV kind that exists in the individuality through analysis and estimate the preventive effect to infecting, for example through pcr analysis and/or hybridization technique, those that for example in WO03014402 and WO9914377, describe are introduced into this paper as a reference.

When immunogenic composition of the present invention comprised HPV16 and 18VLP, non-HPV16/18 cancer types was type 31 or 45 or 52 or its combination.In one aspect, immunogenic composition of the present invention comprises from HPV16,18,31 and 45 VLP.In one aspect, immunogenic composition of the present invention comprises from HPV16,18,31 and 52 VLP.In one aspect, immunogenic composition of the present invention comprises from HPV16,18,45 and 52 VLP.In one aspect, immunogenic composition of the present invention comprises from HPV16,18,31,52 and 45 VLP.When having two or more other cancer types VLP (for example 31 and 45,31 and 52,45 and 52), at least a dosage of these other types is less than the dosage of HPV16 or HPV18.

In one aspect, the dosage of HPV31 is less than the dosage of HPV16.

In one aspect, the dosage of HPV52 is less than the dosage of HPV16.

In one aspect, the dosage of HPV45 is less than the dosage of HPV18.

Aptly; As above the immunogenic composition of definition provides accidental infection and/or the protective effect of persistent infection and/or disease, the for example protective effect of anti-accidental infection that anti-HPV16, HPV18 and one or more above-mentioned listed other HPV types (31,45 or 52) cause.

When immunogenic composition of the present invention comprises HPV16VLP, and when not comprising HPV18VLP, suitable non-HPV16/18VLP cancer types is type 31 and/or type 52.

When immunogenic composition of the present invention comprises HPV18VLP, and when not comprising HPV16VLP, suitable non-HPV16/18VLP cancer types is a type 45.

In one aspect of the invention, said compositions comprises among HPV16 and 18VLP and HPV31 and the HPV45VLP one or both combination.

In one aspect of the invention, compositions comprises the combination of HPV16VLP and HPV31VLP at least.

Compositions of the present invention also can comprise other HPV VLP of any suitable dosage except comprising the VLP that reduces dosage.For example, compositions of the present invention can comprise other " high-risk " cancer types, for example one or more among the HPV33,35,39,51,56,58,59,66 or 68.

In one aspect, compositions can comprise other being called " genital wart " type, HPV6 and/or 11 for example, or be called " skin " type, for example HPV5 and/or 8.In one aspect, other VLP is with HPV16 and/or HPV18 same dose or be higher than its dosage and exist.

In one aspect of the invention, said compositions comprises HPV39 and/or HPV51VLP, and at least a dosage is less than the dosage of HPV16 and/or HPV18 in these.

In one aspect, select the consumption of other VLP arbitrarily, with the infection of anti-other type that same degree is provided or the protective effect of disease.

Some compositions of the present invention individually as follows, comprises the VLP of following dosage:

Composition no	?HPV?16VLP?(μg)	HPV?18 VLP(μg)	HPV?31 VLP(μg)	HPV?45 VLP(μg)
					1	?20	20	10	10
2	?20	30	10	10
					3	?20	30	20	20
4	?30	20	10	10
					5	?30	20	20	20

Aptly; There is not relative influence biology (interference) significantly between the HPV VLP in compositions of the present invention; So, the VLP vaccine of the present invention's combination can provide the protective effect of every kind of HPV VLP type infection describing in the anti-effectively vaccine.Aptly, when independent mensuration, it is to 50%, preferred 100% or basically 100% of identical VLP type immunne response that compositions resists the immunne response of given VLP type.As far as replying of HPV16 and HPV18VLP component, the preferential immune stimulatory of combination-vaccine of the present invention is replied, at least 50% of the replying of providing of HPV16/HPV18VLP vaccine of its combination of serving as reasons.Aptly, the degree that still can see for the protection effect of every kind of VLP type wherein of the immunne response that produces of vaccine of the present invention.Aptly, said immunne response can for example antibody response and the clinical trial described herein of ELISA be measured through using standard technique.

In one aspect, compositions of the present invention does not comprise heatshock protein or its fragment.

In one aspect, compositions of the present invention does not comprise HPV L2 albumen or polypeptide.In yet another aspect, compositions of the present invention comprises HPV L2 albumen or polypeptide.

HPV VLP is well-known in the art with the method for preparing VLP.Typically, VLP by the L1 of virus and randomly the L2 structural protein constitute, referring to for example WO9420137, WO9629413 and WO9405792.The HPV VLP of any suitable can be used for the present invention, for example L1 or L1+L2 VLP.

VLP forms and can estimate through standard techniques for example electric (son is micro-) mirror technology and dynamic laser scattering.

In one aspect of the invention, VLP is for only containing the VLP of L1.

VLP can comprise total length L1 albumen.

In one aspect of the invention, the L1 albumen that is used to form VLP is the L1 albumen of truncate.The HPV L1 albumen of truncate is disclosed in, and for example US6361778 is introduced into this paper as a reference.In one aspect, the nucleus framing signal is removed in truncate.Further, be punctured into the C-terminal truncate.Further, the C-terminal truncate is removed and is less than 50 aminoacid, preferably is less than 40 aminoacid aptly.Aptly, HPV16 L1 sequence starts from second methionine codon, for example in following sequence, show, or in the similar position of other HPV type.In one aspect, when VLP was HPV16 VLP, 34 aminoacid were removed in the C-terminal truncate from HPV16 L1 sequence.In one aspect, when VLP was HPV18 VLP, 35 aminoacid were removed in the C-terminal truncate from HPV18 L1 sequence.

In one aspect, the HPV16 sequence is following sequence:

MSLWLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNNKI 60

LVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGVGISGH 120

PLLNKLDDTENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPIGEHWGKGSPCTNVAV 180

NPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSE 240

PYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDDLYIKGSGSTANLASSNYFPTPSGSMV 300

TSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMSLCAAISTSETTYKNTNF 360

KEYLRHGEEYDLQFIFQLCKITLTADVMTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRF 420

VTSQAIACQKHTPPAPKEDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQ 471

The HPV16 sequence also can be for being disclosed among W09405792 or the US6649167 those, for example, and aptly by truncate.Suitable is punctured in the truncate of the equivalent site of above-mentioned display sequence, as relatively estimating through sequence.

In one aspect, the HPV18 sequence is following sequence:

MALWRPSDNTVYLPPPSVARVVNTDDYVTRTSIFYHAGSSRLLTVGNPYFRVPAGGGNKQ 60

DIPKVSAYQYRVFRVQLPDPNKFGLPDNSIYNPETQRLVWACVGVEIGRGQPLGVGLSGH 120

PFYNKLDDTESSHAATSNVSEDVRDNVSVDYKQTQLCILGCAPAIGEHWAKGTACKSRPL 180

SQGDCPPLELKNTVLEDGDMVDTGYGAMDFSTLQDTKCEVPLDICQSICKYPDYLQMSAD 240

PYGDSMFFCLRREQLFARHFWNRAGTMGDTVPPSLYIKGTGMRASPGSCVYSPSPSGSIV 300

TSDSQLFNKPYWLHKAQGHNNGVCWHNQLFVTVVDTTRSTNLTICASTQSPVPGQYDATK 360

FKQYSRHVEEYDLQFIFQLCTITLTADVMSYIHSMNSSILEDWNFGVPPPPTTSLVDTYR 420

FVQSVAITCQKDAAPAENKDPYDKLKFWNVDLKEKFSLDLDQYPLGRKFLVQ 472

Another kind of HPV 18 sequences are open in WO9629413, and it can be aptly by truncate.Suitable is punctured in the truncate of the equivalent site of above-mentioned display sequence, as relatively estimating through sequence.

Other HPV 16 and HPV 18 sequences are known in the art, applicable to the present invention.

Also can carry out the suitable truncate of HPV 31, HPV 45 and HPV 52, suitable remove the C-terminal part that above-mentioned those L1 albumen is equal to, as through sequence to recently estimating.

The L1 albumen of truncate for example is disclosed among the WO9611272 and US6066324, and this paper is introduced into as a reference.

In one aspect, the L1 albumen of truncate is suitably functional L1 protein derivatives, and it can produce immunne response (if desired, suitably adding auxiliary agent), and said immunne response can be discerned by total length L1 albumen and/or the VLP that forms from the HPV type that L1 albumen obtains.

VLP of the present invention also can contain the functional protein derivant of other type, comprises the HPV L1 albumen of total length mutant or truncate, for example lacks, replaces or insert mutant.L1 albumen or derivant also can be fusion rotein, for example the fusion rotein of L1 albumen and L2 albumen or early protein.L1 albumen or functional protein derivant can form VLP, and the formation of VLP can be estimated through standard techniques such as for example electron microscopy and dynamic laser scatterings.

VLP can use for example yeast cells or insect cell baculovirus cell preparation for example of any suitable cell substrate; And the technology of preparing of VLP is well-known in the art; For example be that WO9913056 and US6245568 reach the technology in the list of references of wherein quoting, this paper is incorporated herein by reference its full content.

In one aspect, VLP prepares through separating assembling and the technology of re-assemblying, and it can provide more stable and/or the human papillomavirus VLP of homogenizing more.People such as McCarthy for example; 1998 " Quantitative Disassembly and Reassembly of Human PapillomavirusType 11 Viruslike Particles in Vitro " J.Virology 72 (1): 33-41 has wherein described separating assembling and re-assemblying the homogeneous preparation with acquisition VLP from insect cell purified recombinant L1 HPV 11 VLP.WO9913056 and US6245568 have also described separating assembling and re-assemblying method of preparation HPVVLP.

In one aspect, HPV VLP of the present invention can prepare like the description among WO9913056 or the US6245568.

VLP of the present invention can mix with auxiliary agent or immunostimulant, and said immunostimulant for example but is not limited to the detoxifcation lipid A in any source and the non-toxic derivant of lipid A, saponin and other reagent that can stimulate the TH1 type to reply.

For a long time, known enterobacteria lipopolysaccharide (LPS) is immune a kind of effective stimulus object, though its application in auxiliary agent is reduced owing to its poisonous effect always.People such as Ribi (1986; Immunology and Immunopharmacology of bacterialendotoxins; Plenum Publ.Corp.; NY, 407-419 page or leaf) described through removing core glycosyl group from the reduction end glycosamine and removing a kind of non-toxic derivant of the LPS that phosphate ester produces: monophosphoryl lipid A (MPL), it has following structure:

A kind of MPL of further detoxifcation form produces through removing acyl chain from 3 of two sugar backbones, is referred to as 3-O-and removes acidylate monophosphoryl lipid A (3D-MPL).It can come purification and preparation with the method for instructing among the GB2122204B, and it also discloses the preparation that two phosphoryl lipid As and 3-O-thereof remove the acidylate variant.

In one aspect, 3D-MPL is the Emulsion form of diameter less than the small particle diameter of 0.2 μ m, and its production method is open in WO94/21292.The aqueous formulation that comprises monophosphoryl lipid A and surfactant has been described in WO9843670A2.

The deutero-auxiliary agent of the bacteria lipopolysaccharide that in the present composition, prepares can be from bacterial origin purification and processing, and perhaps they can be synthetic.For example, in 1986 (referring to preceding text) such as Ribi, described purification monophosphoryl lipid A, removed acidylate monophosphoryl lipid A or two phosphoryl lipid As and in GB2220211 and US4912094, described the 3-O-that derives from Salmonella (Salmonella sp.).The describing of other purification (people such as Hilgers, 1986.Int.Arch.Allergy.Immunol., 79 (4): 392-6 with synthetic lipopolysaccharide is existing; People such as Hilgers, 1987, Immunology, 60 (1): 141-6; With EP 0 549 074 B1).In one aspect, the bacteria lipopolysaccharide auxiliary agent is 3D-MPL.

Therefore, can be used for LPS derivant of the present invention be structurally with similar those immunostimulant of LPS or MPL or 3D-MPL.In another aspect of this invention, the LPS derivant can be an acidylate monosaccharide, and it is the inferior part of MPL said structure.

Saponin is at document Lacaille-Dubois, and M and Wagner H. have instruction in (1996, A review ofthe biological and pharmacological activities of saponins.Phytomedicine the 2nd volume, 363-386 page or leaf).Saponin is steroid or the triterpenes glucosides that in plant kingdom and marine animal circle, extensively distributes.Saponin is celebrated in water, to form colloid solution and deposition cholesterol, can produce foam during said colloid solution jolting.When saponin during near cell membrane, it can form the cavernous structure that causes membranolysis on film.For example, erythrocytic dissolving is exactly an instance of this phenomenon, and this is a definite characteristic of saponin, but is not its whole characteristic.

Known saponin is the auxiliary agent of the vaccine that is used for being administered systemically.Research extensively and profoundly (Lacaille-Dubois and Wagner are referring to preceding text) has been carried out to the auxiliary agent and the hemolytic activity of various saponins in this area.At US 5,057,540 with " Saponins as vaccineadjuvants ", Kensil, C.R., Crit Rev Ther Drug Carrier Syst, 1996,12 (1-2): 1-55; With Quil A (deriving from the bark of South America Quillaia saponaria Quillaja Saponaria Molina) and fraction thereof have been described among the EP 0362279B1.Immunostimulating complex (Immune Stimulating Complexes) the graininess structure (ISCOMS) that is called that comprises Quil A fraction has haemolysis; And the preparation (Morein that has been used for vaccine; B., EP 0109942B1, WO 96/11711, WO 96/33739).Hemolytic saponin QS21 and QS17 (the HPLC purification fraction of Quil A) are described to the efficient system auxiliary agent, and its preparation method is at United States Patent (USP) the 5th, 057, No. 540 with EP 0 362 279 B1 in open.Other saponin that has been used for system vaccination research comprises and derives from the for example saponin (Bomford etc., Vaccine, 10 (9): 572-577,1992) of chalk-plant (Gypsophila) and Soapwort (Saponaria) of other each kind of plant.

A kind of enhanced system comprises the combination of nontoxic lipid A derivant and saponin derivant; The particularly combination of disclosed QS21 and 3D-MPL among the WO 94/00153; Perhaps QS21 by the cholesterol quencher than low reaction originality compositions, as disclosed in WO 96/33739.

In one aspect, the especially effectively auxiliary agent preparation that contain QS21 and 3D-MPL oil-in-water emulsion of auxiliary agent in WO 95/17210, describing.

Therefore, in one embodiment of the invention, it is the compositions of auxiliary agent that the non-toxic derivant with detoxifcation lipid A or lipid A can be provided, and is the compositions of auxiliary agent with monophosphoryl lipid A or derivatives thereof more preferably.

In one aspect, compositions also comprises saponin, more preferably QS21.

In one aspect, the auxiliary agent preparation also comprises oil-in-water emulsion.The present invention also provides the method for preparing bacterin preparation, said method comprise with VLP of the present invention and pharmaceutically acceptable excipient for example 3D-MPL be mixed together.

According to the present invention; The other component that preferably is present in the compositions that adds auxiliary agent comprises the nonionic detergent; For example like Octoxinol described herein and polyoxyethylene ester, particularly uncle's octylphenol polyethylene oxyethanol (Triton X-100) and polyoxyethylene sorbitan monooleate dehydration (Tween 80); With at cholate described herein or chlolic acid derivatives, particularly NaTDC or sodium taurodeoxycholate.In one aspect, the compositions that adds auxiliary agent comprises 3D-MPL, Triton X-100, Tween 80 and NaTDC, and it can provide suitable vaccine with the coupling of L2 antigen preparation.

In one embodiment of the invention, said compositions comprises the blister auxiliary agent preparation that contains cholesterol, saponin and LPS derivant.In this, preferred auxiliary agent preparation can comprise the unilamellar liposome that contains cholesterol, has the lipid bilayer that comprises the dioleoyl phospholipid phatidylcholine aptly, and wherein saponin and LPS derivant combine with lipid bilayer or embed in it.More preferably, these auxiliary agent preparations comprise as the QS21 of saponin with as the 3D-MPL of LPS derivant, and wherein, QS21: the ratio of cholesterol is 1: 1 to 1: 100 w/w, most preferably is 1: 5 w/w.In EP 0 822 831 B, described this analog assistant preparation, its disclosed content has been introduced this paper as a reference.

In one aspect, compositions of the present invention and aluminum are united use, and are fit to adsorb or partly be adsorbed on the aluminum auxiliary agent.In one aspect, aptly, auxiliary agent is an aluminum salt, and itself and 3D MPL unite, for example aluminum phosphate and 3D MPL.In yet another aspect, said auxiliary agent is an aluminium hydroxide, randomly unites with 3D MPL.

In yet another aspect, compositions is the associating of VLP and aluminum salt or aluminum salt+3D MPL.In one aspect of the invention, said aluminum salt is aluminium hydroxide.

Compositions of the present invention also can comprise aluminum or the aluminium compound as stabilizing agent.

The present invention relates generally to the combination of VLP.Yet the key component that is appreciated that VLP is a L1 albumen.L1 albumen is united formation pentamer (capsomers), and its (assembling) forms VLP then.So, what the present invention also relates to as above describe comprises L1 albumen or contains the immunogenic composition of the proteic capsomers of L1, L1 albumen or contain the proteic capsomers of L1 and replaced VLP described herein.Aptly, L1 albumen can the SP protective immune response.Aptly, L1 albumen is that conformation is correct.

For avoiding feeling uncertain, the present invention thereby also relate to the purposes of the functional L1 derivant of as above describing, for example L1 truncate, disappearance, displacement or insert mutant and fusion rotein, can provide and can discern those that the HPV virus immunity replys aptly.For example comprising, these proteic capsomerses are also included among the present invention.For example be disclosed among the WO0204007 as the capsomers of immunogenicity medicament.WO9901557 also discloses the compositions that comprises the HPV capsomers.L1 albumen, derivant is used with the mode identical with the above-mentioned VLP of being used for capsomers.

Therefore; The present invention relates to comprise from HPV16 and 18 with the L1 albumen of at least a other HPV cancer types or the immunogenic composition of its functional derivatives; Other cancer types is selected from HPV type 31,45 and 52, and wherein the dosage of the L1 albumen or derivatives thereof of at least a other cancer types is less than the dosage of HPV16 or 18.

Therefore; The present invention relates to comprise from HPV16 and 18 with the immunogenic composition of the HPV capsomers of at least a other HPV cancer types; Other cancer types is selected from and comprises HPV type 31,45 and 52, and wherein the dosage of at least a other cancer types is less than the dosage of HPV16 or 18.

In one aspect, the immunogenic composition that the present invention relates to as above to discuss and the combination of pharmaceutically acceptable excipient.Suitable excipient is well-known in the art, for example, comprises buffer and water.

Compositions of the present invention and vaccine can be supplied with or send through following any approach: for example oral, local, subcutaneous, mucosa (particularly intravaginal), intravenous, intramuscular, intranasal, Sublingual, intradermal and pass through suppository.

In one aspect of the invention, compositions or vaccine can with HPV EA preparation or co-administered, said EA for example is selected from following antigen: HPV E1, E2, E3, E4, E5, E6, E7 and E8.In yet another aspect, vaccine can not contain the HPV EA, for example is selected from following antigen: HPV E1, E2, E3, E4, E5, E6, E7 and E8.

Randomly, compositions or vaccine also can with non-HPV antigen preparation or co-administered.Aptly, these non--HPV antigens can provide the protective effect of anti-other disease, and said other disease is sexually transmitted disease (STD) most preferably, for example herpes simplex virus, EBV, chlamydia and HIV.We particularly preferably are, and said compositions or vaccine comprise the truncate of gD or its HSV, aptly, are called the C-terminal truncate from HSV-2.So, said composition or vaccine provide the protective effect of anti-HPV and HSV.

The dosage of compositions or vaccine component changes according to the situation of the HPV of disease, sex, age and body weight, route of administration and the vaccine of individuality.Consumption also can change according to the number of VLP type.Aptly, passing dose is to be suitable for producing the amount of vaccine that the immunity protection is replied.Aptly; Every vaccinating agent dosage comprises every kind of VLP 1-100 μ g; Being 5-80 μ g in one aspect, further is every kind of VLP 5-30 μ g, further is every kind of VLP5-20 μ g; Further, every kind of VLP specifically is 5 μ g, 6 μ g, 10 μ g, 15 μ g or 20 μ g.

Typically; Being suitable for human dosage is HPV 16 and the HPV18VLP that comprises 20-40 μ g; With as other HPV cancer types (31,45,52) of the dosage of minimizing described herein; Be suitably the level that every kind of VLP is less than 20 μ g, being suitably can be in that some have inoculated the level that causes protective immune response in the individuality at least.

Other dosage that is suitable in the mankind, using can comprise the HPV 16 and/or 18 than the lowland amount, and the protective effect that this dosage provides in the mankind is measured like the test that can use this paper to list.When VLP of the present invention and for example strong auxiliary agent associating, this dosage possibly suit.

In one aspect; Compositions of the present invention comprises the HPV16 of 20 μ g, HPV18 and the 5-18 μ g of 20 μ g; 5-15 μ g's for example from every kind of VLP of other cancer types (31,45 or 52); And further, be the VLP of 5,6,7,8,9,10,11,12,13,14 or 15 μ g especially from every kind of non-HPV16/18 cancer types.

In one aspect; Compositions of the present invention comprises the HPV16 of 10-15 μ g, the HPV18 of 10-15 μ g and every kind of VLP from other cancer types (31,45 or 52) of 5-9 μ g; And in one aspect, be the VLP of 5,6,7,8 or 9 μ g especially from every kind of non-HPV16/18 cancer types.

In one aspect, the part by weight of HPV16VLP and other cancer types (31,45 or 52) VLP is 1: 0.9-1: 0.1 (HPV 16: other type), and aptly 1: 0.9-1: 0.3, aptly 1: 0.8-1: 0.4.

In one aspect, the part by weight of HPV18VLP and other cancer types (31,45 or 52) VLP is 1: 0.9-1: 0.1, (HPV 18: other type), be suitably 1: 0.-1: 0.3, be suitably 1: 0.8-1: 0.4.

In other words; The dosage that reduces is suitably the 10-90% of HPV 16 or HPV 18VLP dosage; And be the 20-80% of HPV 16 or HPV 18VLP dosage in one aspect, further, be 30-70%; Further be 30-60%, and be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of HPV16 or HPV 18 dosage especially.

In one aspect, compositions is used to prevent one or more aptly: HPV-16 and/or HPV-18 infect, and continue HPV-16 and/or the HPV-18 infection tumor of cervix relevant with HPV-16 and/or HPV-18 and form.

Aptly, the purposes of immunogenic composition of the present invention forms and/or accidental the infection and/or persistent infection for being used for the prevention tumor of cervix relevant with other (non-HPV 16,18) carcinogenic type.

Aptly, immunogenic composition of the present invention is used for the adult or the active immunization of adolescent girls more than 10 years old.As far as all compositionss of the present invention and vaccine, it is 10-15 year that said compositions or vaccine are suitable for the age, the adolescent girls inoculation in preferred 10-13 year.Said compositions or vaccine are after also but the operation of the pathological changes that unusual Pap smear or excision cause by HPV is given in administration, perhaps to the seronegative adult female negative with DNA of HPV cancer types.The women in 10-55 year is another kind of suitable targeting group.In yet another aspect, vaccine can be used for maiden and the women greater than institute's has age of baby, and further, can give boy or man.Further, vaccine can be used to treat the women to HPV serum virus reacting positive.

In one aspect, compositions of the present invention is used to prevent or treat by HPV and infect cervical cancer or CIN 1, CIN II or the CIN III morbid state that causes.

In one aspect, said vaccine is sent by 2 or 3 dosages, for example respectively by 0,1 month scheme or 0,2 month scheme, or 0,2,6 month scheme or 0,1 and 6 months schemes send.Aptly, this vaccination scheme can be after 3 to 10 years, or 5 to 10 years, carry out booster injection after for example preferred 3,4,5,6,7,8,9 or 10 years.

In one aspect, compositions of the present invention or vaccine are liquid vaccine preparation, although vaccine can lyophilization and reconstruct before administration.Also can use, for example topical formulations, for example intravaginal ointment.

This paper comprises that with all lists of references of the application the instruction of patent application and granted patent introduces this paper as a reference.

Compositions of the present invention and vaccine comprise above-mentioned some HPV component of listing.The present invention further aspect, said vaccine comprises said component or be made up of said component basically.

In view of the above, the present invention will set forth with reference to the embodiment of following indefiniteness, and these embodiment relate to the cross-protection of HPV16 and HPV18VLP, and has shown the preparation of HPV VLP:

Embodiment 1

The mixture that uses HPV16L1VLP and HPV18 L1 VLP carries out immunity to the age as the healthy women in 15-25 year.Being tried the women is: 1) HPV-16 and HPV-18 are seronegative; 2) to high-risk HPV cervical infection be negative (detect) through HPV PCR; 3) 6 or still less sex partner are arranged in life; 4) the PAP smear is normal.

The auxiliary agent that comprises 20 μ g HPV-16 L1 VLP, 20 μ gHPV-18 L1 VLP and 500 μ g aluminium hydroxide and 50 μ g 3D MPL in the every 0.5ml dosage of said mixture.Placebo group is only injected the aluminium hydroxide of 500 μ g.

Efficacy of vaccines (V.E.) to high-risk cancer HPV type is estimated, and wherein V.E. representes that vaccine group compares with placebo group, and anti-infective protective effect % increases.

In vaccination group and matched group, can cross-protection be estimated through detecting the existence of various carcinogenic type specific property nucleic acid.For example can use the technology of describing in WO03014402 and the list of references wherein to detect; Particularly use [Journal of Clinical Microbiology (1999) such as WO99/14377 and Kleter; 37 (8): 2508-2517] the LiPA system of describing in; HPV DNA is carried out non-specific amplification and thereafter the DNA type detected, the full content of above-mentioned document is introduced this paper as a reference.

Yet any suitable method may be used to the HPV DNA in the test sample, for example with using the type specific property PCR that every kind of target HPV type is had specific primer.Suitable primer is known for the technical staff, if the sequence of perhaps known carcinogenic HPV type then can easily make up.

To all 12 kinds of high-risk cancer types, HPV-16 kind is relationship type (each group; 31,35 and 58; With 31,33,35,52 and 58) and the HPV-18 kind be that the evaluation of efficacy of vaccines is carried out in the infection of relationship type (45 and 59).

" ITT " (on behalf of all, planned treatment (Intention To Treat) crowd accept the individuality of at least a dosage form vaccine) carried out preliminary analysis.This data show is in table 1.

The result that table 2 and table 3 provide relates to " ATP " (According To Protocol) group, promptly meets those patients of these all standards of test.Table 2 is middle point analysis, and its data are picked up from has at least 50% crowd to be in all patients of 18 months points after the vaccination first.Table 3 has provided final result, and all data are picked up from back 18 months experimenter of vaccination first (0 month).In ATP group, all patients all 0, have accepted the vaccine of 3 dosage in January and 6 months, and seroreaction is negative in the time of 6 months.

As provide in the table 1 data confirmed, carry out immunity with HPV 16VLP and HPV 18VLP mixture, the cross-protection of significantly anti-other HPV type is provided.In this, because of sample size can't provide accurate statistical analysis very little, yet the gained data have but proved positive trend, and advise carrying out immunity with HPV 16VLP and HPV 18VLP, can resist the infection of other HPV type effectively.

When research was goed deep into, this point had obtained confirmation.

The detailed content of EXPERIMENTAL DESIGN further describes in embodiment 3.

Table 2 has confirmed that HPV 16 and 18 pairs one group high-risk cancer types 31,33,35,39,45,51,52,56,58,59,66 and 68 of HPV provide the significance,statistical cross-protection.

Table 3 has confirmed except that HPV-18 relationship type (it shows very strong trend), following group is had significance,statistical cross-protection: HPV 31,35,58; HPV 31,33,35,52,58; With 12 kinds that are estimated high-risk HPV types (non-HPV-16/18 type).

Test data analyzer afterwards be illustrated among the embodiment 1 HPV 16 of the associating of using and the significance,statistical cross-protection of the accidental infection of statistics that 18 vaccines provide anti-HPV 31 (efficacy of vaccines, 75.1%, p=0.007).Though sample size still is not enough to other type is drawn the significance,statistical conclusion; But to other type for example 39,45,51 and 52 data acknowledgement positive trend, and suggestion will resist the infection of other HPV type effectively with HPV 16 and HPV 18VLP immunity.

The data of listing among the embodiment 3 further provide the data that in identical research, reach, and the cross-protection that provides is focused on anti-some particular type.

The 9th, 12,15 and 18 months the time from the patient collected specimens, the HPV of above-mentioned special type infected making an experiment.

Table 2-is the efficacy of vaccines behind three dosage in the accidental heterologous of prevention infects

Table 2: anti-HPV-16 kind is that correlation type, HPV-18 kind are that correlation type, HPV-16 and/or HPV-18 kind are efficacy of vaccines-ATP colony (6-18 month) that correlation type and all high-risk types that do not comprise HPV-16 and HPV-18 infect:

Experimenter's number of N=specific crowd;

Experimenter's number that the accidental HPV of N=infects;

AR=sickness rate=n/N

The 95%CI=95% confidence interval

Lower limit=1-exp (log (arv/arp)+1.96 ^*Sqrt (1/nv-1/Nv+1/np-1/Np))

The upper limit=1-exp (log (arv/arp)-1.96 ^*Sqrt (1/nv-1/Nv+1/np-1/Np))

When vaccine group case=0;

Lower limit ^*=1-exp (log (arv ^*/ arp ^*)+1.96 ^*Sqrt (1/ (nv+0.5)-1/ (Nv+0.5)+1/ (np+0.5)-1/ (Np+0.5)))

The upper limit ^*=1-exp (log (arv ^*/ arp ^*)-1.96 ^*Sqrt (1/ (nv+0.5)-1/ (Nv+0.5)+1/ (np+0.5)-1/ (Np+0.5)))

Wherein:

Arv=vaccine receiver's sickness rate

Arp=placebo receiver's sickness rate

Nv=vaccine receiver's case load

Nv=vaccine receiver's case load or non-case load

Np=placebo receiver's case load

Np=placebo receiver's case load and non-case load

The HPV-16 correlation type: the HPV-16 kind is a correlation type 35,31,58, and does not consider other HPV type

The HPV-16 correlation type ^*: the HPV-16 kind is a correlation type 35,31,58,33,52, and does not consider other HPV type

The HPV-18 correlation type: the HPV-18 kind is a correlation type 45,59, and does not consider other HPV

Type HPV-16 and/or HPV-18 correlation type: HPV-16 and/or HPV-18 kind are correlation type 35,31,58,45,59, and do not consider other HPV type

HPV-16 and/or HPV-18 correlation type ^*: HPV-16 and/or HPV-18 kind are correlation type 35,31,58,33,52,45,59, and do not consider other HPV type

^*=do not comprise the high-risk type of HPV-16 and HPV-18

In the time of 18th month from the patient collected specimens, and the HPV of above-mentioned particular type infected make an experiment.

Embodiment 2

HPV16 and HPV18VLP can prepare according to following method:

Instance 1

This paper describes the combination of HPV16 and HPV18 L1 VLP in detail.Can be from the genotypic L1 albumen of other HPV at an easy rate according to similar approach preparation known in the art.

The preparation of A HPV16/18 L1 VLP

Use code test design for example referring to WO9913056, is carried out the preparation of HPV16 and HPV18VLP.HPV16/18 albumen is at the Trichoplusia ni (HighFive that infects interested HPV16 of coding or 18L1 genetic recombinants Baculovirus (MOI is 0.3) ^TM) (density is～350000cells/ml) middle expression cell.Infect collection of cells after 72 hours.

4.1B cell collection/antigen extracts

Antigen (L1-16/18) is from the Hi5 cell, to divide three one step process: concentrate, extract, purify and extract.Concentration step is removed the culture medium greater than 90%, and it carries out through the tangential filtering flow.Extraction step carries out with hypotonic buffer liquid (Tris 20mM, pH 8.5).Use the volume that equates with volume of culture to extract.Under stable stirring (mooth agitation), used time of contact is minimum to be half an hour.Purify through the tangential filtering flow.

The C purification

At room temperature carry out purification step.Beta-mercaptoethanol (4%w/w) is joined in the extracting solution so that VLP separated the capsomers that is assembled into two antigen LI-16/18.Before adding beta-mercaptoethanol, adding glycerol to concentration is w/w 10%.

All buffer that use all filter through 0.22 μ m filter, are stored in 2 ℃-8 ℃.Before carrying out each purification, to the gel-type vehicle sterilization, and before load sample, with suitable buffer balance.

Provide the purification schemes that is used for from HPV 16 and 18 both separation and purification L1.These schemes are generally similar, comprise step:

Anion-exchange chromatography (dimethylaminoethyl-DMAE),

Anion-exchange chromatography (the front three amino-ethyl-TMAE),

Hydroxyapatite,

Nanofiltration (Planova),

Ultrafiltration,

To HPV 18 usefulness hydrophobic interaction chromatographies (using Octyl Sepharose) or to HPV 16 usefulness anion-exchange chromatographies (DEAE); With

Filtration sterilization.

4.1.1 especially:

4.1.2C1 the antigenic purification of L1-18

4.1.2.1 anion-exchange chromatography DMAE

The extract that purifies (concentration is～albumen of 1g/ml, contain～the L1 albumen of 150mg/ml) is applied to anion-exchange column (dimethylaminoethyl).With (Tris 20mM|NaCl 200mM|4% β-(3-mercaptoethanol BME) buffer, pH 7.9 ± 0.2 carries out eluting.With about 5 column volume buffer solution elution antigens, detect the eluting characteristic at 280nm.

4.1.2.2 anion-exchange chromatography TMAE

H with 1 volume ₂The eluent that O/BME 4% dilution first step obtains.Eluent with dilution is applied to second anion-exchange column (Tri Methyl Amino Ethyl) then.

With (Tris 20mM|NaCl 200mM|4%BME) buffer, pH 7.9 ± 0.2 carries out eluting.With about 4 column volume buffer solution elution antigens, detect the eluting characteristic at 280nm.

4.1.2.3 hydroxyapatite

The resulting eluent of TMAE step is applied to hydroxyapatite (HA) post.

After application of samples, with (the NaH of about 2.5 column volumes ₂PO ₄100mM|NaCl30mM|4%BME) buffer, pH 6.0 ± 0.2 eluting gels.

4.1.2.4 nanofiltration (Planova)

Dilution HA eluent is to reach following condition: (NaH ₂PO ₄25mM|NaCl10mM|4%BME) buffer, pH 7.5 ± 0.2.

Then, use 0.2 μ m prefilter and 0.12m continuously ²Planova 15N filter filter.Filtration is carried out at constant voltage 200mbar ± 20mba.

4.1.2.5 ultrafiltration

With grossflow filtration ultrafiltration system (the Centramate cassette 0.1m that poly (ether sulfone) film is housed ², 100kD) carry out ultrafiltration.

Handle the Planova eluent to reach following condition: (NaH ₂PO ₄100mM I NaCl30Mm|4%BME), pH 6.0 ± 0.2; Then, with its system of packing into, concentrate 5 times, and inject (the NaH of～10 times of initial volumes continuously ₂PO ₄, 20mM I NaCl 500mM) and buffer, pH 6.0 ± 0.2 comes diafiltration.

4.1.2.6 hydrophobic interaction chromatography (Octyl Sepharose)

Ultrafiltration permeate is applied to Octyl Sepharose post.

This chromatographic step is at (the Na that contains 5 volumes of having an appointment ₃PO ₄20mM I NaCl 500mM) buffer carries out in the negative mode of pH 6.0 ± 0.2.

4.1.2.7 aseptic filtration

The L1-18 antigenic solution of purification is through sterilizing with 0.22 μ m membrane filtration.

4.1.3

4.1.4C2L1-16 antigenic purification

4.1.4.1 anion-exchange chromatography DMAE

Clarifying extract is applied to anion-exchange column (dimethylaminoethyl).

With (Tris 20mM|NaCl 180mM|4%BME) buffer, pH 7.9 ± 0.2 carries out eluting.

With about 4 column volume buffer solution elution antigens, detect the eluting characteristic at 280nm.

4.1.4.2 anion-exchange chromatography TMAE

With (20mM Tris|NaCl 180mM|4%BME) buffer, pH 7.9 ± 0.2 carries out eluting.With about 5 column volume buffer solution elution antigens, detect the eluting characteristic at 280nm.

4.1.4.3 hydroxyapatite (HA)

The resulting eluent of TMAE step is applied to the HA post.

After application of samples, with (the NaH of about 3 column volumes ₂PO ₄100mM|NaCl 30mM|4%BME) buffer, pH 6.0 ± 0.2 eluting gels.

4.1.4.4 nanofiltration (Planova)

Dilution HA eluent is to reach following condition: (NaH ₂PO ₄25mM|NaCl 10mM|4%BME) buffer, pH 7.5 ± 0.2.

4.1.4.5 ultrafiltration

Handle the Planova eluent to reach following condition: (NaH ₂PO ₄100mM I NaCl30mM|4%BME), pH 6.0 ± 0.2; Then, with its system of packing into, concentrate 5 times, and inject (the NaH of～10 times of initial volumes continuously ₂PO ₄, 20mM I NaCl 500mM) and buffer, pH 6.0 ± 0.2, diafiltration.

4.1.4.6 anion-exchange chromatography DEAE

Regulate the ultrafiltration eluent and reach level pad (Na ₃PO ₄20mM I NaCl 250mM), the conductivity of pH 6.0 ± 0.2 is applied to anion-exchange column (Di Ethyl AminoEthyl).

With (NaH ₂PO ₄20mM I NaCl 500mM) buffer, pH 6.0 ± 0.2 carries out eluting.With about 3 column volume buffer solution elution antigens, detect the eluting characteristic at 280nm.

4.1.4.7 aseptic filtration

The L1-16 antigenic solution of purification is through sterilizing with 0.22 μ m membrane filtration.

C3

Adsorb every kind of VLP type independently to prepare the single antibacterial (concentrated adsorbed monovalent) of concentrating absorption.

VLP16 concentrates the preparation of the single antibacterial of absorption:

PH be 6.0 ± 0.2 with room temperature under, at 150 μ g from Al (OH) ₃Al ³⁺Last absorption 60 μ g are from the VLP of the purification of HPV 16 one hour, with gentle agitation.This single antibacterial of concentrating absorption is+4 ℃ of preservations.Check absorption through centrifugal preparation and the supernatant VLP of quantification.

The preparation that VLP 8 concentrates the single antibacterial of absorption

PH be 6.0 ± 0.2 with room temperature under, at 150 μ g from Al (OH) ₃Al ³⁺Last absorption 60 μ g are from the VLP of the purification of HPV18 one hour, with gentle agitation.This single antibacterial of concentrating absorption is+4 ℃ of preservations.Check absorption through centrifugal preparation and the supernatant VLP of quantification.

The bacterin preparation that D is final:

The single antibacterial that can mix the concentrated absorption through method for preparing comprises the suspension of every kind of every dosage of VLP of 20 μ g with formation.Final vaccine is kept at+and 4 ℃.

According to the present invention, when suitable, can add the VLP additament from other cancer types of suitable concentration.Such sequence is known in the art, and comprises this proteic VLP and can easily be expressed by those skilled in the art.

The raw material of raw material of said combine adsorption (bulks) or independent absorption can be further with auxiliary agent for example 3D-MPL mix.

Embodiment 3

The accurate detailed content that makes an experiment is people such as Harper, the Lancet.2004Nov13; 364 (9447): provide among the 1757-65.

In a word, the mixture that uses HPV16 L1 VLP and HPV18 L1 VLP is that 15 to 25 years old healthy women carries out immunity to the age.Being tried the women is: 1) HPV-16 and HPV-18 are seronegative; 2) to high-risk HPV cervical infection be negative (detect) through HPV PCR; 3) 6 or still less sex partner are arranged in life; 4) the PAP smear is normal.

HPV16 VLP by 471 aminoacid, C-terminal truncate, form by the HPV L1 albumen of 34 aminoacid deletion.HPV18VLP is by the C-terminal truncate, that 35 aminoacid deletion are arranged, 472 amino acid whose HPV L1 albumen.

At length, reproduced the test portion of Lancet paper below fully:

People such as Harper, the Lancet.2004 Nov 13; 364 (9447): the 1757-65-test

Detailed content

The main purpose of this test be estimate 6th month to 18th month between; Infect the efficacy of vaccines among HPV-16, HPV-18 or both (HPV-16/18) the prevention experimenter; Said experimenter is seronegativity through the ELISA demonstration to HPV-16/18 at first, and through the PCR demonstration HPV-16/18DNA is negative.Second purpose comprises: estimate the efficacy of vaccines in prevention persistent infection HPV-16/18; And estimate between 18 months June to the; Between 27 months June to the, the efficacy of vaccines in the LSIL (CIN1) of damage (HSIL) and histology's confirmation in damage (LSIL) in the low potential malignancy squamous epithelial cancer that the prevention cytology confirms, the high malignancy squamous epithelial cancer, HSIL (CIN 2 or 3) squamous cell cancer or the breast carcinoma relevant with the HPV-16/18 infection.Thus, infecting the relevant cytological prevention of the undetermined ASC of meaning (ASCUS) with HPV-16/18 also joins in the analysis result.We also detect through PCR and have done whole last point (histopathological endpoints) CIN 1 of histopathologist relevant with the HPV-16/18DNA infection in the damaged tissue and 2 detection analysis.Other purpose comprises the evaluation of vaccine immunogenicity, safety and toleration.

The researcher of North America (Canada and the U.S.) and Brazil has been convened the women of current efficacy study through the participant who participates in July, 2000 and the epidemiological study of December HPV cross-sectional view before advertisement or the contact.

For each of 32 research points, public commission for inspecting discipline (institutional reviewboard) has ratified EXPERIMENTAL DESIGN, agreement scheme (consent forms) and correction book (amendments).These women have signed respectively and have participated in research and colposcopic written consent book., must agree and agree with participating in research less than for 18 years old the women for those by its relatives.

Divide two research phases: at the initial stage, vaccination and following up a case by regular visits to was reached a conclusion at 18th month; Follow up a case by regular visits to the extended period with blindness, reached a conclusion at 27th month.

The women who is suitable for initial stage (0-18 month) research comprises that the age is the healthy women in 15-25 year, is no more than 6 sex partner, does not have unusual Pap test history or does not ablate (ablative) or excision treatment cervix uteri, and never treat outside condyloma latum; With before getting into research, be no more than 90 days; The cytology is negative, ELISA detects HPV-16 and HPV-18 antibody are seronegativity, and PCR detects those women that 14 kinds of high-risk types (16,18,31,33,35,39,45,51,52,56,58,59,66 and 68) are the HPV-DNA-feminine gender.

Accomplish the women at the initial stage of studying the earliest and do not have ablation or the Cervical women of excision treatment, or the women who after registration, hysterectomizes, the extended period that suitable participation is studied (18-27 month).

Method

(GlaxoSmithKlineBiologicals, Rixensart Belgium) comprise the HPV-16L1 virus-like particle of 20 μ g and the HPV-18L1 virus-like particle of 20 μ g to the bivalence HPV-16/18 virus sample particle vaccines of every kind of dosage.In Spodopterafrugiperda Sf-9 that contains the AS04 auxiliary agent and Trichoplusia ni Hi-5 cell substrate, prepare every type virus-like particle; It provides with the single dose bottle; Said AS04 auxiliary agent comprises 500 μ g aluminium hydroxide and 50 μ g 3-deacylation monophosphoryl lipid A (MPL; Corixa, Montana, USA).Placebo comprises the aluminium hydroxide of every dosage 500 μ g, and outward appearance and HPV-16/18 vaccine are identical.Every research participant accepts the vaccine or the placebo of 0-5mL dosage in 0 month, January and June.

The healthcare provider is at screening inspection and the 6th, 12 and 18 months, with uterine cervix brush and cervical spatula (in PreservCyt, washed, Cytyc Corporation, Boxborough, MA USA) obtains the cervix uteri sample, is used for cytology and HPV DNA test.The 0th and 6 months, and per subsequently 3 months, the women is used for HPV DNA and tests with two continuous swabs (sequential swabs) (being placed among the PreservCyt) cervical guide sample of asking for.[DMHarper; WW Noll; DR Belloni and BF.Cole; Randomized clinical trial ofPCR-determined human papillomavirus detection methods:self-sampling versus clinician-directed-biologic concordance and women ' spreferences.Am J Obstet Gynecol 186 (2002), pp.365-373].(NJ USA) has reported the cytology result (ThinPrep, Cytyc Corporation) who uses the 1991Bethesda categorizing system for Quest Diagnostics, Teterboro in a central laboratory.

The EXPERIMENTAL DESIGN guide be recommended in twice the report ASCUS after; Or after once report is not confirmed significance atypia glandular cell (atypical glandular cells of undeterminedsignificance), LSIL or HSIL, squamous cell carcinoma, ACIS or adenocarcinoma, carry out colposcopy.These guides are also recommended the biopsy to any suspicious lesions.

The central tissue test chamber has been carried out ID to the formalin-fixed tissue sample that is used for Clinical Processing.Subsequently, one group of three pathologist to have drawn consistent diagnostic result: HPV-16 relevant with the CIN system injury with HPV-18.This consistent diagnostic result also comprises observes the section that the fiber when dissected is gathered, and its PCR that is used for undermined HPV DNA measures.

From tissue samples (MagNaPure Total Nucleic Acid system; RocheDiagnostics; Almere; Netherlands) and from cervix uteri living tissue sample (proteinaseK extraction) isolating HPV DNA is for what increase from all DNA aliquots of purification, and the 65bp of its L1 gene that increased is regional, and all DNA of said purification have SPF10 wide spectrum primer.[Kleter; LJ van Doom; People such as J ter Schegget., Novel short-fragment PCR assay for highly sensitiVe broad-spectrumdetection of anogenital human papillomaviruse.Am JPathol 153 (1998), pp.1731-1739:LJ van Doom; W Quint; People such as B Kleter., Genotyping ofhuman papillomaviruse in liquid cytology cervical specimens by thePGMY line blot assay and the SPF (10) line probe assay.J ClinMicrobiol 40 (2002), pp.979-983 and WG Quint; G Scholte; LJ vanDoom, B Kleter, PH Smits and J.Lindeman; Comparative analysis ofhuman papillomaviruse infections in cervical scrapes and biopsyspecimens by general SPF (I0) PCR and HPV genotyping.J Pathol 194 (2001), pp.51-58].Detect amplified production through the DNA enzyme immunoassay (EIA).With string probe test (LiPA Kit HPV INNO LiPA HPV genotyping assay; SPF-10systemversion 1, Innogenetics, Gent; Belgium; Manufactured by Labo Bio-medical Products, Rijswijk Netherlands) detects 25HPV genotype (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; People such as L Schrauwen.; Development and clinical evaluation of a highly sensitiVe PCR-reverse hybridization line probe assay for detection and identificationof anogenital human papillomavirus.J Clin Microbiol 37 (1999), pp.2508-2517] measure any sample that enzyme immunoassay (EIA) is positive to DNA with type specific property HPV-16 and HPV-18PCR.HPV-16 type specific property PCR primer amplification the 92bp fragment of E6/E7 gene, HPV-18 type specific property PCR primer amplification the 126bp fragment of L1 gene.[MF Baay; WG Quint; People such as J Koudstaal.; ComprehensiVestudy of several general and type-specific primer pairs for detection ofhuman papillomavirus DNA by PCR in paraffin-embedded cervicalcarcinomas.J Clin Microbiol 34 (1996), pp.745-747]

We define accidental cervical infection HPV-16/18 at least a positive to the PCR result of HPV-16 or HPV-18 at duration of test, and definition persistent infection HPV-16/18 is positive at least two kinds of the isolating HPV-DNA PCR tests of identical virogene type at least 6 months.[H Richardson; G Kelsall, people such as P Tellier., The naturalhistory of type-specific human papillomavirus infections in femaleuniversity students.Cancer Epidemiol Biomarkers Prev 12 (2003); Pp.485-490 and AB Moscicki; JH Ellenberg, S Farhat and J.Xu, Persistenceof human papillomavirus infection in HIV-infected and uninfectedadolescent girls:risk factors and differences; By phylogenetic type.JInfect Dis 190 (2004); Pp.37-45] at duration of test,, only divulge cytology and histodiagnosis into the clinical treatment purpose to researcher concealment HPV-DNA result of the test.Analysis comprises the HPV-16/18DNA result of cervix uteri sample and the cervical guide sample of asking for.

We 0,1,6,7,12 and 18 months collection research participants' serum be used to estimate immunogenicity.Carry out the serum appearance test of antibody through ELISA to HPV-16 and HPV-18 virus-like particle.Use recombinant HPV-16 or HPV-18 virus-like particle as the immobilized antigen (referring to web appendix http://image.thelancet.com/extras/04art10103webappendix.pdf) that is used for antibody test.Seropositivity is defined as the titer confirmed when test stops more than or equal to for HPV-16 8ELISA units/mL and as far as HPV-18 7ELISA units/mL.Typical natural titer is through using following blood sample to confirm: derive from aforementioned lemology research and find HPV-16 or HPV-18 are seropositive women through ELISA.

During after women's vaccination 7 days, on the diary card, write down symptoms with three kinds of different symptom strength grades.In addition, they report and have studied after vaccination that the staff has interviewed all adverse events in first 30 days.Serious adverse events and pregnant situation during the collection research.

Statistical method

Suppose that in 12 months the CIR that HPV-16 and HPV-18 type infect is 6%, we estimate that each processed group will have 500 women to provide 80% effectiveness (power) to be used to judge to be higher than the lower limit of zero efficacy of vaccines 95%CI.Our supposition retention rate in 18 months is 80%.Carry out effect, safety and immunogenic intermediate value analysis (interim analyses) and only be used for future studies plan purpose; After underway value is analyzed, and use O ' Brien and Fleming method adjusting final analysis result (whole, α=0.05; Two-way test).[O ' Brien and TR.Fleming, A multiple testing procedure for clinical trials.Biometrics 35 (1979), pp.549-556]

Use concentrated layering, the stage sampling (Stratified, block randomization) that meets the algorithm of checking of international randomization system.Layering is to carry out according to age (15-17,18-21 and 22-25 year) and zone (North America and Brazil).By the information input computer random system of research worker with concrete participant, picked at random participant's quantity is distributed every kind of vaccine.Researcher is concealed treatment configuring condition (Treatmentallocation) with the women who participates in long term follow-up research.

Shown the crowd who wants treatment (intention-to-treat) and Pass Test design in the drawings, the reason of wherein from analyze, getting rid of is listed by rank order; According to the highest ranking standard, meet above a kind of women who gets rid of index and only count once.The combination that we want to treat adding with the participant of Pass Test design analysis is called colony (coho rts), only after following up a case by regular visits to, just can know although be used for limiting the information that destination packet is contained in the Pass Test design.

The efficiency analysis that we have carried out the Pass Test design and have wanted to treat.The result of efficacy of vaccines is the basis with participant's ratio that vaccine group has HPV-16/18 to infect in to placebo group in Pass Test design was analyzed in 18 months.Efficacy of vaccines is defined as 1 and deducts the two ratio; 95%CIs judges the degree of accuracy that effect is estimated.Calculate the P value with two-way Fisher ' s microtest.Corresponding ratio representes that resultful case quantity is divided by adventurous participant.18 months colonies of Pass Test design comprise three tabulations of acceptance dosage vaccines and the women who meets like the EXPERIMENTAL DESIGN of describing among the figure.

Want to treat analyzed in 27 months with the Pass Test design in the calculating of efficacy of vaccines use in to placebo group time that the HPV-16/18 cases of infection are arranged that the Cox proportional hazard model of incidence rate (time-to-occurrence) is the basis with vaccine group.This allow to produce in every group of the control person-time-time data.Efficacy of vaccines recently calculates for use 1 deducts harm, and the p value is calculated for using index series to check.Corresponding ratio representes that the case load amount is divided by all person-times-time.At 0 month, all accept vaccine or the placebo of at least a dosage, high-risk HPV-DNA negative and have the women of valid data to be included in to testing result to want to treat in the colony.27 months colonies of Pass Test design comprise the women who derives from 18 months colonies of Pass Test design and exist in the extended period (18 months to 27 months).

The p value of using the accurate experimental control of Fisher ' s to be used for safety analysis is calculated.The crowd who is used for safety analysis comprises the vaccine or the placebo of at least a dosage of acceptance of all addings, and meets women's (referring to figure below) specific, that minimum EXPERIMENTAL DESIGN requires.

In one group of Pass Test design safety crowd, estimate immunogenicity, it is included in duration of test 0,7 with serology result's women was arranged in 18 months, and it has accepted institute as per the schedule

In one group of Pass Test design safety crowd, estimate immunogenicity; It is included in duration of test 0,7 with serology result's women arranged in 18 months; It has accepted all three Research on dose vaccines or placebo as per the schedule; Press the blood sampling of blood sampling time table, and HPV-16/18-DNA is not shown the positive.

Accurately test the seroprevalence (p＜0.001judged significant) between comparison vaccine group and the placebo group with Fisher ' s.Compare geometric mean titer with ANOVA and Kruskal-Wallis test.(SAS Institute, Cary NorthCarolina) carry out Block stochastic sampling and statistical analysis with SAS version 8.2.

The result

In patent application W02004/056389, listed the initial result who analyzes of cross-protection, this paper is incorporated herein by reference its full content.

Analyze further

" ATP " (according to EXPERIMENTAL DESIGN) organized those patients that meet all experimental conditions to be analyzed.In the ATP group, all patients were 0,1 and accepted 3 dosage in 6 months, 6th month seronegativity.

Like data acknowledgement in the table 4, to compare with matched group, HPV16 and HPV18VLP mixture immunity inoculation provide anti-HPV type 31,52 and the 45 accidental significant cross-protections of infecting of statistics.

In the anti-relevant type of all HPV16 (HPV-31,33,35,52 and 58) group and all high-risk type group; Do not comprise 16 and 18 (HPV31,33,35,39,45,51,52,56,58,59,66 and 68), observe the anti-accidental significant cross-protection of infecting of statistics yet.

In anti-type 31 and 52 (referring to table 5) and the anti-relevant type group of all HPV16 (referring to table 5), also observe the significant cross-protection of statistics of anti-persistent infection.

In the anti-cytological abnormal relevant, also observe the significant cross-protection of statistics, referring to table 6 with HPV52.Anti-relevant with the group of all HPV16 correlation types (HPV-31,33,35,52 and 58); Also observe the remarkable protective effect of statistics in the cytological abnormal relevant, wherein do not comprise 16 and 18 (HPV31,33,35,39,45,51,52,56,58,59,66 and 68) with the group of all high-risk types.

Table 4

Anti-16/18 correlation type ^*The accidental effect that infects

^*Cervical samples; ATP colony

Table 5

Anti-16/18 correlation type ^*The effect of persistent infection

^*All samples; ATP colony

Table 6

Anti-relevant type with 16/18 ^*The effect of relevant cytological abnormal

^*ATP colony

In table 4,5 and 6,

N=specific crowd patient's quantity

AR=sickness rate=n (HPV patient's quantity of accidental infection, persistent infection or cytological abnormal is with suitable (the as appropriate for the table) mutually in the table)/N

The % efficacy of vaccines is 1-(A/B) * 100, regulates according to the relative populations of vaccine and placebo group,

Wherein

The women % that suffers from accidental infection, persistent infection or cytological abnormal in the A=vaccine group is with suiting in the table mutually

The women % that suffers from accidental infection, persistent infection or cytological abnormal in the B=placebo group is with suiting in the table mutually

Claims

1. immunogenic composition; Comprise from HPV 16 and 18 with the VLP or the capsomers of at least a other HPV cancer types; Other cancer types is selected from HPV type 31,45 and 52, and wherein the dosage of the VLP of at least a other cancer types or capsomers is less than the dosage of HPV 16 or 18.

2. according to the compositions of claim 1, wherein other cancer types is HPV 31.

3. according to the compositions of claim 1, wherein other cancer types is HPV 45.

4. according to the compositions of claim 1, wherein other cancer types is HPV 52.

5. according to the compositions of claim 1, wherein other cancer types is HPV 31 and HPV45.

6. according to the compositions of claim 1, wherein other cancer types is HPV 31 and HPV52.

7. according to the compositions of claim 1, wherein other cancer types is HPV 52 and HPV45.

8. according to the compositions of claim 1, wherein other cancer types is HPV 31, HPV45 and HPV 52.

9. according to the immunogenic composition of each aforementioned claim, wherein said compositions comprises VLP or the capsomers from other cancer types of 10 μ g or more HPV 16 and/or HPV 18VLP or capsomers and 2-9 μ g.

10. according to each immunogenic composition among the claim 1-8, wherein said compositions comprises VLP or the capsomers from other cancer types of 20 μ g or more HPV 16 and/or HPV 18VLP or capsomers and 5-15 μ g.

11. according to the immunogenic composition of claim 10, wherein said compositions comprises VLP or the capsomers from other cancer types of 10 μ g.

12. according to each the immunogenic composition and the combination of auxiliary agent among the claim 1-11.

13. according to the compositions of claim 12, wherein said auxiliary agent is an aluminum salt.

14. according to the compositions of claim 13, wherein said auxiliary agent is an aluminium hydroxide.

15. according to the compositions of claim 12, wherein said auxiliary agent is the lipid A derivant.

16. according to the compositions of claim 15, wherein said auxiliary agent is 3D MPL.

17. according to the compositions of claim 16, wherein said auxiliary agent is 3D MPL and aluminium hydroxide.

18. vaccine comprises immunogenic composition and pharmaceutically acceptable excipient according to each aforementioned claim.

19. according to the purposes of each described compositions among the claim 1-17 in the medicine of preparation prevention HPV infection and/or disease.

20. the purposes of vaccine according to claim 18 in the medicine of preparation prevention HPV infection and/or disease.

21. preparation is the method for the immunogenic composition of definition as above; Comprise mixing from HPV16 and 18 and the VLP or the capsomers of at least a other HPV cancer types; Other cancer types is selected from HPV type 31,45 and 52, and wherein the dosage of the VLP of at least a other cancer types or capsomers is less than the dosage of HPV 16 or 18.