WO2006065166A1

WO2006065166A1 - Protein l1 and protein e7 peptide-based composition for treating and preventing a human papillomaviral infection

Info

Publication number: WO2006065166A1
Application number: PCT/RU2005/000391
Authority: WO
Inventors: Rem Viktorovich Petrov; Musa Rakhimovich Khaitov; Sergey Nikolaevich Andreev; Olga Vyacheslavovna Smirnovna; Adilya Rafik Kyzy Chervenko
Original assignee: Obschestvo S Ogranichennoi Otvetstvennostyu 'rusgen'
Priority date: 2005-07-26
Filing date: 2005-07-26
Publication date: 2006-06-22

Abstract

The invention relates to biotechnology, in particular to papillomaviral proteins and peptides used for developing immunoprophylactic and therapeutic vaccines against different variants of a human papillomaviral infection (HPV). The capsid proteins L1 of a human papilloma viral serotypes (16, 18, 31) causing the most aggressive human diseases are obtainable by transforming yeast cells of a plasmid DNA which encodes the L1 proteins from said serotypes. Peptides in the form of transforming E7 HPV protein fragments (E7-peptides) of said serotypes are produced by means of a chemical synthesis. The thus produced yeast strains producers during the cultivation thereof are capable of expressing the L1 proteins in the form of virus-like particles (VLP). Said L1 proteins are extracted, cleaned and used for preparing vaccine compositions which can also contain E7-peptides and adjuvants.

Description

COMPOSITIONS FOR TREATMENT AND WARNINGS

PAPILLOMAVIRUS HUMAN INFECTION ON THE BASIS OF PROTEIN Ll

AND PEPTIDE PROTEIN E7

FIELD OF THE INVENTION

The invention relates to medicine, in particular virology, gynecology, oncology, and relates to a method for producing recombinant structural protein Ll of human papillomavirus (HPV), compositions, vaccines, therapeutic compositions based on it, additionally containing peptides of HPV E7 protein, for prevention and / or treatment of human papillomavirus infection, as well as methods of treating and / or preventing human papillomavirus infection and inducing an immune response in humans.

State of the art

Cervical cancer (cervical) associated with persistent human papillomavirus (HPV) infection is one of the most common and dangerous types of malignant neoplasms in women around the world. According to the WHO, every year 510,000 cases of cervical cancer are diagnosed in the world, of which about half the women are expected to die. In general, more than 630 million people are carriers of human papillomavirus infection, of which 190 million have clinical manifestations. The early diagnosis of HPV is quite complicated, the clinical manifestations of the infection are not detected for a sufficiently long time. The development of mild to moderate dysplasia in severe occurs respectively in 10 and 20% of cases, and severe dysplasia passes into invasive cancer in at least 12% of cases. One of the manifestations of human papillomavirus infection is genital warts, caused mainly by viruses of type 6 and 11. Condylomas are an overgrowth of the mucous membrane and are localized on the external genitalia (sometimes in the urethra, vagina) and in the rectal region. Over 80% of cases of this disease occur in developing countries, where there is neither population screening nor the possibility of optimal treatment. Moreover, modern treatment and, in most cases, modern screening strategies do not take into account the viral etiology of this type of cancer.

The papilloma virus belongs to the family of papoviruses (Parviridae). He has diameter 40 - 55 nm, and its shell, a capsid in the form of an icosahedron, is composed of 72 protein capsomeres consisting of the main protein Ll and minor protein L2. The genome of the virus is presented in the form of a circular double-stranded DNA, which contains 8 early and 2 late genes. To date, more than 100 types of HPV have been discovered, of which more than 30 are capable of infecting genital epithelial cells. Types HPV 6, 11, 34, 39, and a number of others cause the formation of non-malignant genital warts of the genital and respiratory epithelium. Epithelial dysplasia and carcinomas of the cervix, vagina and anal canal are usually associated with HPV-16, 18, 31, 33, and 45.

Recombinant HPV proteins are currently considered promising immunogenic components for prophylactic and therapeutic vaccines. Prophylactic candidate vaccines are based on the recombinant capsid proteins of the Ll virus (major) and L2 (minor). The major Ll protein (55-60 kDa) is able to assemble itself into virus-like particles (VLP), while it has high immunogenicity and induces antibodies that neutralize the infectious virus. In models (rabbit), it was shown that immunization with these proteins expressed in bacteria and the baculovirus system protected animals from viral infection. Well-characterized HPV oncogenic proteins, E6 and E7, are now being considered as therapeutic candidate vaccines. These proteins, as well as their peptide fragments, can be used to induce specific cytotoxic T-lymphocytes in order to eliminate infected cells. Studies in animals and humans demonstrate the possibility of inducing a simultaneous protective response to various types of HPV using multivalent vaccines based on preparations containing a combination of epitopes of several types of HPV Ll proteins. So far, none of the candidate vaccines has passed complete clinical trials.

At the present stage, none of the preventive candidate vaccines against HPV has yet gone beyond the level of clinical evaluation. The National Cancer Institute in the USA (NCI) [http://www.cancer.gov] is testing a monovalent vaccine based on the HPVl 6 VLP protein, which is expressed by insect cells using recombinant baculovirus technology (clinical stage III). Medmmupe has developed the bivalent HPV-16/18 VLP vaccine, which is also based on baculovirus technology (Stage II trials) [http: //www.medirnmune.conVpipeline/hpvaccine.asp]. Company

Mersk [http://www.merck.com/finance/annualreport/ar2002/research_strategy.html; patent RU JV ° 2161651] develops a trivalent (VLP HPV 6, 11, 16) and tetravalent vaccine (VLP HPV-6, 11, 16, and 18) using the expression of HPV Ll proteins in yeast (stage III). The results of preliminary tests, however, on a limited contingent of individuals and terms, indicate that the effectiveness of such vaccines is quite high.

Therapeutic candidate vaccines are also not yet in the development phase. One of them is based on the transformed vaccinia virus genome, where the genes of the oncoproteins E6 and E7 of the type HPVl 6 and 18 (drug “TA-HPV”) are inserted. Such a transformed live virus is supposed to be administered to operated patients after removal of cervical tumors (tests in phase I / II) [http: //www.xenova.co.ukУPressReleases/pr_20030414_02.html]. Chimeric recombinants constructed from HPVl 6 L2, E6 and E7 proteins were also produced in bacterial systems (phase I trials), and preliminary data indicate their good immunogenicity. Trapsgepe created a combinatorial vaccine (“MVA-HPV-EL2”) based on an attenuated vaccinia virus that contains sequences encoding the E6, E7 proteins, as well as the IL-2 cytokine

[http: //wwwДransgene.fr/us/pdl7coпiпiunique_presse/communiques_divers_2004/PR- US_18-03-2004_MVA-HPV-IL2.pdf]. The combination of E7 and heat shock protein Нср was proposed as a vaccine by the company Strassgep

[http://www.stressgen.com/product/hspe7.htm], where the Hsp component must stimulate the immune system to remove infected cells (clinical trials are in phase II). Zusos made a therapeutic DNA vaccine called ZYClOIa (intramuscular injection 3 times at 3-week intervals) and showed a 43% reduction in tumors in the group of patients compared to 23% in the placebo group.

Thus, although vaccines known in the art have a protective effect to one degree or another, they have not yet passed the necessary clinical trials. The claimed vaccines are divided into two types - it is either preventive or therapeutic vaccines. Vaccines combining the properties of both groups are not explicitly described. The above disadvantages are overcome in the present invention.

This invention provides a preventive and therapeutic effect in one vaccine, i.e., it is a combination vaccine.

Disclosure of invention

In its first aspect, the invention provides a method for producing a recombinant human papillomavirus Ll protein selected from serotypes HPVl 6, HPV 18 and HPV31, comprising: a) obtaining a copy of the gene encoding the Ll protein by polymerase chain reaction using clinical material, b) obtaining a plasmid containing a copy of the gene obtained in stage a) and intended for expression of the human papillomavirus Ll protein in host yeast cells, c) transformation of yeast host cells with the plasmid obtained in stage b), d) cultured the presence of transformed yeast host cells under conditions ensuring the efficient expression of recombinant human papillomavirus Ll protein; e) extraction and purification of human papillomavirus Ll protein.

In one embodiment, a plasmid containing a copy of the Ll gene is derived from the yeast plasmid pPDX2.

In a second aspect, the invention provides a recombinant human HPV papillomavirus Ll protein obtained by the method, which is the first aspect of the present invention.

In one embodiment, the recombinant Ll protein is characterized in that it is obtained in the form of virus-like particles.

In a further embodiment, the recombinant Ll protein is useful for the manufacture of a medicament for the prevention of papillomavirus infection.

In a third aspect, the invention provides a composition comprising a purified recombinant Ll protein obtained by the method of the first aspect of the present invention and at least one purified synthetic peptide that is a fragment of an E7 protein human hapillomavirus having a sequence selected from the group represented by:

SEQ ID NO: 1 YMLDLQRETTDLYC,

SEQ ID NO: 2 PAQQERDRANYNIVTFSSK,

SEQ ID NO: 3 QAERDRANYYNIVТFССКСDSТТРРvLСVQSТНVDIR,

SEQ ID NO: 4 QSTHVDIRTLEDLLMGTLGIV,

SEQ ID NO: 5 GQAERDRANYNГVТFССК,

SEQ ID NO: 6 CATLQDГVLНL,

SEQ ID NO: 7 RKATLQDIVLHLERQNЕIРV,

SEQ ID NO: 8 GVNHQHLPA,

SEQ ID NO: 9 RAEPQRHTM,

SEQ ID NO: 10 GQAERDRANYNIVТFССКСDSТLRLСVQSТНVDIR,

SEQ ID NO: 11 VLDLQREATD,

SEQ ID NO: 12 GQAERDTSNYNIVTFCCQ,

SEQ ID NO: 13 GQAERDТSNYNIVТFССQСКSТLRLСVQSTТQVDIR,

SEQ ID NO: 14 ELLMGSFGGVCPNA,

SEQ ID NO: 15 TSNYNTVTF,

SEQ ID NO: 16 DTSNYNГVTF.

In a fourth aspect, the invention provides a vaccine for the prevention and / or treatment of human papillomavirus infection, which comprises an effective amount of a recombinant Ll protein obtained by the method of the first aspect of the present invention.

In one embodiment of the invention, the vaccine further comprises an effective amount of at least one purified synthetic peptide, which is a fragment of human papillomavirus E7 protein, having a sequence selected from the group represented by:

SEQ ID NO: 1 YMLDLQRETTDLYC,

SEQ ID NO: 2 PAQQAERDRANYNГVTFSSK,

SEQ ID NO: 3 QAERDRANYYNГVТFССКСDSТТRLRLСVQSТНVDIR,

SEQ ID NO: 4 QSTHVDIRTLEDLLMGTLGIV,

SEQ ID NO: 5 GQAERDRANYNГVТFССК,

SEQ ID NO: 6 CATLQDГVLНL, SEQ ID NO: 7 RKATLQDIVLHLERQNЕIРV,

SEQ ID NO: 8 GVNHQHLPA,

SEQ ID NO: 9 RAEPQRHTM,

SEQ ID NO: 10 GQAERDRANYNIVТFССКСDSТLRLСVQSТНVDIR,

SEQ ID NO: 11 VLDLQREATD,

SEQ ID NO: 12 GQAERDTSNYNIVTFCCQ,

SEQ ID NO: 13 GQAERDТSNYNIVТFССQСКSТLRLСVQSTТQVDIR,

SEQ ID NO: 14 ELLMGS FGIVCPNA,

SEQ ID NO: 15 TSNYNIVTF,

SEQ ID NO: 16 DTSNYNГVTF.

In a further embodiment of the invention, the vaccine further comprises one or more adjuvants.

In a fifth aspect, the present invention provides a vaccine for the prevention and / or treatment of human papillomavirus infection, which comprises an effective amount of a composition, which is the third aspect of the present invention.

In one of its embodiments, the vaccine for the prevention and / or treatment of human papillomavirus infection additionally contains one or more adjuvants.

In a sixth aspect, the invention provides a therapeutic composition comprising an effective amount of a recombinant Ll protein, which is a second aspect of the invention.

In one of its embodiments, the therapeutic composition further comprises an effective amount of at least one purified synthetic peptide, which is a fragment of the human papillomavirus E7 protein, having a sequence selected from the group represented by:

SEQ ID NO: 1 YMLDLQRETTDLYC,

SEQ ID NO: 2 PAQQAERDRANYNГVTFSSK,

SEQ ID NO: 3 QAERDRANYYNIVТFССКСDSТТRLRLСVQSТНVDIR,

SEQ ID NO: 4 QSTHVDIRTLEDLLMGTLGIV,

SEQ ID NO: 5 GQAERDRANYNГVТFССК,

SEQ ID NO: 6 CATLQDIVLHL,

SEQ ID NO: 7 RKATLQDГVLНLЕРQNЕIРV, SEQ ID NO: 8 GVNHQHLPA,

SEQ ID NO: 9 RAEPQRHTM,

SEQ ID NO: 10 GQAERDRANYNГVТFССКСDSТLRLСVQSТНVDIR,

SEQ ID NO: 11 VLDLQREATD,

SEQ ID NO: 12 GQAERDTSNYNGVTFCCQ,

SEQ ID NO: 13 GQAERDТSNYNIVТFССQСКSТLRLСVQSTТQVDIR,

SEQ ID NO: 14 ELLMGSFGIVCPNA,

SEQ ID NO: 15 TSNYNIVTF,

SEQ ID NO: 16 DTSNYNГVTF.

In one of its embodiments, the therapeutic composition further comprises one or more adjuvants.

A seventh aspect of the present invention is the use of the HPV human papillomavirus Ll protein L, which is the second aspect of the present invention, or the composition, which is the third aspect of the present invention, for the manufacture of a medicament for the prevention and / or treatment of human papillomavirus infection. It should be understood that this aspect also essentially relates to a method of preventing and / or treating human papillomavirus infection, comprising administering a prophylactically or therapeutically effective amount of a recombinant HPV human papillomavirus Ll protein, the second aspect of the present invention, or a composition, which is the third aspect of the present inventions. It should be understood that a method of preventing and / or treating human papillomavirus infection may also include administering an effective amount of a vaccine, which is the fourth or fifth aspect of the present invention, or a therapeutic composition, which is the sixth aspect of the present invention.

Finally, in its seventh aspect, the present invention relates to the use of the recombinant human HPV HPV Ll protein, the second aspect of the invention, or the composition, the third aspect of the invention, for the manufacture of a medicament for inducing an immune response in humans. It should be understood that this aspect also essentially relates to a method of inducing an immune response in humans, comprising prophylactically or therapeutically administering effective amount of recombinant human papillomavirus Ll protein

HPV, the second aspect of the present invention, or a composition, which is the third aspect of the present invention. It should be understood that a method of inducing an immune response in humans may also include administering an effective amount of a vaccine, which is the fourth or fifth aspect of the present invention, or a therapeutic composition, which is the sixth aspect of the present invention.

Brief description of the figures:

FIG. 1. The results of gel electrophoresis of the obtained polynucleotides. 1 and 2 — fragments containing the Ll protein gene of the HPV 16 serotype; 3 and 4 — fragments containing the Ll protein gene of the HPV 18 serotype.

FIG. 2. Plasmid maps (pUC19) with inserts of the HPV Ll gene with restriction sites. A - with the insertion of the Ll gene of HPVl 16, B - with the insertion of the Ll gene of HPVl 8.

FIG. 3. Alignment of the translated amino acid sequences of the sequenced variants of the HPV 16 Ll gene with the published HPV16 Ll protein sequence (Ll-16). Replacements are enclosed in a box.

FIG. 4. Alignment of the translated amino acid sequences of the sequenced variants of the HPVl 8 Ll gene with the published HPV 18 Ll protein sequence (Ll -18). Replacements are enclosed in a box.

FIG. 5. Alignment of the translated amino acid sequences of the sequenced variants of the HPV31 Ll gene with the published HPV31 Ll protein sequence. Replacements are enclosed in a box. The location of the EcoSh website is shown in a darkened box.

FIG. 6. Maps of yeast replicative plasmids designed for expression of HPV Ll protein. A is a map of the plasmid pPDX2-Ll-16 containing the insert of the HPV 16 Ll gene; B is a map of the plasmid pPDX2-Ll-18 containing the insert of the HPV18 Ll gene.

FIG. 7. Polyacrylamide gel electrophoresis of recombinant VLP fractions HPV-16 and 18 after ultracentrifugation of yeast lysates (gel stained with silver solution). Lanes: 1 - Marker pier. weight (upper band - 66 kDa), 2-5 - proteins isolated from strains Y618 / pPDX2-Ll-16 JV ° JV ° l-4, respectively, 6 - proteins isolated from strain Y618 / pPDX2 (negative control), 7, 8 - proteins isolated from strains Y618 / pPDX2-Ll-18 JVaJVaI, 2, respectively. It can be seen that in lanes 2, 3, 5, 8 an additional band appeared in comparison with the negative control, corresponding in weight to Ll - approximately 56 kDa (the level is approximately indicated by the arrow).

FIG. 8. A - Electrophoresis of Ll preparations isolated from the 618 / pPDX2-Ll -18 transformant. Lanes: 1 - marker, 2 - sample after the first gradient centrifugation, 3 - the same sample, purified from sucrose by ultrafiltration on a 10 kDa membrane, 4 - after repeated purification in a gradient of 45% sucrose, the final preparation. B - Electrophoresis of the Ll protein preparation obtained by preparative isolation from the 618 / pPDX2-Ll-16 transformant. Lanes: 1 - negative control (618 / pPDX2), 2 - drug Ll-16, 3 - marker.

FIG. 9. Electron micrograph of recombinant VLP Ll HPVl 6. The marker in the lower left corner corresponds to 100 nm. A - increase x55000, BG - increase hZ 50,000.

The implementation of the invention

The invention relates to recombinant human papillomavirus (HPV) capsid proteins expressed in the yeast system, synthetic peptide fragments of the HPV E7 transforming protein, methods for their preparation, use as prophylactic and therapeutic vaccines against HPV infection, methods for the prevention and treatment of human papillomavirus infections and methods inducing an immune response. Various aspects of the present invention include recombinant HPV DNA molecules, proteins encoded by these molecules, peptide fragments of HPV proteins, compositions containing these molecules, including immunological compositions containing adjuvants, methods of using these compounds and their compositions.

Any currently known heterologous protein expression systems may be used to express Ll protein. Such systems can be both prokaryotic and eukaryotic. As prokaryotic systems can be used, for example, systems based on E. coli (Li et al., Li ML, CP TP,

Estes PA, Lyon MK, Rose RC, Garses RL, Ehrressiop ° F the humap rarillomavirus ture 11 Ll sarsid rroteip Ip Essherishia coli: sharasterizatiop ° F rroteip domaips ipvolved in DNA bipdipg apd sarsid assemblu. J. Virol 71, 2988-95, 1997), B. sibtilis. As eukaryotic systems, for example, systems based on cultured insect tissues (baculovirus systems) can be used (Christepsep ND, Нorfl R, DiAngelo SL, Сdedel NM, Рatriсk SD, Веlsh PA, Budgeon LR, Реd CA, Кreеdеr JW, Аssеlеvl ehrressed humap rarillomavigas ture 11 Ll sarsid rroteip virus- like rartisles are resogpized bu peutgalizipg moposlopal aptibodies apd ipduse high titres ° F peutralizipg aptibodies Gep J. Virol 75, 2271-2276, 1994;.. Shristepsep ND, Kreider JW, Aptibodu-mediated peutralizatiop Ip vivo оf ipfestios pirillomaviruses. J. virol 64, 3151- 3156, 1991; Rose RC, Voppez W, Reixhmap RC, Garcea RL, Expresiop of humap paralomavirus tour 11 Ll rrot ip ip iptest sells: ip viv ap ip vitro assumbled virus-likelist. J. Virol 67, 1936-44, 1993; Kirpuler R, F Chep, DR Loplarparpar Assemblies Ipto virus-likericles thаt аre high high immunity expected by Nat. Acad. USA USA 89: 12180-12184, 1992; Vollers C, P Sсhirmacher, RE Strеesk, M Sarr, Assеmblу оf thе mаjоr аpd thе mіporsarsid rоtеіp оf humap іrіllіmavirus ture 33 іpto virus-like іѕ іrtісерслісплеслпсерплеслплеслпсерплеслплеслпсерплесрплеслперслплеслпслплеслпслплеслтслесрплесртслесрплеслпсерплесл Virology 200: 504-512, 1994), or mammals (vaccine-viral systems) (Zhou J, XY Sup, DJ Stepzel, IH Frazer, Expresiopf HPV 16 Lp apfieplіpіlіpіlіpіlіpііlіpііlеsіpіlіpііlісіpііlе -Liké rartles. Virology 185: 251-257, 1991; Highest ME, N Yaegashi, DA Gallowow, Self-assumble humpelimpoupéloplépoplépoplépoplépoplépoplépoplépoplépoplépopel , 67: 315-322, 1993), methylotrophic yeast P. rasteris, filamentous fungi A. piger, yeast S. cerevisiae (Neerer MP, Hofipapp KJ, Japsep KU, Expessepopperipheporepipepipepipepipe , Gepe 180 1-6, 1996), dividing their yeast S. rombe (Sasagawa T, Pushko P, Sters G, Gсhmeissper SE, Native Hybrid MA, Fixture J, Сrafford L, Тomassіпсепомсіпліспомсіпомситіпоміпсіпомситіпоміпіситіпомситіпоміситіпіситіпіситіпіситіпіситіпіситіпіситіпіситіпіситіпіситіпіситіпістомі Rambe. Virology 206, 126-135, 1995), etc. Preliminary studies on the immunogenic activity of recombinant HPV proteins obtained in the bacterial expression system showed that the titer of antibodies produced by mammals after immunization, low (Ріліпіпski WP, Glassman DL, Крузек RA, Sadowski PL, Robbins AK, Сlopipпd аd exprescіssiоli оf thе bоwipe raіllіmіvigus Ll аpеdоgеpоgоpоgоpоgоpоgоpоgоpоgоpоgоpоgіpоgіpоgіpоgіpоgіpоgіpоgіpоgіpоgіpоgіpоgіpоgіpоgоgоgоgіpоgоgоgоgоgоgоgоgоgоgоgоgоpоgоpоgоgоpоgоpоgоpоgоpogo. The best results were shown by baculovirus and vaccine-viral systems (Hofipashi KJ, Sawk JC, José JG, Brows DR, Sсhultz LD, Görgé Our Rosolowsk M, Fife K, Japsep KU, Séféfusperfumefumf . serevisiae Virologu 209, 506-518., 1995; Zhou J, XY Sup, DJ Stepzel, IH Frazer, Ehrressiop ° F vassipia resombipapt HPV 16 Ll L2 ORF apd rroteips erithelial sells Ip is suffisiept assemblu ° F for HPV viriop-like rartisles Virologu 185 : 251-257, 1991; For more information ME, N Yaegashi, DA Gallow, Self-ass of hum hum ravillomavirus I sarsids exp Lope ruple lupl, 32, 2. , 1993; Kigabauer R.F. In the case of Ll protein expression, it was aggregated into virus-like particles (VLP), and when used as an immunogen, a high level of protective immune response was observed.

The most preferable for creating a vaccine is the use of a system based on the yeast Sasshotus erytes servis because of its cheapness, the ability to produce large amounts of protein in the native conformation, the similarity of the post-translational modification apparatus with that of mammals, and the internationally recognized safety of yeast (Hofmapp KJ, Jock JC, Jouse JG, Jose JG , War DR, Schultz LD, Georget Rosolu M, Fiffe K, Japsep KU, Scepse Determiopf humdapillavirus ture 6a apachelosuccess 5, siclosheluceplo sicloso sslosero selo sélépécole gray visiae are assigned to the group of organisms of the GRAS category (Heperell Regard As Safe).

As plasmids for expression of the target product, there is an extensive arsenal of vectors that differ in origin (from a virus, phage, plasmid, or based on a set of artificially combined control elements), host specificity (vectors that replicate strictly in one host, or shuttle vectors with several replicons), a method of controlling the expression of the target product (various variants of promoters, terminators), the ability to be supported as an independently replicating unit in the host cell (replicative, centromeric plasmids, artificial chromosomes, as well as vectors based on 2 μm yeast plasmid) or integrate into the chromosome (integrative plasmids). Eukaryotic plasmids include, for example, baculovirus vectors (Kirpbauer R, F Wow, N Chepg, DR Low, T Schiller, Parillomavirus Lactum test. USA 89: 12180-12184, 1992; Vollers C, P Sсhirmacher, RE Strеssk, M Sarr, Asеmblу оf thе mаjоr аpd thе miпосарсід ріпеtіпоf humap ріслеслісліtіслісліtіслісtіпуспуспус 504-512, 1994), vaccine virus-based vectors (Zhou J, XY Sup, DJ Stepzel, IH Frezer, Expresitive HPV 16 Ll ap L2 ORF Reserved HPV gu 185: 251-257, 1991; for example ME, N Yaegashi, DA Gallow, Self-ass of hum hum ravillavirus, I sarsids exp, Lopro, it is liberated. -322, 1993), as well as yeast vectors, for example, pGAL-based vectors containing the GAL 1-10 promoter (Hofmashi KJ, Sok JC, Jouse JG, Brow DR, Shchultz LD, Görm Rösol M, Fife K, Japse KU, Sеcepsa dethermiptiop оf humap paralillomavirus tour 6а add-up оf virus-likе ріrtісlеs іn Sassharotusеs serevisiаe. Virology 209, 506-518, 1995). To organize the transcription of structural protein Ll and L2 genes in yeast cells, promoters of different yeast genes, such as GALl, ADHl, ADH2, PGKl, TDHl, CUPl, etc. can be used.

The introduction of the plasmid into the host cell can be carried out in many ways well known to specialists in this field. In the case of prokaryotic host cells, transformation using Ca ⁺ ions, electroporation, transfection, transduction can be used. In the case of eukaryotic cells, electroporation, cationic transformation, and transfection, as well as transformation by DNA fragments sprayed onto particles of colloidal gold, and transformation by protoplasty can also be used.

As a result of the invention, the pPDX2 vector, intended for expression of the Ll protein in yeast cells, was constructed on the basis of plasmids pPDXl, and pUСGАLаtgΛТгøΙ. The first plasmid was used as a vector, and a fragment carrying the GAL1-10 promoter was obtained from the second plasmid. Necessary the plasmid was selected and received the name pPDX2. The resulting plasmid, thus, carries:

• a fragment of the sequence of a 2-micron plasmid of yeast, allowing it to replicate in yeast cells,

• URAB gene, which is a selective marker in the transformation of yeast cells,

• GAL 1-10 gene promoter,

• CYC \ gene terminator,

• between the promoter and the terminator there are unique sites Ххол and ВАТШ, according to which further inclusion in the obtained plasmid of various sequences of the gene sequences of the Ll protein

• the Na gene, which is a selective marker for the transformation of E. coli cells and the origin of replication for bacterial cells.

Thus, genes encoding the Ll protein can be easily transferred to the pPDX2 vector. The efficiency of transformation of yeast cells with plasmid DNA was carried out according to the method of [Ito et al. (1983)] and for the vector pPDX2 is approximately 10 ³ transformants / μg of DNA.

Purification of the recombinant protein may be carried out by any of the methods well known in the art. For purification, such procedures as chromatography (ion exchange, gel permeation) can be used without restriction (Soak JC, Jouse JG, George NA, Schultz LD, Hurni WM, Japse KU, Nerl RW, Ip C, Low RS, Rugifi-virus virus- like ricottles of reso humipapillomavirus tour 11 majo sarsid protein Ll, Roth. Exp. Rur. 17, 477-484, 1999), preparative electrophoresis (capillary, in gel), ultrafiltration (Soock et al., et al. in gradient (Hofipapp KJ, Jok JC, Jos JG, Wropp DR, Schultz LD, George N M Rosol M, F K, Jap KU, Safe Detected Avoid Configu n Sasshothetes servisio.Virolog 209, 506-518, 1995; Sasagawa T, Pushk P, Sters G, Gsmsseper SE, Jagheri MA, Phpssumplessformal Upd Tour 16 IP Fissiop West Chessosassarotus Rotbe. Virology 206, 126-135, 1995).

It is known that the malignancy of HPV-infected epithelial cells is due to the synthesis of three proteins - E5, E6 and E7, encoded by the HPV genome. At the main role in the induction of tumor transformation belongs to the low molecular weight protein E7, which, therefore, is an ideal target for the development of therapeutic vaccines. Such a vaccine should stimulate the production of cytotoxic T-lymphocytes, i.e., E7-specific CD8 + lymphocytes, which can destroy cells expressing E7 protein on their surface. Moreover, both the E7 protein itself and peptides that mimic the T-epitopes of this protein can be used as the main components of the vaccine (Feltkamr M. C. W., HL Smits, MPM WierbOOm, RP Mishiaaag, B. M. de Jopgh, JW Drijfhout, J. Ter Schaggett, C. JM Mélief, and WM Cast. 2242-2249). The length of the peptides can range from 8 to 40 amino acids.

Purified synthetic peptides, which are fragments of the E7 protein, can be obtained by various methods well known to specialists in the field of peptide chemistry, for example, such as Merrifield solid-phase synthesis using commercially available reagents and peptide synthesizers produced by the Percertive BIOSystems, Weightmap, Advapched Chem Labortes AG et al. Various methods can be used to purify synthesized peptides. For the synthesis of relatively short peptides, classical chemistry methods (in solution) can also be used, although they are more laborious. One alternative is peptide synthesis using recombinant technology. In this case, synthetic oligonucleotides are obtained that encode the desired peptide, and this sequence (exon) can be repeated one or more times in one nucleotide chain. Between exons there are special inserts encoding short amino acid sequences recognized by proteases capable of cutting the resulting recombinant product into desired fragments — target peptides.

For greater proteolytic stability of synthetic peptides from the E7 protein, their N- and C-terminal amino acids can be replaced by unnatural D-enantiomeric analogues, or their free N- and C-terminal groups can be blocked by the acyl and amide groups, respectively. For conjugation of peptides with molecules that enhance their immunogenic activity (biopolymers, proteins, adjuvants), functional groups and spacers of aliphatic, aromatic or mixed nature can be introduced into peptides during chemical synthesis.

The methods for purifying peptides can be various, depending on their properties (solubility in various media, molecular charge at neutral pH, hydrophilic-hydrophobic balance), but the most preferred method of purification is gel permeation chromatography on hydrophilic resins (crosslinked dextrans, cellulose derivatives, hydroxylapatite, porous glasses, etc.) and high performance liquid chromatography (HPLC) on reverse phase columns.

Most preferred in accordance with the present invention are the following peptides of the human papillomavirus E7 protein:

Peptides E7 HPV16:

SEQ ID NO: 1 YMLDLQRETTDLYC,

SEQ ID NO: 2 RAQQAERDRANYNWTFSSK,

SEQ ID NO: 3 QАЕРDRАНYNТVТFССКСDSТТRLRLСVQSТНVDIR,

SEQ ID NO: 4 QSTHVDIRTLEDLLMGTLGIV,

SEQ ID NO: 5 GQAERDRANYNIVTFCCC,

Peptides E7 HPVl 8:

SEQ ID NO: 6 CATLQDIVLHL,

SEQ ID NO: 7 RKATLQDIVLHLERQNЕIРV,

SEQ ID NO: 8 GVNHQHLPA,

SEQ ID NO: 9 RAEPQRHTM,

SEQ ID NO: 10 GQAERDRANYNГVТFССКСDSТLRLСVQSТНVDIR,

Peptides E7 HPV31:

SEQ ID NO: 11 VLDLQREATD,

SEQ ID NO: 12 GQAERDTSNYNGVTFCCQ,

SEQ ID NO: 13 GQAERDТSNYNТVТFССQСКSТLRLСVQSTТQVDIR,

SEQ ID NO: 14 ELLMGSFGIVCPNA,

SEQ ID NO: 15 TSNYNГVTF,

SEQ ID NO: 16 DTSNYNГVTF.

Proteins and peptides that are part of the vaccines and compositions of this inventions may be present in salt form, in particular a pharmaceutically acceptable salt. Such salts include, for example, inorganic salts of sodium, potassium, lithium, ammonium, as well as organic salts of primary, secondary or tertiary amines. Various organic salts can also be obtained and used for the purposes of this invention, such as salts of acetic acid, propionic acid, grape acid, maleic acid and other acceptable acids.

Compositions and vaccines of the present invention suitable for use in the prevention and / or treatment of human papillomavirus infection can be prepared in accordance with methods known to those skilled in the art, in particular by mixing with pharmaceutically acceptable excipients. Such compositions will contain an effective amount of Ll protein, as well as, if necessary, at least one peptide that is a fragment of the human papillomavirus E7 protein.

In addition, Ll protein and human papillomavirus E7 protein peptides, either alone or in combination with other active agents or excipients, can be incorporated or encapsulated in the liposome cavity for delivery and to increase the life of compositions based on ex vivo protein and peptides and ip vivo. As is known in the adnal region, liposomes can be assigned to one of several types: multilayer, stable, consisting of several layers, small unilamellar or large unilamellar vesicles. Liposomes can be obtained from various lipid components, both naturally occurring and synthetic, including phosphatidyl ethers and esters such as phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, dimyristoylphosphatidylcholine; steroids such as cholesterol; cerebrosides; sphingomyelin; glycerolipids; and other lipids (see, for example, US Pat. No. 5,833,948).

In the context of this application, “effective amount)) means such an amount of the active principle (protein, peptide) of the present invention that is sufficient to provide the desired effect in relation to the condition in connection with which they are administered to the individual. The exact amount will depend on the specific circumstances and can be estimated by a person skilled in the art using known techniques. Typically, the amount should be able to have a prophylactic effect on the development of papillomavirus infection or therapeutic effect if present, and also induce an immune response in the person to whom it is administered. The specialist will understand that the effective amount will depend on the type of papillomavirus, the mode of administration, whether the composition or vaccine is administered alone or in combination with other drugs, the general state of health of the individual, the reactivity of his immune system. Typically, the compositions and vaccines of the present invention will be administered in doses ranging from about 1 μg to about 1 mg.

Under ((pharmaceutically acceptable)) in the context of the present description refers to such an excipient, for example, a carrier, a solvent, an immunomodulator, an adjuvant or any other targeted additive that does not cause undesirable side effects in the individual to whom they are administered and do not interfere with the implementation of the target biological function when the introduction of a vaccine into the body of an individual. Such pharmaceutically and physiologically acceptable means well known in the art (see, e.g., Remington's Rharmaseutisal Ssiepses 18 ^W editiop, Gepparo A.R., Ed, Mask Rublishipg Somrapu - 1990;.. Napdbook Rharmaseutisal Ehsiriepts ° F, G ^rd editiop, A Kibbe, Ed., Pharmacutral Pres - 2000). A variety of excipients that may be included in the compositions or vaccines of the present invention are well known to those skilled in the art. Such excipients may include buffers, dispersants, preservatives, stabilizers.

To maintain osmotic pressure, vaccines may contain tonicity agents such as sucrose, glucose, sodium chloride, polyglucin, reopoliglukin, as well as polyhydric sugar alcohols such as glycerol, erythritol, arabitol, xylitol, sorbitol, or mannitol.

To maintain physiological pH, the vaccines of the present invention may contain physiologically acceptable buffering agents, such as Heps, phosphate, citrate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate or histidine buffers, as well as combinations thereof.

Since the vaccines of the present invention are administered parenterally, one skilled in the art will recognize that the vaccines must be in sterile form. Various methods known in the art can be used to ensure sterility, such as filtering through a sterilizing a filter with a pore size of 0.22 microns. To prevent bacterial infection, preservatives, for example, thimerosal, phenol, may also be included in the vaccines.

To increase the immunogenicity, the vaccines of the present invention may contain a variety of adjuvants, such as CPG, aluminum phosphate or hydroxide, stearyl tyrosine, MDP, GMDP, polyoxidonium, extracts of fragments of the bacterial cell wall (see FR Vogel, MF Travel, C. R. Alviпg. And Сomrepdium оf Vassipе Аdjuvapts аpd Exciriepts, 1999, 2nd Editiop).

The immunological efficacy of the obtained VLPs, i.e., good humoral and cellular immune responses, as a necessary condition for the protective activity of the vaccine, is demonstrated in various tests on animal models. Such tests are well known in the art and are recognized by experts as adequate [Breitbur et al. J. Virol., 1995.69 3959-3963; Kirpbauer et al. Virology, 1996, 219, 37-44; Suzish et al. Roc. Natl. Acad. Sсi. USA, 1995, 92, 11553-11557; M. F. Duggap-Kep, M. D. Browp, S. N. Staseu, P. L. Sterp. Rarillomavirus vassipes. Framers ip BIOSIPSE, 3, dl 192-1208, 1, 1998; Schoell WM, Mirshashmi R., Liu V., Japan MF, ER ER, Repalver MA, AvtoRette N.E. Gerapatiop of tumo-rescuerto ispulertulopredeposito HP: Of Servisal Sapser. Hupesol Opsoel 74: 448-455, 1999].

These include:

• the induction of high titers of anti-VLP antibodies obtained after immunization of laboratory animals with the corresponding Ll VLP without adjuvant,

• reactivity of monoclonal HPV virus neutralizing reference antibodies (commercial) in enzyme immunoassay reactions (ELISA) with the obtained Ll VLP,

• electron micrograph data (spherical particles about 50 nm in size should be visible),

• the ability to produce, as a result of immunization with Ll drugs, VLP-specific T-helper lymphocytes, • ability to produce as a result of immunization with Ll

VLP-specific cytotoxic lymphocytes.

The immunological effectiveness of the obtained peptides from the E7 protein is demonstrated by the following tests:

• the induction of anti-peptide antibodies obtained after immunization of laboratory animals with peptides from E7 protein with the appropriate adjuvants,

• the ability to produce specific T-helper lymphocytes as a result of immunization with E7 peptides

• the ability to produce, as a result of immunization with Ll drugs, VLP-specific cytotoxic lymphocytes

High titers of antibodies to the obtained recombinant Ll VLP are achieved by immunizing laboratory mice with compositions based on Ll VLP16, 18, or 31 preparations. The immunizing composition may include the Ll protein itself, saline solution, adjuvants, protein stabilizers, and other components, for example, immunoactive synthetic peptides that mimic the T-cell epitopes of the HPV E7 protein and create T-cell immunity. Compositions containing Ll VLP are considered prophylactic vaccines. Compositions containing, in addition to Ll VLP peptides, HPV protein E7 16, 18, 31 can be used as both prophylactic and therapeutic vaccines.

Vaccines and compositions of the present invention can be administered in various ways, for example (without limitation) subcutaneously, topically, intramuscularly, orally, intravenously, transmucosally, vaginally, rectally. Vaccines and compositions may be administered as a single dose or as multiple doses. The administration schedule can be evaluated by the attending physician taking into account, in particular, factors such as the type of papillomavirus, age, gender, general condition of the patient, the severity of the disease or condition to be treated or prevented, the route of administration, and the particular vaccine or composition. Within the competence of the physician, choose the dosage of the administered composition or vaccine.

In one embodiment, the Ll protein and peptides of the human papillomavirus E7 protein are introduced in a solution or suspension in a suitable aqueous medium. Besides in addition, the vaccine compositions of the present invention for injection can be prepared in lipophilic solvents, such as (without limitation) oils such as vegetable, olive, peanut, palm, safflower, corn or soybean; synthetic fatty acid esters such as ethyl oleate or triglycerides; cholesterol derivatives such as cholesterol oleate, cholesterol linoleate, cholesterol myristylate. The compositions can be prepared directly in a lipophilic solvent or, preferably, in an oil / water emulsion (see, for example, Liu F. et al., Pharm. Res. 12: 1060-1064 (1995), Prankerd RJ, J. Rept. Ssi Tes. 44: 139-149 (1990)).

The invention will now be illustrated with reference to examples, however, it should be understood that the examples are provided solely for the purpose of clarifying the essence of the claimed invention and are not intended to limit it.

Example 1. Design primers

In order to clone the HPV Ll protein genes of serotypes 16 and 18, a computer search was made for the sequences of Ll protein genes of various subtypes of serotypes 16 and 18 of HPV in the open databases of Expu, PubMed. Based on the sequences found, primers were designed and synthesized to amplify the target genes, as shown in Table 1.

Table 1. Sequences of primers designed for amplification of Ll protein genes of serotypes 16 and 18 of HPV.

Underlined are the restriction enzyme sites Xhol and BgIII

Non-complementary regions containing six-membered sequences recognized by the restriction enzymes Xhol (in the primers to the 3 'end) and BgIII (in the primers to the 5'-end) were added to the primers from the 5' ends.

SHEET Example 2. Polymerase chain reaction (PCR)

The annealing temperature during the reaction was calculated by the formula T = 2 (A + T) +4 (G + C), where T is the temperature, A, T, G, C is the number of corresponding nucleotides in the primer. The working solutions for PCR were: 1Ox reaction buffer (600 mM Tris-Hcl, pH 8.5, 250 mM KCL, 15 mM MgCl ₂ , 100 mM mercaptoethanol, 1% Triton X-100), 1.25 mM mixture of dNTP, Taq -polymerase 10 u / μl, solution of primers, template DNA (in serum), H ₂ O (supQ). For PCR, 25 pmol of each of the primers was taken. Ready for the reaction, the mixture was carefully mixed, mineral oil was layered on top. The reaction was set at 20 μl. Microcentrifuge tubes were placed in a thermal cycler, which was programmed as follows: Cycle 1: primary melting 94 ° C 1.5 min; Cycle 2: basic, melting 94 ⁰ C 20 s. Primer annealing at a specific temperature (in our case, 67 ⁰ C) 5 s, synthesis 72 ⁰ C 30 s, repeat 5 times, melting 94 ⁰ C 5 s, anneal primers at 67 ⁰ C 5 s, synthesis 72 ⁰ C 30 s Repeat: 40 times. Cycle 3: storage at 10 ⁰ C

Thus, using PCR based on clinical materials, the required sequences of 1511 (serotype 16) and 1521 (serotype 18) nucleotides were synthesized. Each amplification of a single serotype gene was based on two different samples, in four replicates. In FIG. Figure 1 shows a photograph of gel electrophoresis of fragments obtained by PCR.

Example 3. Cloning of the Ll protein gene of HPV 16 and 18 into wιazmid. Fragments obtained by PCR were cloned into plasmid pUC19, taken from the laboratory collection. Plasmid pUC19 contained the lacZ gene encoding β-galactosidase. Inside the lacZ gene are numerous unique restriction sites. When any DNA fragments are inserted at these sites, the reading frame of the lacZ gene is usually violated, and β-galactosidase is not synthesized. The presence and absence of functional β-galactosidase is easily determined using a reagent called X-gal, which is added to the microorganism culture medium. If functional β-galactosidase is present in the cells, the colony of microorganisms turns blue.

When cloning DNA fragments containing the human papillomavirus Ll protein genes of serotypes 16 and 18, ligation was used according to "Typym" ends. For this, the fragments obtained by PCR were phosphorylated by polynucleotide kinase, since fragments without phosphate at the end cannot be ligated. The kinase mixture was as follows: 1/1 OV 1OmM ATP, 1/1 OV special buffer (buffer "A"), polynucleotide kinase, water bidistyl. to the required volume. The reaction was carried out for an hour at 37 ⁰ C.

The pUC 19 vector was treated with Smal restriction endonuclease that recognizes and cleaves the CCC | GGG site. Thus, the restriction enzyme Smal left a “loose” ends. Restriction was performed according to the following protocol. The number of enzyme units was calculated based on the size of DNA, its concentration and the number of restriction sites. The selection of reaction conditions was carried out in accordance with the catalog of the manufacturer (Fermeptas, Lithuania). The reaction mixture was collected according to the formula: DNA + 1Ox buffer + enzyme + H ₂ O bidistyl., The enzyme was added last. After an incubation of 60 min at a temperature optimal for the operation of this enzyme, the result was evaluated by the pattern of electrophoresis separation of restriction products on an agarose gel. DNA electrophoresis was performed in 50 mM Tris-acetate buffer in a 1% agarose gel, as described in Maniatis et al., 1984. The electrophoresis bath was poured with electrode buffer, agarose was dissolved in an amount of 1% by heating in the same buffer, and gel was added ethidium bromide 1-2 μg / ml. The gel at a temperature of 60 ⁰ C was poured into a plate with combs (the usual number of holes is 14, the usual volume of the gel is 20 ml). After waiting for the gel to harden, samples were added. Samples were pre-mixed with DNA buffer containing dye bromphenol blue. Electrophoresis was performed at a voltage of 10 V / cm. After sufficient passage of the phoresis marked by the leading bromophenol blue, the position of DNA in the UV light was recorded.

Using electrophoresis, we verified the complete passage of restriction to the Smal site, as well as the presence of fragments of the desired size in the kinase mixture, after which the Smal restriction enzyme in the reaction mixture with pUC19 vector DNA was inactivated by heating at 65 ^° C for 20 minutes; similarly treated with kinase in a reaction mixture with PCR fragments. The restriction enzyme-treated vector with PCR fragments was then ligated. Approximately the same number of fragments and vector were taken for ligation. Ligation was carried out as follows: 1 / 10V 10 mM ATP, 1 / 10V ligase buffer, 5-10 units. T4-DHK- ligases, 0.2 μg DNA, bidistyl. water up to 20 μl. The reaction was carried out for 16 hours at +4 ⁰ C.

The bacteria Escherichia coli strain TG were transformed with a ligase mixture. For this, 50 μl of an overnight culture of E. coli cells was added to 5 ml of fresh LB medium (1% peptone, 0.5% yeast extract, 1% NaCl) and cultured for 1.5 hours at 37 ^° C. Then the cells were cooled for 10 minutes. in an ice bath and was separated from the medium by centrifugation in microcentrifuge tubes for 15 seconds at 12,000 rpm. The cell pellet was washed with 200 μl of 100 mM CaCl ₂ , then suspended in 1 ml of fresh 100 mM CaCl ₂ and incubated on ice for 2 hours. After incubation in 100 mM CaCl _{2, the} cells were precipitated by centrifugation for 15 seconds at 12,000 rpm, then the pellet was resuspended in 400 μl of 100 mM CaCl ₂ . Next, 200 μl of the cell suspension was mixed with the ligase mixture and incubated for 40 min in an ice bath. After that, heat shock was carried out for 2 min at 42 ^° C and cooled in an ice bath for 5 min. Then 800 μl of LB was added, incubated for 60 min at 37 ^° C, centrifuged for 20 sec at 12,000 rpm, excess fluid was discarded, and cells were plated on an LA plate with antibiotic, X-GAL and IPTG using a spatula. When grown on selective medium, the transformed cells were white. Received 5 types of transformants carrying the plasmid pUC19, ligated with 3 existing fragments containing the Ll gene of the HPVl serotype 16, and with 2 fragments containing the Ll gene of the HPVl 8 serotype.

For analysis, plasmid DNA was isolated from 500 obtained white transformants (with a broken reading frame of the lacZ gene) according to the following protocol. Cells from 3 ml of overnight culture (in LB medium with antibiotic) were collected by centrifugation for 15 sec at 12000 rpm in microcentrifuge tubes, suspended in GET buffer (per 100 ml: 0.92 g of glucose; 0.37 g of EDTA; 2.5 ml Tris, pH 7.5) - 100 μl per Vortex. 200 μl of fresh lysis buffer (NaOH 0.25 g; 2.5 ml SDS 10%; 22.5 ml H ₂ O) was added to the suspension, the mixture was stirred by inversion and incubated at +4 ⁰ C for 10 -12 min, then 140 μl ZM sodium acetate, pH 5.0. After mixing in test tubes, a white precipitate (proteins, chromosomes) fell out. Sediment flakes were removed by centrifugation for 2 min at 12,000 rpm, after which 10 μl of the supernatant was applied to preliminary electrophoresis to separate the resulting plasmids by size. Of the 300 total analyzed clones, 20 were selected, which were the procedure for further DNA extraction. The supernatant was transferred to microcentrifuge tubes, to which 200 μl of 10 M LiCl was added to precipitate RNA, and the mixture was incubated at -2O ⁰ C for 40-60 min. The incubate was centrifuged for 5 min at 12,000 rpm, the supernatant was transferred into microcentrifuge tubes.

600 μl of isopropanol was added to the solution, the mixture was stirred (brief incubation at + 4 ^° C), then centrifuged for 5 min at 12,000 rpm and the aqueous phase was removed by a water-jet pump. The precipitate was washed twice with 200 μl of C ₂ H ₅ OH 70%, centrifuging for 15 seconds and removing alcohol with a water-jet pump. The resulting precipitate was dissolved in 50 μl bidistilled water.

Restriction of the isolated plasmids was carried out at the PvuII site and plasmids carrying the desired insert were selected according to the formation of fragments of the calculated size during electrophoretic separation. To confirm the correctness of the cloned fragments, an additional analysis was performed using BamHI / EcoRI restriction. Verification of the selected plasmids was carried out using restriction sites BAM. With this restriction variant, the orientation of the insert was determined. In the case of pUC19-Ll-16, if the orientation is straight (as shown in Fig. 2A), fragments of 521 and 3683 nucleotides and 1018 and 3186 nucleotides are cleaved if the orientation is reversed. In the case of pUC19-Ll-18, fragments of 1324 and 2886 nucleotides are cleaved if the orientation is straight (as shown in Fig. 2B), and 221 and 3989 nucleotides if the orientation is reversed. As a result, the orientation of the inserts in plasmids pUC-Ll-18-1, pUC-Ll-18-2 and pUC-Ll-16-2 is defined as a straight line (as shown in Fig. 2), for plasmids pUC-Ll-16-1 , 3 and 4 - as the opposite.

Example 4. Sequencing and comparison of the nucleotide sequences of the Ll gene and the predicted amino acid sequences of the Ll protein

The mixture for automatic sequencing was compiled in microcentrifuge tubes for the NDP from the matrix, primer and bidistilled water. The amount of the matrix was calculated according to the requirements of the manufacturer of the sequencer. For pUC19 carrying the insert of the Ll gene, this amount is 0.1-0.2 μg / reaction. The amount of primer was 4 pmol / reaction. Using bidistilled water, the mixture was brought to a volume of 12 μl. After sequencing of plasmids in the nucleotide sequences of the Ll gene of serotype 16, a total of 22 mismatches were observed, of which 5 were natural (that is, common for all PCR copies), 16 PCR errors, and 1 error in the primer (5'-terminal). In similar sequences of the Ll gene of serotype 18, a total of 10 mismatches are observed, of which 8 are natural and 2 are PCR errors. Representative sequences of the Ll protein gene of each of the HPV16, HPV18 and HPV31 serotypes obtained by sequencing are shown in the Sequence Listing Numbers SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 186, respectively.

Since the genetic code is degenerate, some base substitutions in DNA do not lead to a substitution of amino acids in the primary protein sequence, therefore, the obtained sequences were translated using the DNA-SUN program, and the amino acid sequences were compared with published ones. The alignment results of the predicted amino acid sequences of the HPV Ll protein of serotypes 16, 18 and 31 are shown in FIG. 3-5.

Thus, in the amino acid sequences of the Ll gene of serotype 16, a total of 12 discrepancies are observed, of which 4 are natural (that is, common to all translated variants), 7 PCR errors, and 1 replacement resulting from an error in the primer. All natural substitutions are published. Of the substitutions that result from PCR errors, 4 belong to option 16-1, 2 to option 16-2, and option 16-4 carries only one replacement.

In the amino acid sequences of the Ll gene of serotype 18, 5 inconsistencies are observed, of which 4 are natural and 1 is PCR error. Of the natural substitutions, 3 are published (it should be noted that for serotype 18 only 3 complete amino acid sequences of the Ll protein are published). The only amino acid substitution resulting from PCR error belongs to variant 18-1. Thus, option 18-2 is completely error-free in amino acid sequence.

Example 5. Construction of yeast vectors expressing Ll proteins

The pPDX2 vector, designed for expression of the Ll protein in yeast cells, is constructed on the basis of pPDXl plasmids, after which fragments containing the genes encoding the Ll protein are transferred to this vector. The result is plasmid constructs containing the corresponding Ll genes (plasmids pPDX2-Ll-16, pPDX2-Ll-18, etc.) (Fig. 6).

The result was six yeast replicative plasmids designed for expression of the human papillomavirus Ll Ll protein of serotypes 16 and 18. Of these, two for serotype 18 and four for serotype 16. These plasmids were named pPDX2-Ll-16 JVeJVe 1-4 and pPDX2-Ll-18 JVsJfel, 2.

Example 6. Transformation of yeast cells Sasshotus servisisae

The transformation of S. yeast cells with plasmid DNA was carried out in two stages: on the first, the preparation of competent cells, on the second, the actual procedure.

The yeast cell culture grown on _YPD medium and in the logarithmic phase was centrifuged for 2 min at 4000 rpm, the pellet was washed with 5-10 ml of TE buffer (10 mM Tris-Hcl pH 7.6; 0.1 mM EDTA) . After centrifugation, 5 ml of TE + LiAc (10 mM Tris-Hcl pH 7.6; 0.1 mM EDTA; LiAc 0, 1 M), 20 μl mercaptoethanol were added to the pellet, and incubation was performed for 60 min at 28 ^° C. Next, the cells were besieged by centrifugation for 2 min at 4000 rpm, and the precipitate was washed with TE + LiAc. TE + LiAc was added to the cell pellet at the rate of 10 ⁸ -10 ⁹ cells / ml.

Prepared competent cells in an amount of 2x10 -1x10 were mixed with 5-10 μg of DNA in microcentrifuge tubes. The mixture was incubated for 30 min at 3O ⁰ C, an equal volume of PEG-4000 (50% PEG-4000; 50% TE + LiAc) was added to all tubes, then incubated for 60 min at 3O ⁰ C. Then, heat shock was carried out for 15 min at 42 ° C, after which the cells were plated on plates with selective medium (SCD with appropriate amino acid additives).

Example 7. Production Ll VLP

Yeast cells were grown for 68-72 hours in complete medium with 2% glucose, and at the end of the logarithmic phase, 2% galactose was added to the cells. Cells were harvested by centrifugation, PEN buffer (20 mM Na ₃ PO ₄ , pH 7.2, 100 mM NaCl, 1.7 mM EDTA) and PMSF were added to the cell pellet in an equal volume to a final concentration of 2 mM. Cells were destroyed using glass beads with a diameter of 0.6 - 0.8 mm. The lysate thus obtained was clarified by centrifugation at 5000 g for 20 min and applied to a gradient of 45% sucrose dissolved in PEN buffer. In this case, the gradient contained a substrate of 70% sucrose dissolved in PEN buffer. Test tubes were laid on a 4-hour centrifugation at 10000Og. In this case, virus-like particles passed through 45% sucrose, lingering at the 45/70% sucrose interface.

After ultracentrifugation, the upper phase up to half of 45% sucrose was removed from the tubes using a water jet pump. From the remaining sucrose, 3 ml were taken from the bottom using a syringe. The resulting preparation was subjected to a tangential flow ultrafiltration on a 10 kDa membrane for purification from sucrose. A buffer of 0.2 M MOPS, pH 7.0, 0.4 M NaCl was used for ultrafiltration.

The sucrose-free Ll VLP preparation was centrifuged at 500Og for 20 minutes and re-applied to the sucrose gradient. Samples taken from the second sucrose gradient were re-purified from sucrose by ultrafiltration on a membrane. Protein Ll VLP is usually retained at the 45/70% sucrose interface. The desired fraction was subjected to ultrafiltration on a membrane with a pore size of 10 kDa, clarification (5000 g), repeated ultracentrifugation (10000 Og) and ultrafiltration on a membrane of 100 kDa, thus removing (monomeric) proteins not included in the Ll VLP complex. An example of the production of Ll VLP HPVl 8 according to electrophoresis is shown in FIG. 7. The time to reach the maximum concentration of VLP in the yeast cell depends on the serotype of the virus (40-80 hours) and the composition of the nutrient medium, carbon source. For preparative isolation of VLP, producer cell cultures are grown in culture medium with a carbon source specific for each serotype. For example, for serotype 18 - 3% glucose + 3% glycerol, and for serotype 16 - 0.5% glucose + 1.5% ethanol + 3% glycerol. At the end of the logarithmic phase, 2% galactose is added to the medium.

Example 8. The measurement of protein according to the method of Bradford

Protein samples were added to the wells of 96-well plates in an amount of 20 μl, a dye solution from the Bradford set (Sigma) in an amount of 180 μl was added to them. After a 5-minute incubation, the optical density of the mixtures was measured on a MultiSap reader using a λ = 620 nm filter. A calibration curve was constructed using prepared protein standards based on BSA (bovine serum albumin) with concentrations of 0, 50, 100, 150, 300 and 500 μg / ml.

Example 9. Acrylamide Gel Denaturing Electrophoresis (SDS-PAGE) A protein electrophoresis chamber was assembled, 2 ml was poured into it. separating gel as a substrate. To visualize proteins of the same size as Ll protein (~ 55 kDa) it is necessary to use a separating gel with an acrylamide content of 8% (20% solution of 40% acrylamide, 10.5% solution of 2% bisacrylamide, 1% solution 10% sodium dodecyl sulfate, 37.5% Tris-HCl IM, pH 8.8, H ₂ O). TEMED in an amount of 1/1000 volume and ammonium persulfate in an amount of 8/1000 volume were added to the gel immediately before polymerization. After the substrate solidified, an additional 8 ml of separation gel was poured into the chamber, and 1 ml of water was added from above. After the polymerization of the separating gel was complete, the water merged, the surface was dried with filter paper. 2.5 ml of a concentrating gel (10% solution of 40% acrylamide, 5.3% solution of 2% bisacrylamide, 12.5% Tris-Hcl IM, pH 6.9, 1% solution of 10% - were poured into the chamber sodium dodecyl sulfate, H ₂ O TEMED in an amount of 1/1000 volume and ammonium persulfate in an amount of 8/1000 volume were added to the gel immediately before polymerization). A comb of 10 wells was inserted into a concentrating gel (30 μl per well).

After complete polymerization of the concentrating gel, the comb was removed, the wells were washed with distilled water. The chamber was immersed in an electrophoresis bath and poured with buffer (per 1 liter of buffer: Tris - 3 g, glycine - 14.4 g, sodium dodecyl sulfate - 1 g). Protein solutions diluted so that the load on each lane is the same, mixed in 1.5 ml microcentrifuge tubes with equal sample paint volumes (per 100 ml: Tris 1.51 g, glycerol 20 ml, pH up to 6.75 , sodium dodecyl sulfate 4 g, β-mercaptoethanol 10 ml, water up to 100 ml, bromophenol blue 2 mg) and boiled for 60 s. After cooling the tubes to room temperature, the samples were introduced into the wells using a microsyringe. Electrophoresis was carried out at a constant current of 0.33 mA / cm ² for an hour. After electrophoresis, marked by the leading dye bromphenol blue, the camera was disassembled, the gel was preparing for staining.

Example 10. Silver staining of acrylamide gels after protein electrophoresis

If the amount of protein in the lane is less than 1 μg, after protein electrophoresis, the gel was silver stained. The gel was immersed in a fixing solution (25% 2-isopropanol, 10% glacial acetic acid) for 15 minutes, then washed for 15 minutes in distilled water, 15 minutes in 50% ethanol, and 15 minutes again in distilled water. After this, the gel was poured into 200 ml of a pre-prepared AgNO ₃ solution (100 ml of NaOH, 3.5 ml of 30% ammonia per 200 ml; 1.6 g of AgNO _{3 was} dissolved in 10 ml of distilled water, then added to the main volume; the solution was titrated with ammonia until the precipitate disappeared) and stained for 30 min in the dark. The stained gel was rinsed with distilled water 3-5 times for 5 minutes and 50 ml of the developing solution were poured (0.2% by volume of sodium citrate, 0.2% formaldehyde). After development, the gel was washed with water and photographed.

In FIG. 7 shows the results of electrophoretic analysis in polyacrylamide gel of several recombinant VLP samples of HPV-16 and 18 after ultracentrifugation of lysates obtained from several clones of yeast strain Y618 transformed with pPDX2-Ll-16 (lanes 2-5) and pPDX2-Ll-18 (lanes 7 -8). It can be seen that in lanes 2, 3, 5, 8 an additional band appeared in comparison with the negative control (lane 6), corresponding to Ll in weight of approximately 56 kDa.

Example 11. Coloring acrylamide gels after protein electrophoresis

Coomassie

When staining Coomassie, the gel was immersed for 5 min in a solution containing Coomassie brilliant blue - 0.25% by volume, isopropanol - 25%, glacial acetic acid - 10%, H ₂ O. The dye was pre-heated to 60 ⁰ C. The stained gel was washed warm 10% acetic acid until the bands appear.

Photographs of electrophoregrams are given in FIG. 8. It can be seen that in both photographs there is a band corresponding in molecular weight to the full-sized Ll protein (approximately 56 kDa). There is also a minor band corresponding in size to the degraded Ll protein (approximately 45 kDa). According to published data, a degraded protein is released during any purification [Soket et al., 1999]. Finally, there is a band corresponding in size to the endogenous Ti particles of the yeast (about 100 kDa).

Example 12. Enzyme-linked immunosorbent assay (ELISA)

Antigen solutions (Ll VLP proteins, peptides) in carbonate-bicarbonate buffer (KBB) with a pH of 9 (3 μg / ml) are applied at 50 μl / well in the Costa ELISA microplates and incubated for one hour at room temperature and overnight in the refrigerator (+4 °). After remove solution from the plate, buffer (50 μl / well) is added to the wells with adsorbed antigen to dilute the serum and conjugate (0.02 M, pH 7.2 PBS / 0.05% Tween 20 / 0.5% BSA) and incubated hour at 37 °. Wells are washed 4 times with 200 μl of buffer (0.02 M, pH 7.2 PBS / 0.05% Tween 20). Antibody solutions (monoclonal antibodies or polyclonal serum) are added at 50 μl / well, incubated for 1 hour at 37 ° and washed 4 times with 200 μl of buffer (0.02 M, pH 7.2 PBS / 0.05% Tween 20). Contribute anti-mouse conjugate (apti-IgG-HPP, Amersham) in a working dilution (1/2500) of 50 μl / well. Wash the wells as described above. Add a substrate solution (pH 5.0, 0.1 M FCB, 0.04% RPD, 0.2% hydrogen peroxide 3%) 50 μl / well and incubate at room temperature with a shade of 15 minutes. An alternative solution of TMB (Sigma) is also used as an alternative substrate. The reaction is blocked with 2 N sulfuric acid and the wells are scanned at a wavelength of 492 nm using a Multisap reader.

Example 13. Confirmation of the structure of VLP by ELISA. For the analysis of each protein Ll VLP specific monoclonal antibodies were used, obtained from the company US Biological and from prof. Christepsep (Theme Miltop S. Hershéu Medicinal Septer, Nershu Reppsulvia) (see Christepsep et al, 2001), as well as polyclonal anti-mouse antibodies to synthetic peptides from Ll VLP, corresponding to the dominant B-epitopes of this Table 4 (see epitope. The results of ELISA are shown in table 1.

eighteen.

Thus, the analysis data confirm the expected immunoreactivity of the obtained Ll VLP proteins with the indicated antibodies. Moreover, monoclonal neutralizing antibodies Hl 6 U4 S / N 9 and Hl 8 J4 S / N (Christepsep et al, 2001) react with VLP 16 and 18 reference preparations (obtained from Prof. Christepsep,) and with recombinant Ll VLP16 and 18. Since these monoclonal antibodies are specific only for conformational epitopes of Ll VLP, the results confirm the correct assembly of the obtained recombinant proteins into the capsid structure of VLP characteristic of the native virus. This structure is necessary for the generation of neutralizing antibodies when vaccinated with such drugs.

Example 14. Confirmation of the structure of VLP by Western blot.

Ll VLP preparations were also subjected to Western Blot (WB) assays using the above monoclonal antibodies from US Biological for recognition. WB data demonstrate the presence of a specifically recognizable band with a molecular weight corresponding to a full-sized Ll protein (approximately 56 kDa). A minor band corresponding in size to the degraded Ll protein (approximately 45 kDa) is also recognized by these antibodies. The band corresponding in size to the endogenous Ti particles of the yeast (about 100 kDa) is not recognized by antibodies.

Example 15. The confirmation of the structure of Ll VLP by electron microscopy.

The final proof of the structure of the Ll VLP proteins was obtained by transmission electron microscopy (see Fig. 9). Microphotographs confirm that the proteins synthesized in yeast are assembled into virus-like structures of the required size, and they fully correspond to the photographs previously obtained for Ll VLP published in the world literature.

Example 16. Immunological efficacy of recombinant Ll VLP - humoral response.

Assessment of the immunogenicity of recombinant Ll VLP is carried out by triple immunization of laboratory mice, serum antibody titers are evaluated by ELISA (table 2). The table shows the results of immunization of mice with Ll VLP 18 protein. It is seen that the titer of antibodies after the first immunization is quite high, with the second immunization the titer almost reaches the ceiling, and the addition of an adjuvant does not significantly stimulate the antibody response. The same picture is observed in the case of immunization with the protein Ll VLP 16 (table 3).

Table 2. Antibody titer to recombinant Ll VLP 18 after immunization of hybrid Fl mice

Table 3. Antibody titer to recombinant Ll VLP 16 after immunization of hybrid Fl mice.

Example 17. Immunological efficacy of recombinant Ll VLP - cell response.

The proliferative T-cell response (spleen cells) after vaccination with Ll VLP proteins and synthetic peptides is analyzed by ELISPOT, i.e., the titer of gamma-interferon secreted by activated T-cells is determined in a microplate version. For this purpose, peptides are used that simulate T-cell epitopes of proteins with a chain length of 9-25 amino acids (table 4). Such peptides are capable of causing antigen-specific activation of immune T cells. An indicator of the activation of cytotoxic (CTL) and T helper cells (ThI) cells is their secretion of interferon-gamma (IFN-γ). Determination of antigen-specific IFN-γ-producing cells and counting of their number was carried out using ELISPOT equipment on commercial kits from BD BIOSIPSEPS. Cells of immunized mice (splenocytes) are placed in 96 well plates at the bottom of which anti-mouse IFN-γ antibodies are adsorbed, incubated in complete culture medium with peptides for 18 hours at 37 ° in a 5% CO ₂ atmosphere. At the end of the incubation, the cells are removed and antibodies against mouse IFN-γ labeled with biotin are introduced. After incubation with labeled streptavidin, stained cells are analyzed on a special scanner (AELVIS (EES) - Eli.Sap). The results are presented in table 5.

Table 4. Synthetic peptides HPV Ll HPVl 6

Ll HPV18

E7 HPV16

E7 HPV31

(A) - Alanine residue introduced for the convenience of peptide synthesis: The Acm protecting group for the cysteine mercapto group (C) was introduced to simulate the blocking of this group in the Ll protein

Table 5. The number of IFN-γ producing cells after 18-hour incubation with the studied peptides (average values are given in three parallels).

Thus, it is seen that a strong T-cell immune response develops to recombinant Ll VLP 16 (the numbers are shown in bold), which indicates the presence of not only preventive, but also therapeutic potential in the developed drugs. From the data obtained, it can be seen that immunization with NC-725 + PAL-15 causes a noticeable activation of IFN-γ producing cells. Thus, the number of cells activated by the peptide was approximately 85, and spontaneously activated 9. The peptide without the adjuvant is not able to elicit a T-cell response.

Example 18. Immunological efficacy of recombinant Ll VLP - cell response in a population enriched in cytotoxic CD8 + lymphocytes.

In another series of immunizations, the data in Example 17 found further confirmation in tests with T-cell selection (a subpopulation of CD4 + cells was removed to detect the reactivity of cytotoxic CD8 + cells) (table 6).

Table 6. The number of IFN-γ producing cells after 18-hour incubation with the studied peptides (average values are given in three parallels). The active zones are highlighted in red and blue.

Thus, Ll VLP 16 shows high activity, as in the previous experiment. Of particular importance is that when using the pool of CD8 + lymphocytes (CD4 + cells removed), the response was stronger due to the enrichment of this population, which indicates a high cytotoxic potential of this immunogen for infected cells. The synthetic peptides P53.236 and NC-725, containing both CTL and Thl epitopes (both of the E7 transforming protein HPV 16 and HPV31) also showed marked activity, but only in combination with adjuvant. Thus, the resulting recombinant proteins and peptides have a strong prophylactic and therapeutic potential in relation to human papillomavirus infection.

Although the invention has been described above with reference to its preferred embodiments, it will be clear to a person skilled in the art that various changes and modifications of the described specific embodiments are possible without deviating from a wide inventive concept. Any changes and modifications of the invention that will function in the same way and ensure the achievement of the same result will fall within the scope of protection of the invention, which is determined by the claims below.

Claims

CLAIM

1. A method for producing a recombinant human papillomavirus Ll Ll protein selected from serotypes HPV16, HPV18 and HPV31, comprising: a) obtaining a copy of the gene encoding the Ll protein by polymerase chain reaction using clinical material, b) obtaining a plasmid containing a copy of the gene obtained in step a) and for expression of the human papillomavirus Ll Ll protein in host yeast cells, c) transformation of the host yeast cells with the plasmid obtained in step b), d) culturing the transformed host yeast cells under via ensuring efficient expression of the recombinant papillomavirus protein Ll; e) extraction and purification of papillomavirus protein Ll.

2. The method of claim 1, wherein the plasmid containing a copy of the Ll gene is derived from the yeast plasmid pPDX2.

3. Recombinant HPV human papillomavirus Ll protein obtained by the method according to any one of claims. 1 or 2.

4. The protein according to claim 3, characterized in that it is obtained in the form of virus-like particles.

5. The protein according to any one of paragraphs. 3-4, useful for the manufacture of a medicament for the prevention of papillomavirus infection.

6. A composition comprising purified Ll protein according to any one of paragraphs. 3-5 and at least one purified synthetic peptide, which is a fragment of human papillomavirus E7 protein, having a sequence selected from the group represented by:

SEQ ID NO: 1 YMLDLQRETTDLYC,

SEQ ID NO: 2 RAQQAERDRANYNWTFSSK,

SEQ ID NO: 3 QAERDRANYYNIVТFССКСDSТТRLRLСVQSТНVDIR,

SEQ ID NO: 4 QSTHVDIRTLEDLLMGTLGIV,

SEQ ID NO: 5 GQAERDRANYNIVTFCCC, SEQ ID NO: 6 CATLQDIVLHL,

SEQ ID NO: 7 RKATLQDIVLHLERQNЕIРV,

SEQ ID NO: 8 GVNHQHLPA,

SEQ ID NO: 9 RAEPQRHTM,

SEQ ID NO: 10 GQAERDRANYNIVТFССКСDSТLRLСVQSТНVDIR,

SEQ ID NO: 11 VLDLQREATD,

SEQ ID NO: 12 GQAERDTSNYNIVTFCCQ,

SEQ ID NO: 13 GQАЕРDТSNYNГVТFССQСКSТLRLСVQSTТQVDIR,

SEQ ID NO: 14 ELLMGSFGIVCPNA,

SEQ ID NO: 15 TSNYNIVTF,

SEQ ID NO: 16 DTSNYNIVTF.

7. A vaccine for the prevention and / or treatment of papillomavirus infection, comprising an effective amount of protein according to any one of paragraphs. 3-5.

8. The vaccine according to claim 7, additionally containing an effective amount of at least one purified synthetic peptide, which is a fragment of the human papillomavirus E7 protein, having a sequence selected from the group represented by:

SEQ ID NO: 1 YMLDLQRETTDLYC,

SEQ ID NO: 2 PAGQAEPDRAHYNГVTFCCK,

SEQ ID NO: 3 QAERDRANYYNГVТFССКСDSТТRLRLСVQSТНVDIR,

SEQ ID NO: 4 QSTHVDIRTLEDLLMGTLGIV,

SEQ ID NO: 5 GQAERDRANYNIVTFCCC,

SEQ ID NO: 6 CATLQDГVLНL,

SEQ ID NO: 7 RKATLQDГVLНLЕРQNЕIРV,

SEQ ID NO: 8 GVNHQHLPA,

SEQ ID NO: 9 RAEPQRHTM,

SEQ ID NO: 10 GQAERDRANYNIVТFССКСDSТLRLСVQSТНVDIR,

SEQ ID NO: 11 VLDLQREATD,

SEQ ID NO: 12 GQAERDTSNYNGVTFCCQ,

SEQ ID NO: 13 GQAERDТSNYNIVТFССQСКSТLRLСVQSTТQVDIR,

SEQ ID NO: 14 ELLMGSFGIVCPNA,

SEQ ID NO: 15 TSNYNГVTF,

SEQ ID NO: 16 DTSNYNГVTF.

9. The vaccine according to any one of paragraphs. 7 or 8, optionally containing one or more adjuvants.

10. A vaccine for the prevention and / or treatment of papillomavirus infection, comprising an effective amount of a composition according to claim 6.

11. The vaccine of claim 10, further comprising one or more adjuvants.

12. A therapeutic composition comprising an effective amount of protein according to any one of claims. 3-5.

13. The therapeutic composition of claim 12, further comprising an effective amount of at least one purified synthetic peptide that is a fragment of human papillomavirus E7 protein having a sequence selected from the group represented by:

SEQ ID NO: 1 YMLDLQRETTDLYC,

SEQ ID NO: 2 PAQQAERDRANYNГVTFSSK,

SEQ ID NO: 3 QAERDRANYYNГVТFССКСDSТТRLRLСVQSТНVDIR,

SEQ ID NO: 4 QSTHVDIRTLEDLLMGTLGIV,

SEQ ID NO: 5 GQAERDRANYNГVТFССК,

SEQ ID NO: 6 CATLQDIVŠL,

SEQ ID NO: 7 RKATLQDIVLHLERQNЕIРV,

SEQ ID NO: 8 GVNHQHLPA,

SEQ ID NO: 9 RAEPQRHTM,

SEQ ID NO: 10 GQAERDRANYNГVТFССКСDSТLRLСVQSТНVDIR,

SEQ ID NO: 11 VLDLQREATD,

SEQ ID NO: 12 GQAERDTSNYNGVTFCCQ,

SEQ ID NO: 13

SEQ ID NO: 14 ELLMGSFGIVCPNA,

SEQ ID NO: 15 TSNYNTVTF,

SEQ ID NO: 16 DTSNYNГVTF.

14. The therapeutic composition according to any one of paragraphs. 12 or 13, optionally containing one or more adjuvants.

15. The use of the Ll protein of human papillomavirus HPV according to any one of paragraphs. 3-5 or a composition according to claim 6 for the manufacture of a medicament for the prevention and / or treatment of human papillomavirus infection.

16. The use of the Ll protein of human papillomavirus HPV according to any one of paragraphs. 3-5 or a composition according to claim 6 for the manufacture of a medicament for inducing an immune response in humans.