WO2006065166A1 - Composition de traitement et de prevention de l'infection de l'humain par le papillomavirus sur la base de la proteine l1 et des peptides de la proteine e7 - Google Patents

Composition de traitement et de prevention de l'infection de l'humain par le papillomavirus sur la base de la proteine l1 et des peptides de la proteine e7 Download PDF

Info

Publication number
WO2006065166A1
WO2006065166A1 PCT/RU2005/000391 RU2005000391W WO2006065166A1 WO 2006065166 A1 WO2006065166 A1 WO 2006065166A1 RU 2005000391 W RU2005000391 W RU 2005000391W WO 2006065166 A1 WO2006065166 A1 WO 2006065166A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
protein
hpv
human papillomavirus
vaccine
Prior art date
Application number
PCT/RU2005/000391
Other languages
English (en)
Russian (ru)
Inventor
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 (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Obschestvo S Ogranichennoi Otvetstvennostyu 'rusgen' filed Critical Obschestvo S Ogranichennoi Otvetstvennostyu 'rusgen'
Priority to PCT/RU2005/000391 priority Critical patent/WO2006065166A1/fr
Publication of WO2006065166A1 publication Critical patent/WO2006065166A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • 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.
  • HPV human papillomavirus
  • 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.
  • 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.
  • 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
  • VLP virus-like particles
  • 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.
  • TA-HPV drug
  • 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.uk ⁇ PressReleases/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
  • 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.
  • This invention provides a preventive and therapeutic effect in one vaccine, i.e., it is a combination vaccine.
  • 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.
  • a plasmid containing a copy of the Ll gene is derived from the yeast plasmid pPDX2.
  • the invention provides a recombinant human HPV papillomavirus Ll protein obtained by the method, which is the first aspect of the present invention.
  • the recombinant Ll protein is characterized in that it is obtained in the form of virus-like particles.
  • the recombinant Ll protein is useful for the manufacture of a medicament for the prevention of papillomavirus infection.
  • 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:
  • 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.
  • 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:
  • the vaccine further comprises one or more adjuvants.
  • 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.
  • the vaccine for the prevention and / or treatment of human papillomavirus infection additionally contains one or more adjuvants.
  • the invention provides a therapeutic composition comprising an effective amount of a recombinant Ll protein, which is a second aspect of the invention.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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
  • a composition which is the third aspect of the present invention.
  • 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.
  • 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 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.
  • HPV human papillomavirus
  • 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.
  • prokaryotic systems can be used, for example, systems based on E. coli (Li et al., Li ML, CP TP,
  • eukaryotic systems for example, systems based on cultured insect tissues (baculovirus systems) can be used (Christepsep ND, ⁇ orfl R, DiAngelo SL, ⁇ dedel NM, ⁇ atriantek SD, ⁇ réellelsh PA, Budgeon LR, ⁇ réelled CA, ⁇ restockedd Canalr JW, ⁇ ss speechl Sciencesvl ehrressed humap rarillomavigas ture 11 Ll sarsid rroteip virus- like rartisles are resogpized bu
  • 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 sakipcoast gray visiae are assigned to the group of organisms of the GRAS category (Heperell Regard As Safe).
  • vectors 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 ⁇ m yeast plasmid) or integrate into the chromosome (integrative plasmids).
  • vaccine virus-based vectors Zahou 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
  • 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, S Spurcepsa dethermiptiop réellef humap paralillomavirus tour 6 réelle add-up schreibf virus-lik
  • the introduction of the plasmid into the host cell can be carried out in many ways well known to specialists in this field.
  • transformation using Ca + ions transformation using Ca + ions, electroporation, transfection, transduction can be used.
  • 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.
  • the pPDX2 vector intended for expression of the Ll protein in yeast cells, was constructed on the basis of plasmids pPDXl, and pU ⁇ G ⁇ L Facilitytg ⁇ .
  • 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:
  • URAB gene which is a selective marker in the transformation of yeast cells
  • 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 / ⁇ g of DNA.
  • Purification of the recombinant protein may be carried out by any of the methods well known in the art.
  • 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.
  • 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.
  • classical chemistry methods in solution
  • One alternative is peptide synthesis using recombinant technology.
  • 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.
  • 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.
  • acyl and amide groups can be blocked by the acyl and amide groups, respectively.
  • 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.
  • hydrophilic resins crosslinked dextrans, cellulose derivatives, hydroxylapatite, porous glasses, etc.
  • HPLC high performance liquid chromatography
  • 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.
  • 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.
  • Ll protein and human papillomavirus E7 protein peptides 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.
  • 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).
  • 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).
  • “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.
  • 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.
  • the compositions and vaccines of the present invention will be administered in doses ranging from about 1 ⁇ g to about 1 mg.
  • compositions 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.
  • 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;..
  • compositions or vaccines of the present invention may include buffers, dispersants, preservatives, stabilizers.
  • 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.
  • 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.
  • 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.
  • physiologically acceptable buffering agents such as Heps, phosphate, citrate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate or histidine buffers, as well as combinations thereof.
  • 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.
  • preservatives for example, thimerosal, phenol, may also be included in the vaccines.
  • 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. Alvi ⁇ g. And ⁇ omrepdium schreibf Vassip Canal ⁇ djuvapts réellepd Exciriepts, 1999, 2nd Editiop).
  • adjuvants such as CPG, aluminum phosphate or hydroxide, stearyl tyrosine, MDP, GMDP, polyoxidonium, extracts of fragments of the bacterial cell wall
  • VLPs i.e., good humoral and cellular immune responses
  • 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. Sretei. USA, 1995, 92, 11553-11557; M. F. Duggap-Kep, M. D. Browp, S. N. Staseu, P. L. Sterp.
  • VLP-specific cytotoxic lymphocytes 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.
  • the Ll protein and peptides of the human papillomavirus E7 protein are introduced in a solution or suspension in a suitable aqueous medium.
  • 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)).
  • 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.
  • 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 / ⁇ l, solution of primers, template DNA (in serum), H 2 O (supQ). For PCR, 25 pmol of each of the primers was taken.
  • Cycle 1 primary melting 94 ° C 1.5 min
  • Cycle 2 basic, melting 94 0 C 20 s.
  • Cycle 3 storage at 10 0 C
  • 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 w ⁇ azmid. Fragments obtained by PCR were cloned into plasmid pUC19, taken from the laboratory collection. Plasmid pUC19 contained the lacZ gene encoding ⁇ -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 ⁇ -galactosidase is not synthesized. The presence and absence of functional ⁇ -galactosidase is easily determined using a reagent called X-gal, which is added to the microorganism culture medium. If functional ⁇ -galactosidase is present in the cells, the colony of microorganisms turns blue.
  • X-gal reagent
  • the pUC 19 vector was treated with Smal restriction endonuclease that recognizes and cleaves the CCC
  • 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 ⁇ g / 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.
  • the bacteria Escherichia coli strain TG were transformed with a ligase mixture. For this, 50 ⁇ l 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 ⁇ l of 100 mM CaCl 2 , then suspended in 1 ml of fresh 100 mM CaCl 2 and incubated on ice for 2 hours.
  • fresh LB medium 1% peptone, 0.5% yeast extract, 1% NaCl
  • the cells were precipitated by centrifugation for 15 seconds at 12,000 rpm, then the pellet was resuspended in 400 ⁇ l of 100 mM CaCl 2 .
  • 200 ⁇ l 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.
  • 800 ⁇ l 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.
  • the transformed cells 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.
  • 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 ⁇ l per Vortex.
  • the supernatant was transferred to microcentrifuge tubes, to which 200 ⁇ l 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.
  • 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 ⁇ g / reaction.
  • the amount of primer was 4 pmol / reaction.
  • the mixture was brought to a volume of 12 ⁇ l. 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).
  • 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).
  • 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.
  • S. yeast cells with plasmid DNA 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 ⁇ l 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 2% galactose is added to the medium.
  • BSA bovine serum albumin
  • 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.
  • SDS-PAGE Acrylamide Gel Denaturing Electrophoresis
  • 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).
  • Example 10 Silver staining of acrylamide gels 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.
  • 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.
  • 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.
  • the gel 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).
  • Antigen solutions (Ll VLP proteins, peptides) in carbonate-bicarbonate buffer (KBB) with a pH of 9 (3 ⁇ g / ml) are applied at 50 ⁇ l / 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 ⁇ l / 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 ⁇ l of buffer (0.02 M, pH 7.2 PBS / 0.05% Tween 20).
  • Antibody solutions are added at 50 ⁇ l / well, incubated for 1 hour at 37 ° and washed 4 times with 200 ⁇ l 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 ⁇ l / 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 ⁇ l / 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.
  • 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.
  • the analysis data confirm the expected immunoreactivity of the obtained Ll VLP proteins with the indicated antibodies.
  • monoclonal neutralizing antibodies Hl 6 U4 S / N 9 and Hl 8 J4 S / N 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.
  • 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 is also recognized by these antibodies.
  • the band corresponding in size to the endogenous Ti particles of the yeast is not recognized by antibodies.
  • Example 15 The confirmation of the structure of Ll VLP by electron microscopy.
  • Example 16 Immunological efficacy of recombinant Ll VLP - humoral response.
  • 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.
  • ELISPOT i.e., the titer of gamma-interferon secreted by activated T-cells is determined in a microplate version.
  • 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- ⁇ ).
  • Example 18 Immunological efficacy of recombinant Ll VLP - cell response in a population enriched in cytotoxic CD8 + lymphocytes.
  • 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).
  • Ll VLP 16 shows high activity, as in the previous experiment.
  • 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
  • the resulting recombinant proteins and peptides have a strong prophylactic and therapeutic potential in relation to human papillomavirus infection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • General Chemical & Material Sciences (AREA)

Abstract

L'invention appartient au domaine de la biotechnologie et concerne des protéines de papillomavirus ainsi que des peptides utilisés pour la création de vaccins prophylactiques et thérapeutiques dirigés contre les différentes variantes de l'infection par le papillomavirus chez l'humain (HPV). Les protéines capsides L1 des sérotypes viraux 16, 18 et 31 papillomes humains, qui provoquent les formes les plus agressives des maladies chez l'homme, sont obtenues par la transformation des cellules de levure par un ADN plasmidique codant pour les protéines L1 des sérotypes mentionnés. Les peptides, qui représentent des fragments de la protéine de transformation E7 HPV (peptides E7) des sérotypes mentionnés ici, sont obtenus par synthèse chimique. Les souches productrices à base de levure, créées selon l'invention, sont capables d'exprimer pendant leur culture les protéines L1 sous la forme de pseudo-particules virales (VLP). Les protéines L1 sont extraites, purifiées et utilisées pour la préparation de compositions de vaccins qui peuvent comprendre dans leur composition des peptides E7 et des adjuvants.
PCT/RU2005/000391 2005-07-26 2005-07-26 Composition de traitement et de prevention de l'infection de l'humain par le papillomavirus sur la base de la proteine l1 et des peptides de la proteine e7 WO2006065166A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/RU2005/000391 WO2006065166A1 (fr) 2005-07-26 2005-07-26 Composition de traitement et de prevention de l'infection de l'humain par le papillomavirus sur la base de la proteine l1 et des peptides de la proteine e7

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/RU2005/000391 WO2006065166A1 (fr) 2005-07-26 2005-07-26 Composition de traitement et de prevention de l'infection de l'humain par le papillomavirus sur la base de la proteine l1 et des peptides de la proteine e7

Publications (1)

Publication Number Publication Date
WO2006065166A1 true WO2006065166A1 (fr) 2006-06-22

Family

ID=36588138

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/RU2005/000391 WO2006065166A1 (fr) 2005-07-26 2005-07-26 Composition de traitement et de prevention de l'infection de l'humain par le papillomavirus sur la base de la proteine l1 et des peptides de la proteine e7

Country Status (1)

Country Link
WO (1) WO2006065166A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090117140A1 (en) * 2007-09-26 2009-05-07 Mayumi Nakagawa Human papilloma virus dominant CD4 T cell epitopes and uses thereof
CN102552897A (zh) * 2012-01-18 2012-07-11 广东华南联合疫苗开发院有限公司 一种宫颈癌预防性vlp疫苗
RU2545909C2 (ru) * 2013-03-19 2015-04-10 Федеральное государственное бюджетное учреждение науки Институт биохимии имени А.Н. Баха РАН Российской академии наук (ИНБИ РАН) Способ иммунохроматографического определения специфических антител

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994020137A1 (fr) * 1993-03-09 1994-09-15 University Of Rochester Production de proteine de capside de virus du papillome humain et particules de type viral
CN1353114A (zh) * 2000-11-03 2002-06-12 浙江大学医学院附属第二医院 人类乳头瘤病毒11l1－e7重组蛋白及其应用
WO2003018624A1 (fr) * 2001-08-31 2003-03-06 University Of Cape Town Vecteurs, produits de synthese et plantes transgeniques pour proteine capside du hpv-11 et du hpv-16

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994020137A1 (fr) * 1993-03-09 1994-09-15 University Of Rochester Production de proteine de capside de virus du papillome humain et particules de type viral
CN1353114A (zh) * 2000-11-03 2002-06-12 浙江大学医学院附属第二医院 人类乳头瘤病毒11l1－e7重组蛋白及其应用
WO2003018624A1 (fr) * 2001-08-31 2003-03-06 University Of Cape Town Vecteurs, produits de synthese et plantes transgeniques pour proteine capside du hpv-11 et du hpv-16

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090117140A1 (en) * 2007-09-26 2009-05-07 Mayumi Nakagawa Human papilloma virus dominant CD4 T cell epitopes and uses thereof
CN102552897A (zh) * 2012-01-18 2012-07-11 广东华南联合疫苗开发院有限公司 一种宫颈癌预防性vlp疫苗
RU2545909C2 (ru) * 2013-03-19 2015-04-10 Федеральное государственное бюджетное учреждение науки Институт биохимии имени А.Н. Баха РАН Российской академии наук (ИНБИ РАН) Способ иммунохроматографического определения специфических антител

Similar Documents

Publication Publication Date Title
RU2206608C2 (ru) Папилломавирусные вакцины
ES2337492T3 (es) Formulaciones y formas de uso de la vacuna capsomero de virus de papiloma.
ES2263405T3 (es) Producción de proteína de capside del papilomavirus humano y partículas de tipo virus.
US10736954B2 (en) L2 peptide immunogenicity
JP2010263899A (ja) Ｌ２ペプチドを含むキメラ・ヒト・パピローマウイルス１６ｌ１タンパク質、それから調製したウイルス様粒子および該粒子の調製方法
EP2377879A1 (fr) Protéines de fusion HPV E7 à terminaison N
BRPI0818957A2 (pt) "genes que codificam proteínas l1 da capa proteica do capsídeo de papilomavírus humano"
ES2273376T3 (es) Formulaciones de vacunas recombinantes frente a papilomas.
WO2006065166A1 (fr) Composition de traitement et de prevention de l'infection de l'humain par le papillomavirus sur la base de la proteine l1 et des peptides de la proteine e7
US7182947B2 (en) Papillomavirus truncated L1 protein and fusion protein constructs
Kuck et al. Efficiency of HPV 16 L1/E7 DNA immunization: influence of cellular localization and capsid assembly
ES2325053T3 (es) Metodo in vitro para desensamblaje/reensamblaje de particulas similares a virus de papilomavirus (vlp).
JP4945730B2 (ja) 粘膜指向性ヒトパピローマウイルス群の感染予防ワクチン抗原
RU2546243C1 (ru) Рекомбинантная вакцина для профилактики папилломавирусной инфекции человека и способ ее получения
US8715681B2 (en) Minimal motifs of linear B-cell epitopes in L1 protein from human papillomavirus type 58 and their applications
US11547753B2 (en) Cutaneous papilloma virus vaccine
RU2681174C1 (ru) Способ получения рекомбинантной вакцины для профилактики папилломавирусной инфекции человека, рекомбинантная вакцина
Deng et al. The preparation of human papillomavirus type 58 vaccine and exploring its biological activity and immunogenicity in vitro
Naupu The Immunogenicity of Plant-produced Human Papillomavirus (HPV) Virus-like particles (VLPs) in Mice
Burger Production and immunogenicity of chimaeric human papillomavirus-like particle vaccines
WO1998010790A1 (fr) Formule d'un vaccin contre le papillomavirus
BR102014032991A2 (pt) processo de obtenção e recuperação de vlps recombinantes obtidas do papilomavírus bovino tipo 2 expressas em células de mamíferos e o uso para composição vacinal

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2008106481

Country of ref document: RU

122 Ep: pct application non-entry in european phase

Ref document number: 05851081

Country of ref document: EP

Kind code of ref document: A1