Mucosal vaccine for immune therapy of diseases caused by human papilloma viruses, and related therapy (versions)

FIELD: medicine.

SUBSTANCE: invention refers to genetic engineering and can be used in medicine. The mucosal vaccine contains effective amount of hybrid protein consisting of oncoprotein E7 of human papilloma virus fused with heat-shock protein of mycobacteria Hsp70, chitosan related to hybrid protein 1:0.1-10 and additives pharmaceutically acceptable manufacturing of suppositories. The mucosal vaccine is used in therapy of the diseases associated with human papilloma virus.

EFFECT: possibility for multiple improvement of clinical effectiveness of diseases associated with human papilloma virus, considerable reduction of treatment cost in comparison with common techniques of treating cervical carcinoma and "РПК"; elimination of injection by-effects undesirable and extremely dangerous for the patent's life, eg anaphylactic shock, owing to local application; simplification of medical process - the patient can receive medical treatment out of clinic by independent introduction of the preparation.

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The technical field of the present invention

The invention relates to the field of genetic engineering, specifically, it relates to compositions of recombinant proteins on the basis of the E7 oncoprotein of human papilloma virus, fused with heat shock protein of mycobacteria Hsp70 in combination with chitosan and pharmacologically acceptable for the manufacture of suppositories additives. The invention relates to vaccine therapy of diseases caused by human papilloma virus.

The prior art of the present invention

In the mid 70-ies was first suggested the possible involvement of human papillomavirus (HPV) in the pathogenesis of cervical cancer and studies have been conducted to identify the virus in biopsy material. It was found that in malignant tumors of the cervix are detected most frequently two types of viruses - HPV-16 and HPV-18. And in benign lesions are present, mainly viruses 6 and 11 types. Upon further study of the role of HPV in neoplastic processes all serotypes were divided into three groups:

low oncogenic risk: 6, 11, 42, 43, 44;

- the average degree of carcinogenic risk: 31, 33, 51, 52, 58;

- high degree of carcinogenic risk: 16, 18, 45, 56.

Currently, a number of studies have shown that HPV DNA is detected in the average 98,9% and 94.7% of the samples ostracode the cases of genital warts and cervical cancer, respectively. In populations in healthy women HPV detected in 35% of cases, with 6, 11, 16 and 18 types - only 21.6 per cent. Interesting for further study was the fact that the majority of HPV-16 DNA in the cells of genital warts is in episomal form, and in samples of cervical cancer - is integrated into the cellular genome. In recent years, in connection with the introduction of prophylactic vaccines against HPV have been several large-scale epidemiological studies on the geographical distribution of different types of HPV in cervical samples from patients with invasive cervical cancer (cervical cancer). It turned out that in most cases around the world (65-77%) detected HPV 16/18 types, but the ranking of HPV types has distinct geographical features. For example, in China these are followed by the HPV 58 and 52 types of, in Africa - HPV type 45, Central / South America - HPV type 31. (Bao Y., Li N., Smith J. and Y. Qiao, Human papillomavirus type-distribution in the cervix of Chinese women: a meta-analysis. Int. Journal of STD &AIDS, 19, 2, 106-111; Nubia M., Xavier C., Xavier .et al., Against which papillomavirus types shall we vaccinate and screen? The international perspective. Int. Journal of Cancer, 2004, 11, 2, 278-285; C. Ihekweazu, Worldwide distribution of HPV types in women with normal cervical cytology and in women with cervical adenocarcinoma, Eurosurveillance, 2006, 11, Issue 12.)

The spread of the infection is predominantly sexually transmitted diseases, reaching a maximum of infection up to 30 years. More the 50% of sexually active population of the world during the life of the infected with human papilloma virus, and this is the primary event in the pathogenesis of cervical cancer. Most infections ends of spontaneous recovery. However, in some cases, developing persistent infection that can trigger the mechanisms of cell transformation of epithelial cells. So, when CIN 1 there is active viral replication and asymptomatic selection. The transformation of CIN 1 in invasive cancer occurs with great frequency and, as a rule, is accompanied by the integration of viral DNA into the genome of the host cell (Kiselev V.I. the human papilloma Virus in cervical cancer. M.: Dimitra Graph Of Groups®", 2004).

HPV high degree of carcinogenic risk (mainly 16 and 18 types) found in 50-80% of the samples of moderate and severe dysplasia of the squamous epithelium of the cervix and in 90% of invasive cancer. An important fact is that the clinical manifestations of HPV infection not detected for quite a long time, but the effects of persistence of the virus do not get easier. The development of mild and moderate dysplasia in heavy is respectively 10 and 20% of cases. Such statistics is not fatal: the transition from one stage of atypia of the epithelium to another is quite long, which gives great opportunities in terms of diagnosis and treatment. In addition, it is important that the factor is, that being HPV is not for life: according to who (2001) in the absence of aggravating factors during the 3 years squamous intraepithelial lesion low-grade gravity, containing HPV, are regression

in the 50-62% of the observations. According to scientists at the University of California at 70% of young HPV-positive women HPV DNA is detected for 24 months. The rate of elimination is significantly reduced during infection with multiple HPV types, as well as a history of warts of the vulva. At the same time, among women who had at least 3 times were the positive results of HPV, the risk of severe cancer lesions of the epithelium was increased 14-fold. Every year in the world there are about 500,000 cases of cervical cancer, of which about 50% fatal. In the Russian Federation according to the Ministry of health in 2002 was registered 11320 cases of cervical cancer (Molochkov VA, Kiselev V.I., Rudich IV, Scherbo S.N. Papilloma virus infection: clinical features, diagnosis, treatment, guidelines for doctors. M.: Publishing house "Russian doctor", 2004).

It should be noted that in recent years significantly increased the number of diagnoses of colorectal cancer is also caused by the human papilloma virus. Special distribution of this type of PA is sociology received among men with non-traditional sexual orientation. Recorded cases of cancer of the oral cavity when having oral sex with a partner infected with HPV (News.com // in the world // 18 November 2004. Oral sex causes cancer). In most cases, cancer is preceded by the formation of warts mucous membrane of the rectum, which is accompanied by bleeding during defecation, inflammatory processes and fissures of the anus.

Current views on the mechanism of carcinogenesis due to HPV

HPV infects and replicates in the epithelium, as evidenced by the detection of episomal viral genome in the cells of the basal layer of the epithelium. The life cycle of the virus is closely associated with the differentiation of the host cell. The viral DNA replication and synthesis of capsid proteins of the virus occurs in the most differentiated layers of the epithelium, and the virus has a whole set of mechanisms, subordinating their interests livelihoods infected cells. Virus DNA encodes the synthesis of two proteins E6 and E7, which induce the transition of differentiated cells in S-phase of the cell cycle. At the stage of active reproduction of the virus genes E6 and E7 are regulated by the E2 gene product, which is a repressor of transcription of these genes. Therefore, while the virus is in the episomal state, there is a benign process the si expansion of infected tissues. A key event in the malignant transformation of cells is the integration of the virus into the genome of the cells, which is accompanied by a deletion of the E2 gene. This event has two important implications:

1) in the epithelial cells with the integrated form of HPV is logged overexpression of genes E6 and E7, as in the process of integration is lost E2 gene encoding the repressor of transcription of these genes,

2) any antiviral drugs are powerless to stop the process of neoplastic transformation, as infected cells do not contain the virus in the traditional sense, and all therapeutic measures should be aimed at the elimination of cells with the integrated form of HPV genome.

Control of the cell cycle and cell differentiation is carried out by proteins E6 and E7 through their interaction and inactivation of these key regulator proteins proliferative activity and apoptosis, as p53 and retinoblastoma protein (pRB). Uncontrolled proliferation of infected cells leads to the accumulation of genetic damage and, ultimately, to the malignancy. The oncoprotein E7 uses multiple paths to cell cycle regulation. Found that E7 is capable of forming a stable complex with the protein pRB, causing its degradation, which leads to the release of transcription factor E2F, which stimulates the transcription of genes, neo is required for DNA replication and S-phase of the cell cycle. E7 also affects the activity of a number of proteins of the cell cycle, as a and E cyclina, cdk2 kinase and inhibitors of cyclin-dependent kinases P21 and P27 (Kiselev V.I., Kiselev, I. Etiologic role of human papillomavirus in cervical cancer: genetic and pathogenetic mechanisms. Cytokines and inflammation. 2003, 2, 4, 31-38; W.C. Phelps, Bames J.A. and Loba D.C., Human papillomavirus: molecular targets and prospects for antiviral therapy. International Antiviral News, 1999, 7, 4-8).

Animal studies indicate that humoral immunity plays a role in protection from HIV infection. Model virus-like particles (virus-like particles - the VLP), self-assembling of the main capsid protein of HPV-L1, is widely used to study the protective role of neutralizing antibodies. Immunization of animals the VLP protects against experimental infection with homologous virus. Passive transfer of serum from mice immunized with the VLP, other experimental animals also has a protective effect, which confirms the protective role of neutralizing antibodies (Tindle R.W., Immune evasion in human papillomavirus-associated cervical cancer. Nature Reviews, 2002, Cancer, 2, 1-7; Zhou j, Liu WJ, Peng S.W. et al. Papillomavirus capsid protein expression level depends on the match between codon usage and tRNA availability, J. Virol., 1999, 73, 4972-4982; Schwarts S. Regulation of human papillomavirus late gene expression, Ups. J. Med. Ski, 2000, 105, 171-192). In the case of experimental infection of immunized mice HPV type other protective EF is known not registered, which indicates the specificity of the observed phenomena and the absence of cross-reactions between different types of HPV. Moreover, the denaturation of the VLP resulted in the loss of protective effects, which indicates that the role of the intact protective epitopes in the induction of antitelomerase. Another late in the gene encoding the minor capsid protein L2, was also investigated as a potential candidate for prophylactic vaccination. It was found that immunization of animals hybrid recombinant protein L2 induces high titers of neutralizing antibodies and protects against experimental papilloma-virus infection. Thus, accumulated to date suggests that capsid proteins of HPV are promising candidates for the creation of preventive vaccines (Nees M. et al. Papillomavirus type 16 oncogenes down-regulate expression of interferon-responsive genes and up-regulate proliferation-associated and NF-kB - responsive genes in cervical keratinocytes. J. Vir., 2001, 75, 4283-4296; Barnard P., Payne E. & McMillan, N.A. The human papillomavirus E7 protein is able to inhibit the antiviral and anti-growth functions of interferon-a. Karpova et al., 2000, 277,

411-419). However, when using these proteins for therapeutic immunization with established, chronic papilloma-virus infection failed to get any significant effect. Apparently, for therapeutic immunization requires a different strategy, namely the induction of antigen-specificheskogo the T-cell immune response. The available data suggest that stimulation of the cellular component of immunity with respect to the non-structural viral proteins may lead to regression of the developing tumor.

Observations confirm the validity of this assumption can be divided into four groups:

1. The incidence of HPV - associated diseases is significantly higher among patients transplant clinics (J.S. Park et al. Inactivation of interferon regulatory factor-1 tumor suppressor protein by HPV E7 oncoprotein. Memorandum for

the E7-medated immune evasion mechanism in cervical carcinogenesis. J. Biol. Chem 2000, 275, 6764-6769) and HIV-positive, i.e. in cases with impaired cellular immunity (Frazer I., et al. Systemic administration of bacterial products dosage host specific protective immunity to an epithelial tumour antigen. J Immunol., 2000, 167, 6180-6187, Doan T. et al. Human Papillomavirus Type 16 E7 Oncoprotein Expressed in the Peripheral Epithelium Tolerizes E7-Directed Cytotoxic T-Lymphocyte Precursors Restricted through Human (and Mouse) Major Histocompatibility Complex Class I Alleles. J. Virol., 1999, 73, 6166-6170).

2. Experimental animals immunized with non-structural proteins of HPV, is effectively protected against HPV infection and the development of neoplastic processes (Gtannini S. et al. Cytokine expression in squamous intraepithelial lesions of the uterine cervix: implications for the generation of local immunosuppression. din. Exp. Immunol., 1998, 113, 183-189).

3. Spontaneous regression of genital warts showing extensive infiltration of the surrounding tissues CD4+(T-helpers) and CD8+(cytotoxic) T-cells (Ressing M.E. t al. Human CTL epitopes encoded by human papillomavims type 16 E6 and E7 identifed through in vitro and in vivo immunogenicity studies of

HLA-A 0201-binding peptides. J. Immunol., 1995, 154, 5934-5943).

4. Cutaneous lesions associated with HPV in patients receiving immunosuppressive therapy, often disappear when you cancel a course of treatment (Lee. S.J. et at. Both E6 and E7 oncoproteins of human papillomavims 16 inhibit IL-18-induced IFN-y production in human peripheral blood mononuclear and NK cells. J Immunol.,

2001, 167, 497-504).

The data presented demonstrate in favor of that specific stimulation of T-cell immunity may lead to complete remission of HPV-associated diseases. In connection with this practical task when creating a therapeutic vaccine is to stimulate T-cell immunity against non-structural proteins of HPV, such as proteins E6 and E7.

The main requirement for therapeutic vaccines is that they should stimulate cellular component specific immunity and to block the development of neoplastic processes. Theoretically, cellular response should be directed to viral and cellular proteins involved in malignant transformation of the epithelium. However, despite intensive research, little is known about the cellular targets in tumor transformation, so most experimental vaccines are aimed at the products of the genes E6 and E7 of HPV, whose role in oncogenesis proven unequivocally.

Pokolyubichi E6 and E7 constitutively expressed in the majority of cervical cancer cells and absent in normal tissue, these viral oncoproteins are considered as the most promising target for the development of vaccines for therapy of HPV-associated tumors (Straght S.W., Herman C. & D.J. McCance The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes. J Virol, 1995, 69, 3185-3192).

Target for immunotherapy of tumors usually are normal or mutant cellular proteins, whereas proteins E6 and E7 are absolutely alien to the immune system of the host and therefore must be highly immunogenic (Przepiorka D. and Srivastava P., Heat Shock Proteins - Peptide complexes as immunotherapy for human cancer. Molecular medicine today, 1998, 11, 478-484).

The next reason why proteins E7 and E6 are considered as promising candidates for vaccination, is that many of the tumor during immunotherapy may lose the target proteins, dodging, thus, immune from attack and retaining its transformed status, then the development of cervical cancer is uniquely determined by the expression of proteins E6 and E7 and their neutralization will result in regression of the tumor.

Development of peptide vaccines is hampered by the fact that up to the present time is not yet exactly defined epitopes in proteins E6 and E7, are able to induce cytotoxic immunity. Therefore, the main efforts are concentrated on constructing protein vaccines using recombinant proteins E7 and E6.

The last IP the surveys show, what is the preferred target for development of vaccines is a protein E7 of HPV 16 and 18 types for cervical cancer and E7 of HPV type 6 for anal warts, as it has the smallest structural variability in different geographical isolates of HPV and has no splicing variants (Swan D.C., Rajeevan M., Tortolero-Luna G., et al. Human papillomavirus type 16 E2 and E6/E7 variants, Gynecol. Oncol. 2005, 96, (3) 695-700).

It is shown that the recombinant protein E7 of HPV type 16 is able to induce cytotoxic immunity, and the injection of this protein in combination with various adjuvants enhances the cytotoxic response.

In recent years, especially popular new strategy to enhance immunogenicity of E7, consisting in the construction of hybrid proteins. In particular, we have studied the immunological properties of the hybrid protein containing the full sequence of the heat shock protein Hsp70 and E7 protein of HPV. Immunogenicity and protectively this design was significantly higher, probably due to the fact that Hsp 70 M. tuberculosis provides effective presentation of antigens (Becker T., Harti F., and F. Wieland, CD40, an increasing interest among receptor for binding and uptake of Hsp70 - peptide complexes, J.Cell Biology, 2002, 158, 1277-1285).

It is known that E7 is synthesized in small quantities in the reproductive phase of the life cycle of HPV. However, its synthesis is greatly increased after integration of the viral DNA into the cellular genome, as this process is, how great the ILO accompanied by the loss of the E2 gene encoding the repressor gene E7. Despite this body is not observed induction of specific cellular immunity against the protein E7. Some experimental models allow us to shed light on the amazing ability of oncoprotein E7 to induce tolerance.

If a skin graft from transgenic mice that synthesize E7 in the epithelial cells, transplanted genetically related mice, the expected graft rejection does not occur. If mouse enter stimulating the formation of anti-inflammatory cytokines (killed bacteria or endotoxins), then comes a rapid graft rejection. However, rejection of the graft against the background of the introduction of inducers of cytokines occurs only in the period of engraftment, and re-transplantation of the graft, the same mouse leads to rejection without additional induction of cytokines. There are different assumptions about the mechanisms of this phenomenon, but they all agree on one thing - antigenpresenting cells, absorbing E7 in the area of the cervical canal, for some reasons can not go through all the necessary stages of maturation, to migrate to the lymph nodes and induce T-cell immune response against this protein without additional stimulation of the cytokine cascade (Gross G., Joblonska S., Pfister H. enital papillomavirus infections, 1989; I. Frazer et al. Systemic administration of bacterial products dosage host specific protective immunity to an epithelial tumour antigen, JImmunol., 2001, 167, 6180-6187).

Continuous synthesis of oncoproteins necessary for the maintenance of the tumor phenotype of the cells. Obviously, the advent in the body cells bearing foreign antigens (E6 or E7), should induce the appropriate immune response. However, patients with cervical carcinomas have a very low level of T-cell immunity against E6 and E7.

When you vaccinate these proteins also not observed severe reactions despite the good features of the immune status. These observations and experiments on animals show that the oncoprotein E7, synthesized in the epithelial cells, is able to "escape" or even supressive immune response against itself.

When creating any tumor vaccine it is necessary to solve several fundamental problems. First, identify the target against which it is necessary to induce the immune response of the body. In the case of HPV infection in the General opinion of such a target is a protein E7 (Kiselev V.I., human papilloma Virus in cervical cancer. - M.: Dimitra Graph Of Groups®", 2004).

Not less important is the choice of adjuvant for the induction of sustained protective immunity and method of vaccine administration. The latter is the circumstance, great attention is paid, because I found that the method of introduction of the vaccine significantly depends on its effectiveness. As already mentioned, HPV infections affect the mucous membranes as in the case of cervical cancer, and at higher elevations of DRD and rectal cancer. However, currently developed vaccine preparations are intended for subcutaneous injection, that is aimed at specific stimulation of the systemic immune system and ignore the mucosal immune response (Patent US 6338952 B1, 2002. Stress proteins and uses therefore; RF Patent №2229307, Fingers M.A., E.S. Severin, I. Kiselev, Kiselev V.I., Sveshnikov p. g Composition of recombinant proteins, the method of obtaining such a composition, the pharmaceutical kit of reagents for immune therapy and preventive vaccination of tumor diseases anogenital areas, method of immunotherapy and preventive vaccination based on it). Based on U.S. patent company "Nventa develops injectable form of the vaccine preparation containing the protein E7, "merged" with the heat shock protein of M. tuberculosis with a molecular mass of 65 kDa. From our point of view, a significant disadvantage of this design is the choice of the protein of 65 kDa, as there are convincing data on induction of autoimmune reactions in humans at the introduction of this protein. In addition, this heat shock protein inferior to their adjuvant properties of protein Hsp70 M tubercuosis (Zugel, U. and Kaufmann, S., Role of heat shock protein in protection from and pathogenesis of infectious deseases, Clinical microbiology reviews, 1999, 12, 1, 19-39).

In the patent of Russian Federation №2229307 described the development of a vaccine based on a hybrid protein E7, Hsp70 M tuberculosis. The authors describe injectable dosage form, which can achieve regression of tumors induced by the virus in experimental models with transplantable tumors. Demonstrated the efficacy of vaccination in these models confirms the correctness of the choice E7 protein as a molecular target against which the induced immune response. However, when cervical dysplasia developing in women due to chronic papilloma-virus infection of the cervix, induction of systemic immunity cannot guarantee regression processes of malignancy, because the cervix has a certain isolation from the system of immunity in the regulation of local immune responses. In the development of anal warts E7 protein also plays a major role in the processes of malignancy and the suppression of local immune reactions. Therefore, mucosal applique vaccine should lead to the neutralization of E7 protein and to induce regression of pathological processes. Besides, now it becomes apparent that to achieve a sustained protective immunity using recombinant vaccines can is about only with repeated immunization. This creates significant logistical challenges, as many times the patient will need to visit the clinic for vaccination. In the case of vaginal vaccination, the patient can do it yourself. Another problem associated with injectable vaccination, is that with repeated intramuscular introduction of foreign protein is a high probability of anaphylactic shock, which is excluded by vaginal or rectal route of administration of the drug.

Finally, another advantage of mucosal vaccines is the preferential induction of local mucosal immunity, which is required when the pathologies that affect the mucous membranes, as in the case of papilloma-virus infection.

As dosage forms for mucosal application of vaccines can be considered suppositories or "sprays"that allow you to cause mucous vaccine preparation in the form of lyophilized powder.

The disclosure of the present invention

The authors proposed an invention based on years of research papillomatosis came to the conclusion that the optimal vaccine for the treatment of diseases associated with human papilloma virus, must meet the following requirements:

1. The method of introduction should include the stimulation of local adaptive and innate immunity, namely, when the cervical cancer learn - route of administration vaginal; in the case of rectal cancer - route of administration rectal.

2. Given the low immunogenicity of the protein E7 in the composition of the recombinant protein, which is the active ingredient of the vaccine of the drug must be present amino acid sequences of proteins with immune modulating effect on the formation of cytokines and improves the presentation of E7 antigen.

3. Dosage form vaccine product must contain ingredients that can increase the adhesion to mucous membranes and affinity of protein components of the vaccine by antigen-presenting cells.

To solve the tasks in this work were developed in two dosage forms of the vaccine for immunotherapy of HPV-associated pathologies. So, for the treatment of diseases anogenital areas developed mucosal vaccines in the form of suppositories.

For the treatment of cervical cancer have been developed suppositories containing as major antigens hybrid proteins E7 type 16 or type 18, merged with Hsp70 M tuberculosis. The hybrid protein is a single polypeptide chain consisting of a first protein E7 of HPV type 16 or 18. As already installed currently, the oncoprotein E7 is the major inducer of processes malignancy of epithelial cells infected with human papilloma virus (Yin E.K. and J.S. Park, The rle of HPV E6 and E7 oncoproteins in HPV-associated cervical carcinogenesis, Cancer Res. Treat., 2005, 37, 6, 319-324). In addition to the oncogenic potential of E7 protein possesses immunosuppressive properties. Thanks squirrel E7 transformed cells can "escape" from the immune reactions of the organism (Kanoda S., Fahey L.M. and W.M. Kast, Mechanism used by papillomaviruses to escape the host immune response. Current Cancer Drug Targets, 2007, 7,

79-89). It is for these reasons E7 protein is the optimal target for immunotherapy. Another part of this protein is represented by a heat shock protein of M. tuberculosis, with high affinity to antigen-presenting cells and is a potent inducer of cytokine synthesis (Przepiorka D. and Srivastava P., Heat Shock Proteins - peptide complexes as immunotherapy for human cancer. Molecular medicine today, 1998, 478-484; T. Becker, Hard F.LL, and F. Wieland, CD40, an extractllular receptor for binding and uptake of Hsp70 - peptide complexes. The Journal of cell biology, 2002, 158, 7, 1277-1285; Srivastava P., Interaction of Heat Shock Proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses, Annu. Rev. Immunology, 2002, 20, 395-425; H.J. Lachmann, Strangeways L., Vyakarmam A and Evans G. Raising antibodies by coupling peptides PPD and immunizing BCG-sensitized animals. Ciba Found. Symp., 1986, 119, 25-27; Del Giudici G. Hsp70: a carrier molecule with built-in adjuvanticity, Experimentia, 1994, 50, 1061-1066).

In the case of rectal cancer, it is advisable to use as antigen E7 protein type 6, which more often than other types of HPV occurs in lesions of the mucous membrane of the rectum.

We conducted experimental studies of the immunogenicity of the hybrid proteins in their application to mucous is showed in isolation, these proteins are not able to induce a pronounced protective immunity against E7 proteins. Our assumption was made that the reason for the low immunogenicity of the polypeptides is their inability to absorb on the epithelium of mucous, so that they can undergo rapid proteolytic degradation. To solve this problem it was decided to use the protein complexes with chitosan. In the experimental verification of this assumption was that the complex hybrid proteins, chitosan, expressed mucoadhesive property and promote active sorption antigens on the cell surface, effectively induces both humoral and cellular component of immunity in respect of E7 proteins.

Based on this part of the vaccine as an adhesive means, facilitating penetration of antigens through the mucous membranes, and strong non-specific stimulator of the immune reactions included chitosan in the ratio of protein:

chitosan 1-1 to 1-10. The optimal ratio of protein and chitosan in the composition of the dosage form was studied experimentally. It is established that the maximum effect in the induction of immunological responses against antigenic determinants of proteins was observed when using a ratio in the range of 1-1 to 1-10. By reducing or HC is the chances of improving the concentration of chitosan in relation to a fixed dosage of the protein was observed a decrease in the efficiency of induction of immunological reactions.

For the manufacture of suppositories used the following pharmacologically acceptable additives: Witepsol, cocoa butter or tallow type And as lipophilic bases, polyethylene glycols of various molecular weights as the hydrophilic base, Nipagin, nipazol or aminoglycosides as antimicrobial agents, succinic acid and sodium benzoate as stabilizers or preservatives, thimerosal and thiomersal as stabilizers and antimicrobial agents (patent RU 2224542 C2, AC 39/116, 9/02, OR 13/00; the RF government decree No. 885 from 02.08.1999,; patent RU 2150268, AK 9/02, AK 35/74, OR 15/02; "Thimerosal and vaccination against hepatitis b virus", the journal of Medicine for all", №1, 2001; Patrizi, A., Rizzoli L., S. Vincenzi et al. Sensirization to thimerosal in atopic children. Contact Dermatitis, 1999, 40(2), 974-979).

The present invention relates also to methods for treating diseases associated with human papilloma virus, providing local application of mucosal vaccines. As an option for treatment of cervical dysplasia caused by HPV, used hybrid protein E7

type 16-Hsp70 or type 18 E7-Hsp70 in combination with chitosan at a ratio of 1:0.1 to 10, and pharmacologically acceptable additives. Made suppository administered intrawaginalno. Depending on stages of the disease suppositories prescribed 2-4 times every week is Liu for mild cervical dysplasia and 4-8 times with an interval of one week with dysplasia 2-nd and 3-rd degree. For the treatment of anal warts use a hybrid protein of the type 16 E7-Hsp70 or E7 type 6-Hsp70 in combination with chitosan at a ratio of 1:0.1 to 10, and pharmacologically acceptable additives. Made suppository is administered rectally. Suppositories prescribed at night once a week. Maximum clinical effect is achieved by assigning 4-8 suppositories.

Regional variations of vaginal suppositories for the treatment of cervical cancer

Taking into account geographical distribution and to maximize the effectiveness of regional options for the treatment of cervical cancer should contain the following combinations of recombinant hybrid proteins E7-Hsp70 in the composition of vaginal suppository:

1. Global pentavalent based E, E, E, E, E, merged with Hsp70.

2. Chinese tetravalent vaccine based E, E, E, E, merged with Hsp70.

3. Latin American trivalent vaccine based on E, E, E, merged with Hsp70.

4. African trivalent vaccine based on E, E, E, merged with Hsp70.

5. For the rest of the world (Europe, North America, Australia) bivalent option based E, E, merged with Hsp70.

Thus, the proposed invention allows to raise efficiency of treatment of diseases associated with human papilloma virus. The resulting effect is as the expense of primary induction of local immunological reactions due to mucosal application of recombinant proteins in complex with chitosan. It is this combination of active components of the vaccine ensures their high affinity for epithelial cells of the mucous infected with HPV, the active recognition of antigens by the immune system and, as a consequence, the induction of local immunity against E7 proteins, which play a key role in the development of pathological processes. The use of the proposed mucosal vaccine can significantly reduce the cost of treatment in comparison with the known methods of treatment of cervical cancer and RPK. The offered vaccine is intended for local application that allows you to eliminate unwanted and extremely dangerous for the life of the patient the side effects of injectable drugs, such as anaphylactic shock. The proposed method of treatment greatly simplifies the treatment process the patient can undergo treatment outside the clinic, independently introducing the drug.

The invention is illustrated in the following graphics:

Figure 1. Dynamics of serum IgG immune response in different ways immunization of mice RSL E76-Hsp70.

Figure 2. The magnitude of mucosal normalized IgA or antibody-based test response in intranasal and subcutaneous immunization of mice RSL E76-Hsp70.

Figure 3. The level of secretion of IFN-gamma(1) and IL-4(2) lymphocytes from inguinal lymph nodes (A) and spleen (B), stimulated in culture with a mixture of recombinant proteins A and A

Figure 4. The level of secretion of IFN-gamma(1) and IL-4(2) lymphocytes from inguinal lymph nodes stimulated in culture with a mixture of recombinant proteins A and E.

The implementation of the present invention

Example 1

The preparation of vaccines based on recombinant proteins for mucosal application of chitosan

Recombinant hybrid proteins E716-Hsp70 (SEQ ID NO:2), E718-Hsp70 (SEQ ID NO:4), E76-Hsp70 (SEQ ID NO:6) was purified from Taurus the incorporation by solubilization in 8M urea followed metallogenetic and ion exchange chromatography to a homogeneous state. The degree of purity of the protein was monitored by electrophoresis in 10% polyacrylamide gel under denaturing conditions, the protein concentration was determined by the method of Lowry using bovine serum albumin as standard. Before cooking vaccine recombinant hybrid protein (RSL) was subjected to dialysis to remove preservatives and stabilizers against phosphate-saline buffer solution (PBS, pH 7.2), was diluted or concentrated to the desired concentration and spent sterilizing filtration. After these procedures, the solutions of recombinant proteins was mixed with a solution of chitosan in different proportions and used for the manufacture of suppositories.

Example 2

Immunization of mice and sampling

Used adult females thinking is th (age 6-8 weeks) Balb/c (Nursery RAMS "High") in the amount of 5 pieces per group. Immunization produced three times at intervals of 4 weeks (0-28-56 day). When conducting intranasal immunization the mice were injected 20 μl of drug RSL with chitosan (10 μl in each nostril) using a micropipette under using anesthesia. For vaginal and rectal immunization with the help of micropipette was administered 10 μg protein in complex with chitosan in a volume of 20 µl. Subcutaneous immunization was performed in the withers mice by injection of 200 μl of the suspension RSL with Alhydrogel. (For subcutaneous (s.c) vaccination drugs RSL was adsorbing to 0.5% (weight/weight) aluminum hydroxide gel (Alhydrogel, Brenntag Biosector, Denmark) during the night.) In both cases, the dose of RSL for each immunization was 10 µg protein per mouse. Control group mice were immunized similarly with the use of 0.5% (weight/volume) solution of chitosan and 0.5% (weight/weight) solution Alhydrogel for intranasal and subcutaneous route of administration, respectively.

Blood samples for determination of specific IgG was taken from retroorbital vein of each mouse using a Pasteur pipette in a volume of 0.1 ml of 10 days after the first, second and third immunization. After the retraction of the clot serum was separated by low speed centrifugation and stored at -20°C till analysis in ELISA.

Nasopharyngeal swabs for determination of specific IgA were obtained 14 days after the third immun the organizations. With this purpose, mice were killed by method of cervical dislocation, were deceptional and washed the nasopharynx from the trachea using 0.5 ml of PBS. Nasopharyngeal swabs were stored at -20°C till analysis in ELISA. To study T-cell immune response to recombinant proteins and peptide epitopes mice were killed by method of cervical dislocation after 14 days after the third immunization and aseptically extracted lungs, spleen and lymph nodes of the mediastinum. Culture of lymphocytes was obtained by gentle mechanical grinding tissue, lysis of erythrocytes and triple washing of the cells by low-speed centrifugation in medium RPMI 1640 with the addition of 10% fetal serum. 20 mm L-glutamine and antibiotics.

Example 3

Comparison of specific IgG and IgA immune response or antibody-based test with intranasal and subcutaneous immunization RSL E76-Hsp70

Mice were immunized RSL E76-Hsp70 intranasally and subcutaneously in accordance with the Protocol and dosage described in example No. 3. Geometric mean titres (BHT) IgG and IgA antibodies specific for recombinant protein A in serum and nasopharyngeal swabs obtained according to the procedure of example 2 was determined using enzyme-linked immunosorbent assay (ELISA). To detect serum IgG 96-hole tablet was immobilized recombinant protein A of Rast is ora with a concentration of 5 μg/ml in PBS overnight, spent the titration of mouse sera by three-fold serial dilutions and made peroxidase conjugate goat antibodies against IgG (H) mouse. As the substrate used TMB, the optical density (OD) was recorded after 15 min at a wavelength of 450 nm. The titre of specific IgG antibodies in each serum was determined by end point, which corresponded to the dilution of sera with OD>0.2. The results are shown in figure 1.

Figure 1 displays the dynamics of serum IgG immune response in different ways immunization of mice RSL E76-Hsp70:

Group 1 - E76-Hsp70+chitosan, intranasal;

Group 2 (control) - chitosan, intranasal;

Group 3 (control) - aluminum hydroxide, subcutaneously;

Group 4 - E-s70+aluminum hydroxide, subcutaneously. The ordinate axis pending the logarithm of the geometric mean titer IgG antibodies.

To determine the titer of IgA in nasopharyngeal swabs used ELISA, similar to those described above, but instead of anti-IgG(H) conjugate was used peroxidase conjugate goat antibodies against IgA (H) mouse. End point titration for each rinse was determined as the highest dilution of the sample at which the OD>0.1. To minimize variations associated with the procedure for obtaining a nasopharyngeal swabs in each sample was determined by the total content of IgA using a commercial ELISA kit. In konecne is m the value of mucosal IgA immune response was expressed as the ratio of the titer specific to A IgA antibodies to total IgA content (expressed in micrograms) in the same sample. The results are shown in figure 2.

Figure 2 shows the magnitude of mucosal normalized IgA or antibody-based test response in intranasal and subcutaneous immunization of mice RSL E76-Hsp70.

Group 1 - E76-Hsp70+chitosan, intranasal;

Group 2 (control) - chitosan, intranasal;

Group 3 (control) - aluminum hydroxide, subcutaneously;

Group 4 - E76-Hsp70+aluminum hydroxide, subcutaneously.

Example 4

Comparison of specific T-cell response during intravaginal and subcutaneous immunization with a mixture of RSL E716-Hsp70 and E718-Hsp70

Used adult female mice (age 8-10 weeks) Balb/c in the amount of 5 pieces per group. Immunization was carried out 3 times with an interval of 10 days under the scheme: 0-10-20 days. For intravaginal immunization (i.vag.) mice were administered 20 μg of a mixture of RSL 10 µg E716-Hsp70 and E718-Hsp70, respectively, in 16 μl of 0.5% (weight/volume) solution of chitosan (Chitosan, Selectchemie AG, Switzerland) using a micropipette into the vagina. Subcutaneous immunization (s.c.) held at the withers mice by injection of 200 μl of the suspension mixture RGB (20 μg) with 0.5% (weight/weight) aluminum hydroxide gel (Alhydrogel, Brenntag Biosector, Denmark). Control group mice were immunized similarly with the use of 0.5% (weight/volume) solution of chitosan and 0.5% (weight/weight) solution Alhydrogel for intravaginal and subcutaneous route of administration, respectively. 14 days after the third immunization of mice mind is ruleli and aseptically removed the spleen and inguinal lymph nodes. Culture of lymphocytes was obtained by gentle mechanical grinding tissue, lysis of erythrocytes and triple washing of the cells by low-speed centrifugation in medium RPMI 1640 with the addition of 10% fetal serum, 20 mm L-glutamine and antibiotics. Culture of lymphocytes obtained from the spleen and inguinal lymph nodes of mice immunized with a mixture of RSL E716-Hsp70 and E718-Hsp70 and control groups, examined for the secretion of interferon gamma (IFN-gamma) and interleukin 4 (IL-4) after stimulation with recombinant proteins E716-Hsp70 and E718-Hsp70 using ELISPOT kits (BD Biosciences, Oxford, UK). The data shown in figure 3.

Figure 3 reflects the level of secretion of IFN-gamma(1) and IL-4(2) lymphocytes from inguinal lymph nodes (A) and spleen (B), stimulated in culture with a mixture of recombinant proteins A and E. Mice were immunized:

Grey columns E-S70+E-S70+chitosan, intrawaginalno;

Black columns - chitosan, intrawaginalno;

White columns - aluminum hydroxide, subcutaneously;

Hatched columns E-S70+E-S-70+aluminum hydroxide, subcutaneously. The ordinate axis pending the number of cytokine-secreting lymphocytes at 1 million cells.

Example 5

Comparison of specific T-cell response in rectal and subcutaneous immunization with a mixture of RSL E716-Hsp70 and E76-Hsp70.

Used adult female mice (age 8-10 weeks) line alb/c in the amount of 5 pieces per group. Immunization was carried out 3 times with an interval of 10 days under the scheme: 0-10-20 days. For rectal immunization (i.R.) mice were administered 20 μg of a mixture of RSL 10 µg E716-Hsp70 and E76-Hsp70, respectively, in 16 μl of 0.5% (weight/volume) solution of chitosan (Chitosan, Selectchemie AG, Switzerland) using a micropipette into the rectum. Subcutaneous immunization (s.c.) held at the withers mice by injection of 200 μl of the suspension mixture RGB (20 μg) with 0.5% (weight/weight) aluminum hydroxide gel (Alhydrogel, Brenntag Biosector, Denmark). Control group mice were immunized similarly with the use of 0.5% (weight/volume) solution of chitosan and 0.5% (weight/weight) solution Alhydrogel for rectal and subcutaneous route of administration, respectively.

14 days after the third immunization, mice were killed and aseptically extracted inguinal lymph nodes. Culture of lymphocytes was obtained by gentle mechanical grinding tissue, lysis of erythrocytes and triple washing of the cells by low-speed centrifugation in medium RPMI 1640 with the addition of 10% fetal serum, 20 mm L-glutamine and antibiotics. Culture of lymphocytes obtained from inguinal lymph nodes of mice immunized with a mixture of

RSL E716-Hsp70 and E76-Hsp70 and control groups, examined for the secretion of interferon gamma (IFN-gamma) and interleukin 4 (IL-4) after stimulation with recombinant proteins E716-Hsp70 and E76-Hsp70 using ELISPOT kits (BD Biosciences, Oxford, Velikova the project). The data shown in figure 4.

Figure 4 reflects the level of secretion of IFN-gamma(l) and IL-4(2) lymphocytes from inguinal lymph nodes stimulated in culture with a mixture of recombinant proteins A and E. Mice were immunized:

Grey columns E-S70+E-S70+chitosan, rectal;

Black columns - chitosan, rectal;

White columns - aluminum hydroxide, subcutaneously;

Hatched columns E-S70+E-S-70+aluminum hydroxide, subcutaneously. The ordinate axis pending the number of cytokine-secreting lymphocytes at 1 million cells.

Example 6

Adjuvant effect of Hsp70 M tuberculosis on cells of the immune system in vitro

Immature dendritic cells (DC) were obtained by perfusion hollow bones of the hind limbs of mice CBA using sterile isotonic by the standard method. Cells were washed three times and cultured in the matrix with the addition of thermoinactivation fetal cattle serum in RPMI medium 1640 (150,000 cells/ml) for 48 hours without stimulation, and in the presence of Hsp70 in a concentration of 25 μg/ml Obtained by centrifuging the culture fluid was analyzed for production of murine cytokines using ELISA kits (R&D Systems (USA) Expression differencirovannyh markers DK investigated by the method of flow cytofluorometry using monoclonal anti the l manufactured by R& D Systems (USA). The results of determination of cytokine production (conventional units) and differencirovannyh markers DK (% cells) are shown in table 1:

Table 1
CytokinesImmature DCStimulation of Hsp70, 25 μg/ml, 48 h
IL-1 beta513.8679.0
IL-20.07.3
IL-60.0130.9
IL-100.06.3
IL-1251.6262.6
TNF-alpha29.0118.0
IFN-gamma14.623.0
TGF-beta286.5229.0
Differencirovanie markers
CD83 9.8±0.127.3±0.6
CD385.43±0.1484.5±1.5
CD864.00±0.111.44±0.04
I-AK (MHC2)19.1±0.634.7±0.3
CD11C43.1±0.548.6±0.9
CD400.70±0.141.21±0.09
TLR214.8±0.335.4±0.3
TLR41.5±0.18.0±0.2

These data indicate that Hsp70 is induced in the DC production of proinflammatory cytokines: IL-1 beta, IL-6, IL-12 and TNF-alpha, indicating a pronounced adjuvant effect of Hsp70. The study of changes in phenotype of DC in stimulation of Hsp70 showed that the proportion of cells expressing markers of Mature DC (CD38, CD40, CD83 molecules MHC2), increases. The spectrum of markers of Mature DC will not increase the expression of co-stimulatory molecules CD86 and CD11. It is unclear also change the level of TLR2 and TLR4, more typical of immature DC.

Example 7

Manufacturer megacomponents the x vaginal suppositories for the treatment and prevention of human papillomavirus

Components:

1) Solution of recombinant proteins, E716-Hsp70, E718-Hsp70

2) Chitosan company TAI ZHOU CANDORLY BATCH No. 06020301, the degree of diacetylmorphine 86%

3) Polyethylenglycol 1500 firm APPLI SNEM CAS-NO.: 25322-68-3

4) solid fat type And

5) witepsol HW

6) TRIS firms MP Biomedicals Inc. cat. No. 195605.

Part 1 suppository:

A solution of recombinant proteins E716-Hsp70, E718-Hsp70

(C=1 mg/ml) in 0.01 M TRIS0.1-0.5 ml
Chitosan0,00165 g - 0.03 g
Basis (witepsol/tallow type (a)to 3.0 g

Method for producing suppositories

Suppositories were prepared by the method of pouring.

The calculation of the components was carried out on 100 suppositories 3.0,

A solution of recombinant protein E7 16 and 18 types

(C=1 mg/ml) in 0,01M TRIS50 ml
Chitosan0,165 g
PEG 150045,0 g
Cocoa butter255,0 g

To a solution of recombinant protein E7 of types 16 and 18 (C=1 mg/ml) in 50 ml of 0,01M TRIS add 0,165 g of chitosan and stirred for 10 minutes, then filtered through filter paper (filter with blue stripe). In obtained after filtration the solution is injected pre-molten PEO 1500 and stirred for 5 minutes. To the mixture gradually add pre-molten hydrophobic base (witepsol, fat type (a) and stirred for 30 minutes. The obtained suspension is poured into pre-cooled suppozitornoj form and placed in the refrigerator at -4°C.

Example 8

Fabrication of multicomponent rectal suppositories for the treatment and prevention of human papillomavirus

Components:

1) Solution of recombinant protein E7-16-Hsp70, E7-6-Hsp70

2) Chitosan company TAI ZHOU CANDORLY BATCH No. 06020301, the degree of diacetylmorphine 86%

3) Polyethylenglycol 1500 firm APPLI CHEM CAS-NO.: 25322-68-3

4) solid fat type And

5) witepsol HW

6) tween-80 to 5%

7) TRIS firms MP Biomedicals Inc. cat. No. 195605.

Part 1 suppository:

A solution of recombinant proteins E716-Hsp70, E76-Hsp70

(C=1 mg/ml) in 0,01M TRIS0.1-0.5 ml
Chitosan0,00165 g - 0.03 g
Basis (witepsol/tallow type (a)to 3.0 g

Method for producing suppositories

Suppositories were prepared by the method of pouring.

Computation of PR who drove 100 suppositories 3.0,

A solution of recombinant protein E7 16 and 18 types

(C=1 mg/ml) in 0.01 M TRIS50 ml
Chitosan0,165 g
PEG 150045,0 g
Cocoa butter255,0 g

To a solution of recombinant protein E7 of types 16 and 18 (C=1 mg/ml) in 50 ml of 0.01 M TRIS was added 0,165 g of chitosan and stirred for 5-10 minutes, then filtered through a paper filter (filter with blue stripe). In obtained after filtration the solution is injected pre-molten PEO 1500 and stirred for 5 minutes. To the mixture gradually add pre-molten hydrophobic base (witepsol, fat type (a) and stirred for 30 minutes. The obtained suspension is poured into pre-cooled suppozitornoj form and placed in the refrigerator at -4°C.

Example 9

The study of clinical effectiveness of the developed mucosal vaccine in patients with cervical dysplasia caused by human papilloma virus type 16 and 18

Clinical trials were selected three groups of patients with a diagnosis of cervical dysplasia (CIN I) and (CIN II). Each group consisted of 10 people. The diagnosis of cervical dysplasia was mouth is oflen on the basis of clinical examination and laboratory analysis of samples of cervical epithelium on the carriage of HPV 16 and 18 types. Each patient was assigned treatment with vaginal suppositories of different composition 1 time per week for 4 weeks. In some cases, the treatment was carried out for 8-12 weeks, keeping the appointment of the drug on 1 suppository for the night once a week. After completion of treatment, patients were examined with colposcopy, conducted a morphological analysis of cells of the cervix and laboratory analysis of PCR for the presence of DNA of HPV 16 and 18 types.

Table 2
The number of patients diagnosed with CIN I CIN IIPurpose: Vaginal suppositories containingThe results of treatment (number of patients)
ClinicMorphologyPCR HPV DNA
NormaPathologyNormaPathologyNormaPathology
10Recombinant Proteins Hsp-E7-16, Hsp-E7-18+chitosan8 28273
10Recombinant Proteins Hsp-E7-16, Hsp-E7-18464637
10Recombinant Protein Hsp70191919

Example 10

The study of clinical effectiveness of the developed mucosal vaccine in patients with a diagnosis of condyloma rectum.

For the clinical study consisted of 20 patients proctological Department with a diagnosis of Recurrent condyloma rectum". Patients complained of pain during the act of delicacie, traces of blood in the stool. During visual inspection of the mucous membrane of the rectum revealed HPV size 0.2-0.5 see, the mucosa is inflamed. Some patients found cracks in the anus.

Patients assigned to monotherapy rectal suppositories containing the described ingredients 1 candle at night, once a week for the of 4 weeks. In some cases, the treatment was carried out for 8-12 weeks, keeping the appointment of the drug on 1 suppository for the night once a week. After the course of treatment in 16 patients achieved complete clinical recovery, which was accompanied by the disappearance of the warts and all signs of inflammation. 4 patients after treatment was observed in reducing the number of warts with no sign of inflammation. Offer mucosal vaccine in the form of rectal suppositories very effective in these pathologies.

1. Mucosal vaccine against diseases associated with human papilloma virus, which contains an effective amount of a hybrid protein consisting of the E7 oncoprotein of human papilloma virus, fused with heat shock protein of mycobacteria Hsp70, chitosan in relation to the hybrid protein of 1:0.1 to 10 and pharmacological acceptable for the manufacture of suppositories supplements.

2. Mucosal vaccine according to claim 1, characterized in that for the treatment of cervical dysplasia caused by HPV, used hybrid protein of the type 16 E7-Hsp70 or type 18 E7-Hsp70.

3. Mucosal vaccine according to claim 1, characterized in that for the treatment of anal warts using the fast hybrid protein of the type 16 E7-Hsp70 or E7 type 6-Hsp70.

4. A method of treating diseases associated with human papilloma virus, providing local application of mucosal vaccine according to any one of claims 1 to 3 in the form of a suppository.

5. The method of treatment of cervical dysplasia caused by HPV, providing intravaginal introduction effective amount of mucosal vaccine of claim 2.

6. A method of treating anal warts, providing for rectal administration of an effective amount of mucosal vaccine according to claim 3.



 

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SUBSTANCE: invention is related to preparation of protein, binding tumour necrosis factor (TNF), and may be used in medicine. Strain-producer of baculovirus BvG2RIgG is created with the help of recombinant plasmid DNA pFastBac-G2R-IgG with size of 6444 p.n. and molecular mass 4.18 mDa, which bears fragment of smallpox virus genome of strain India-1967, which codes protein that binds TNF, and fragment of human genome, which codes fragment of heavy chain of human antibody G. Produced strain produces soluble chimeric protein, which consists of smallpoz virus protein, which binds TNF, and fragment of heavy chain of human antibody G.

EFFECT: wider spectrum of new generation preparations intended for treatment of human diseases related to hyperproduction of tumour necrosis factor.

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FIELD: medicine.

SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.

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FIELD: medicine.

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4 cl, 15 dwg, 3 ex

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Fused proteins il-7 // 2369616

FIELD: medicine.

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13 cl, 5 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns virology and medicine area. The synthetic DNA-molecule coding protein L1 HPV45 is presented. Thus DNA-molecule was Codonum-optimised for high-level protein expression in a yeast cell. The given synthetic molecules can be used for reception of virus-like particles (VLP) HPV45 and for reception of vaccines and the pharmaceutical compositions containing VLP-particles HPV45.

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FIELD: medicine.

SUBSTANCE: invention concerns protein NMB1870 which represents common surface protein Neisseria meningitidis expressed with all Neisseria serogroups. Protein is subdivided into three separate families. The whey induced against antigen of a certain family, has bactericidal effect within this family, but is inactive concerning strains expressing antigens of one of other two families, i.e. there is a cross protection within family, but not between families. It is established, that NMB1870 can be subdivided into domains and that antigen domains can be recovered from NMB1870 of all three families and expressed as a polypeptide chain that is implemented by the method disclosed in the invention. Also it is discovered that NMB1870 expresses some epitopes in surface loops located between alpha spirals, and that epitope substitution of the loop of one family with that of the other family enables making chimeric NMB1870 of antigenicity characteristic for proteins of several families. In the invention there are disclosed chimeric proteins NMB1870 (versions) partially containing NMB1870 of various families.

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EFFECT: reception of active recombinant protein LF on the simplified technology and with a high output of synthesised protein of the lethal factor.

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