WO2010056148A1 - Mucosal vaccine for immunotherapy of diseases caused by human papillomaviruses - Google Patents

Abstract

The invention relates to the field of genetic engineering and can be used in the treatment of diseases associated with the human papillomavirus.  The mucosal vaccine contains, in an effective amount, a hybrid protein consisting of the human papillomavirus oncoprotein E7 fused with the mycobacterial heat shock protein Hsp70, chitosan in a ratio with the hybrid protein of 1:0.1-10, and pharmacologically acceptable additives for the manufacture of suppositories.  The proposed invention makes it possible to increase by many times the effectiveness of the treatment of diseases associated with the human papillomavirus, significantly reduce the cost of treatment compared to known methods for treating cervical cancer and rectal cancer, eliminate undesirable and life-threatening side effects to the patient of injection preparations, for example anaphylactic shock, due to topical application, and simplify the treatment process since the patient can carry out the course of treatment as an outpatient, administering the preparation independently.

Description

 Mucosal vaccine for immunotherapy of diseases caused by human papilloma viruses

FIELD OF TECHNOLOGY

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

BACKGROUND

In the mid-70s, it was first suggested that human papillomavirus (HPV) could be involved in

- the pathogenesis of cervical cancer and studies have begun to identify the virus in biopsy material. It was found that in malignant tumors of the cervix, the most frequently detected are two types of viruses - HPV 16 and HPV 18. And in benign lesions, mainly viruses of types 6 and 11 are present. Upon further study of the role of HPV in neoplastic processes, all serotypes were divided into three groups:

• Low degree of oncogenic risk: 6.11, 42.43, 44

• Average degree of oncogenic risk: 31, 33.51, 52.58

• High degree of oncogenic risk: 16,18,45,56

Currently, several studies have shown that HPV DNA is detected on average in 98.9% and 94.7% of genital warts and cervical cancer samples, respectively. In the population of healthy women, HPV is found in 35% of cases, with 6, 11, lb and 18 types - only in 21.6%. Interesting for further study, it turned out that the majority of HPV 16 DNA in genital warts cells is in episomal form, and in cervical cancer samples it is integrated into the cellular genome. In recent years, in connection with the start of the use of preventive vaccines against HPV, several large-scale epidemiological studies have been carried out on the geographical distribution of various types of HPV in cervical samples from patients with invasive cervical cancer (Cervical cancer). It turned out that in most cases all over the world (65-77%) HPV types 16/18 are detected, however, further ranking of HPV types has pronounced geographical features. For example, in China, the next most important HPV types are 58 and 52, in Africa - HPV type 45, in Central / South America - HPV type 31. (Wao Y, Li N., Smith J. apd Qiao Y., Nshpap paillomavirus tour-distributiop ip tеrvіh оf Сіхіпееs womep: а meta-apаlussis, InL Jоіpal оf STD & AIDS, 19, 2, 106-111; Nubia M., Havier V., Haviere S. et al., Agavirus whit shall we vassipate apd ssreep The ipterpatiopal rersrestive, Ipt Joirpal ° F Sapser, 2004, 11, 2, 278-285;?. Ihekweazu C, ° F Worldwide distributiop HPV tures Ip womep with pormal servisal sutologu apd Ip womep with servisal adeposarsipoma, Eirosirveillapse 2006 , 11, Issue 12).

The spread of infection occurs mainly through sexual contact, reaching a maximum of infection up to 30 years. More than 50% of the sexually active population of the world is infected with the human papillomavirus during their lifetime, and this is a “primary” event in the pathogenesis of cervical cancer. Most infections result in spontaneous recovery. However, in some cases, a persistent infection develops that can trigger the mechanisms of cellular transformation of epithelial cells. So with CESf 1 there is an active replication of the virus and its asymptomatic isolation. The conversion of CIN 1 to invasive cancer occurs with a high frequency and, as a rule, is accompanied by the integration of viral DNA into the genome of the host cell (V. Kiselev, Human papillomaviruses in the development of cervical cancer. M .: Dumumpeid Graph Gripp® Company, 2004). HPV of a high degree of oncogenic risk (mainly

16 and 18 types) are found in 50 - 80% of samples of moderate and severe dysplasia of the squamous uterine cervix and in 90% of invasive cancer. An important circumstance is that the clinical manifestations of HPV infection are not detected for a sufficiently long time, however, the consequences of the persistence of the virus from this do not become easier. The development of mild and moderate dysplasia in severe occurs, respectively, in 10 and 20% of cases. Such statistics is not fatal: the transition of one stage of epithelial atypia to another takes a long time, which gives great opportunities in terms of diagnosis and treatment. In addition, the fact that HPV carriage is not life-long is important: according to the WHO (2001), in the absence of aggravating factors for 3 years, low-severity squamous cell epithelial lesions containing HPV are regressed in 50 - 62% of cases . According to scientists at the University of California, in 70% of young HPV-infected women, HPV DNA ceases to be detected for 24 months. Speed elimination is significantly reduced when infected with several types of HPV, as well as in the presence of a history of genital warts. At the same time, among women with at least 3 times positive results of HPV determination, the risk of severe oncological lesions of the epithelium increased by 14 times. About 500,000 cases of cervical cancer are registered annually in the world, of which about 50% are fatal. According to the Ministry of Health, in 2002, 11320 cases of cervical cancer were recorded in the Russian Federation (BA Molochkov, B. Kiselev, I. Rudykh, S. H. Scherbo, Papillomavirus infection, clinic, diagnosis, treatment, manual for doctors, M. : Publishing house "Russian doctor", 2004).

It should be noted that in recent years the number of diagnoses of colorectal cancer, also caused by human papilloma viruses, has significantly increased. This type of pathology is especially widespread among men with non-traditional sexual orientation. Cases of the development of oral cancer with oral sex with a partner infected with HPV have been reported (News.com // in the world // November 18, 2004, Oral sex leads to cancer). In most cases of oncological pathology, the formation of condylomas of the rectal mucosa is preceded, which is accompanied by bleeding during the act of deformation, inflammatory processes and anal fissures. Current understanding of the mechanism of carcinogenesis due to HPV HPV infects and replicates in the epithelium, as evidenced by the detection of an episomal viral genome in the cells of the basal layer of the epithelium. The life cycle of the virus is closely related to the differentiation of the host cell. Replication of viral DNA and synthesis of capsid proteins of the virus occurs in the most differentiated layers of the epithelium, and the virus has a whole range of mechanisms that subordinate the vital activity of the infected cell to its interests. Virus DNA encodes the synthesis of two proteins - E6 and E7, which induce the transition of differentiated cells into the S phase of the cell cycle. At the stage of active reproduction of the virus, the expression of the E6 and E7 genes is regulated by the product of the E2 gene, which is a repressor of the transcription of these genes. That is why, while the virus is in an episomal state, benign processes of growth of infected tissues are observed. A key event in the malignancy of cells is the integration of the virus into the cell genome, which is accompanied by a deletion of the E2 gene. This event has two important consequences:

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

2) any antiviral drugs are powerless to stop the process of tumor transformation, since infected cells do not contain the virus in the traditional sense, and all therapeutic measures should be aimed at eliminating cells with an integrated form of the HPV genome. Cell cycle control and cell differentiation is carried out by E6 and E7 proteins through their interaction and inactivation of such “key” proteins-regulators of proliferative activity and apoptosis of cells as p53 and retinoblastoma protein (pRB). Uncontrolled proliferation of infected cells leads to the accumulation of genetic damage and, ultimately account, to malignancy. Oncoprotein E7 uses several pathways to regulate the cell cycle. It was found that E7 is able to form a stable complex with the pRB protein, causing its degradation, which leads to the release of the transcription factor E2F, which stimulates the transcription of genes necessary for DNA replication and S-phase of the cell cycle. E7 also affects the activity of a number of cell cycle proteins, such as A and E cyclins, cdk2 kinase and inhibitors of cyclin-dependent kinase p21 and p27 (Kiselev B.I., Kiselev O.I., Etiological role of human papillomavirus in the development of cervical cancer uterus: genetic and pathogenetic mechanisms, Cytokines and inflammations, 2003, 2, 4, 31-38; РHelrs WC, Varpes J.A. apd Loba DC, Humaparillotavigas: molotargetgeps aprpresult forpolitelaprilapriltapl, 1999, 7, 4-8).

Animal studies show that humoral immunity plays a role in protecting against HIV infection. A model of virus-like particles (vigas-likelihoods-VLP), self-assembled from the main HPV-Ll capsid protein, is widely used to study the protective role of virus-neutralizing antibodies. Immunization of animals with VLP protects them against experimental infection homologous viruses. The passive transfer of serum from mice immunized with VLP _{5 to} other experimental animals also has a protective effect, which confirms the protective role of virus-neutralizing antibodies (Type RW, Impeve evaciop, Hpap parillomavis-asserceper, Neper, 2002, 2; Zhou J., Liu WJ., Repg SW et al. Rarillomagic sarsid rotelep relaps on the matte beta tpnapalit, J.Verpent, 1999, 49 Expresiop, Urs. J. Med. Ski, 2000, 105, 171-192). In the case of infection of experimental immunized mice with HPV of a different type, the protective effect was not recorded, which indicates the specificity of the observed phenomenon and the absence of cross-reactions between different types of HPV. Moreover, denaturation of VLP led to the loss of protective effects, which indicates the role of intact protective epitopes in the induction of antibody production. Another late gene encoding a minor L2 capsid protein has also been investigated as a potential candidate for prophylactic vaccination. It was found that immunization of animals with the hybrid recombinant protein L2 induces a high titer of neutralizing antibodies and protects against experimental human papillomavirus infection. Thus, the data accumulated to date indicate that HPV capsid proteins are promising candidates for the creation of a prophylactic vaccine. NF-kB- Resource Gepes IP Servisal keratiposutes. J. Ig., 2001, 75, 4283-4296; Bamard R, Payne E. & MsMShap N.A. That hshpap rarilomavigas E7 rote is also available to iptivit apd apti-grwth fiсtiops оf ipterferop. Virology, 2000, 277, 411-419). However, when using these proteins for therapeutic immunization with an established, chronic papillomavirus infection, it was not possible to obtain any significant effect. Apparently, another strategy is needed for therapeutic immunization, namely, the induction of an antigen-specific T-cell immune response. Available data suggest that the stimulation of the cellular component of immunity against non-structural viral proteins can lead to regression of the developing tumor.

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

1. Occurrence HPV - associated disease is substantially higher among patients with transplant clinics (Rark JS et al Ipastivatiop ° F ipterferop regulatoru factor-1 tumor surrressor rroteip bu HPV E7 opsorroteip Imrlisatiop for the E7- medated immupe evasiop meshapism Ip servisal sarsipogepesis J. Viol... Chet 2000, 275, 6764-6769) and HIV-infected, i.e. in cases with deep impaired cellular immunity (Frazer L, et al. Sustemis admipistratiop ° F basterial rrodusts ipduses host rrotestive sresifis immshiitu to an erithelial tumour aptigep. J Immupol., 2000, 167, 6180-6187, Doap T. et al. Nshpap Rarillomavigas Toure 16 E7 Opsorrotep Expres Ip Rerihl Erithelishp Toloriz E7-Directed Sutotokhis T-Lumrhosute Resursurs Restored nshpap (apd Mousе) Major Nistosomratibilitu Сomplex Class I Аllеles. /. ViroL, 1999, 73, 6166-6170).

2. Experimental animals immunized with the non-structural proteins of HPV, are effectively protected against HPV infection and the development of neoplastic processes (Gtashiipi S. et al Sutokme ehrressiop Ip squamous iptraerithelial lesiops ° F the uteripe servih:. Imrlisatiops for the geperatiop ° F losal immshiosurrressiop CHn Exp IttshoL... 1998, 113, 183-189).

3. During spontaneous regression of genital warts, there is an intense infiltration of surrounding tissue CD ₄₊

(T-helpers) and CDs ₊ (cytotoxic) T-cells (RESPIgE M.E. et al. Human CTL adapters epsompid humidivirus iptivodipodivpoditivpolitompivodimpulit bipdiag peptides. J. J. L, 1995, 154. 5934-5943). 4. The skin lesions associated with HPV in patients receiving immunosuppressive therapy often disappear when the treatment course is canceled (Le. SJ. Et at. E6 apd E7 optorotepiofs of humaprilomavigas 16 iphibit IL-18-ipfpu humid rerherall blodopopuslear-apd NK sells. Jtipol, 2001, 167, 497-504).

The data presented indicate that specific stimulation of T-cell immunity can lead to complete remission of HPV-associated diseases. In this regard, the practical task when creating a therapeutic vaccine is to stimulate T-cell immunity in relation to non-structural HPV proteins, such as proteins E6 and E7.

The main requirement for therapeutic vaccines is that they must stimulate the cellular component specific immunity and block the development of neoplastic processes. Theoretically, the cellular reaction should be directed to viral and cellular proteins involved in malignancy of the epithelium. However, despite intensive studies, little is known about cell targets during tumor transformation; therefore, the majority of experimental vaccines are aimed at the products of the HPVT genes E6 and E7, whose role in oncogenesis has been unequivocally proven.

Since the E6 and E7 proteins are constitutively expressed in most PIIIM cells and are absent in normal tissue, these viral oncoproteins are considered the most promising targets for the development of vaccines for the treatment of HPV-associated tumors (Straght SW, Herman B. & McCance DJ. The E5 opsorfotip of humap raillomavirus tour 16 iphibits the adidifiсtiop оf iposomes ip humap keratosputes. J Virol, 1995, 69, 3185-3192).

As a rule, the target for immunotherapy of tumors is normal or mutant cell proteins, while E6 and E7 are completely foreign to the host’s immune system, and therefore should be very immunogenic (Przeriorka D. apd. Srivastava R., Non Shock Protei - Rertide comlexes as immüütäraru for humap sapser. Molesilartisipipe Todau, 1998, 11, 478-484).

The next reason that the E7 and E6 proteins are considered as promising candidates for vaccination is that many tumors can lose target proteins during immunotherapy, thus escaping the immune attack and preserving their transformed status, then the development of cervical cancer is uniquely determined by the expression of E6 and E7 proteins and their neutralization will lead to tumor regression.

The development of peptide vaccines is constrained by the fact that, until now, the epitopes in proteins E6 and E7 capable of inducing cytotoxic immunity have not yet been precisely determined. Therefore, the main efforts are focused on the construction of protein vaccines using recombinant proteins E7 and E6.

Recent studies show that a more preferred target for vaccines is E7 protein.

HPV types 16 and 18 for cervical cancer, and E7 HPV type 6 for anal condylomas, since it has the smallest structural variability in different geographical isolates of HPV and does not have splicing options (Swap DC, Rajeevap M., Tortolero-Lshia G. , et al. Numap paralomavigures tour 16 E2 app E6 / E7 variapts,

Gupesol. Optol 2005, 96, (3) 695-700).

It was shown that recombinant HPV protein E7 type 16 is capable of inducing cytotoxic immunity, and injection of this protein in combination with various adjuvants enhances the cytotoxic reaction.

In recent years, a new strategy for increasing the immunogenicity of E7, which consists in the construction of hybrid proteins, has been especially popular. In particular, the immunological properties of a fusion protein containing the complete sequence of the heat shock protein Hsp70 and HPV protein E7 have been studied. The immunogenicity and protectiveness of such a design was significantly higher, probably due to the fact that Нср 70 M. tuberculosis provides an effective presentation of antigens. (Vesker T., Hartl R, and Wieland F., CD40, Up Excelper Forp BipDipg Apd Utake Off Hsp70 - Repert Comlex, J. Cell Biogu, 2002, 158, 1277-1285).

It is known that E7 is synthesized in small quantities during the reproductive phase of the HPV life cycle. However, its synthesis increases significantly after the integration of viral DNA into the cellular genome, since this process, as a rule, is accompanied by the loss of the E2 gene encoding the repressor of the E7 gene. Despite this, the induction of specific cellular immunity against the E7 protein is not observed in the body. Some experimental models shed light on the amazing ability of Oncoprotein E7 to induce tolerance.

If a skin transplant from a transgenic mouse synthesizing E7 in epithelial cells is transplanted with a genetically related mouse, then the expected transplant rejection does not occur. If the mice are introduced stimulating formation of anti-inflammatory cytokines (killed bacteria or endotoxins), then the graft quickly rejects. However, transplant rejection against the background of the introduction of cytokine inducers occurs only during the graft engraftment, and a second transplant of the same mouse transplant results in rejection without additional induction of cytokines. Different assumptions are made about the mechanisms of this phenomenon, however, they all agree on one thing - antigen-presenting cells that absorb E7 in the cervical canal, for some reason cannot go through all the necessary stages of maturation, migrate to the lymph nodes and induce a T-cell immune response against this protein without additional stimulation of the cytokine cascade (Gröss G., Jöblopska S., Pfister H. Gepital rappilomirus virus, 1989, Frezzer I. et al. rotational resistance with immunity to ap erithelial tumour aptigep, Jtipol (2001, 167, 6180-6187).

The constant synthesis of oncoproteins is necessary to maintain the tumor phenotype of cells. Obviously, the appearance in the body of cells carrying foreign antigens (E6 or E7) should induce appropriate immune responses. However, patients with cervical carcinomas have a very low T-cell immunity against E6 and E7.

When vaccinated with these proteins, there is also no pronounced reaction, despite the good characteristics of the immune status. These observations: as well as experiments on animals show that the oncoprotein E7, synthesized in epithelial cells, is able to "slip" or even suppress immune reactions in relation to itself.

When creating any antitumor vaccine, it is necessary to solve several fundamental problems. First, determine the target against which it is necessary to induce the body's immune responses. In the case of HPV infection, it is generally believed that such a target is E7 protein (Kiselev V.I., Human papillomaviruses in the development of cervical cancer.-M: Dumumpeid Graph Gripp® Company, 2004).

An equally important task is the choice of adjuvant for the induction of stable protective immunity and the method of administration of the vaccine preparation. Last circumstance much attention is paid, since it turned out that its effectiveness depends on the method of administration of the vaccine. As already mentioned, HPV infections affect the mucous membranes both in the case of cervical cancer, and in RRS, and cancer of the rectum. However, vaccine preparations developed to date are intended for subcutaneous administration, that is, they are aimed at specific stimulation of systemic immunity and ignore mucosal immune responses (US Pat. No. 6,338,952 Bl 2002 Strothers and USPs; Terféoré; Patent PFN ° 2229307, Finger EC A., Finger M. A., , Kiselev O.I., Kiselev V.I., Sveshnikov P.G., Recombinant protein composition, method for preparing such a composition, pharmaceutical kit for immunotherapy and prophylactic vaccination of tumor diseases of the anogenital sphere, method and mmunotherapy and preventive vaccination based on it). On the basis of the American patent, the company Nvepta is developing an injectable form of a vaccine preparation containing the E7 protein, "fused" to the heat shock protein M. tuberculosis with a molecular weight of 65 IdDa. From our point of view, a significant drawback of this design is the choice of 65 Sha protein, since there is convincing data on the possibility of inducing autoimmune reactions in humans with the introduction of this protein. In addition, this heat shock protein is inferior in its adjuvant properties to the Hsp 70 M. tibercylosis protein. (Zugel U. apd Kaufmapp S., RoIe оf hеаt shоk рroteіp іp rоrеstiop grоm apd раtоgepesis оf ipfestiosus desеsesеs, Сіliпісіl тісробіологу ревеве, 1939, Реве). In the patent of the Russian Federation N ° 2229307 describes the development of a vaccine based on the hybrid proteins E7 and Hp 70 M.tibersilosis. The authors describe an injectable dosage form with which it is possible to achieve regression of tumors induced by the virus in experimental models with transplantable tumors. The demonstrated vaccination efficacy in these models confirms the correct selection of the E7 protein as the molecular target against which immune responses are induced. However, with cervical dysplasias developing in women due to chronic papillomavirus infection of the cervix uteri, the induction of systemic immunity cannot guarantee the regression of malignancy processes, since the cervix has a certain isolation from systemic immunity in the regulation of local immunological reactions. In the development of anal warts, the E 7 protein also plays a major role in the processes of malignancy and the suppression of local immunological reactions. Therefore, mucosal application of the vaccine should lead to the neutralization of the E7 protein and to induce a regression of pathological processes. In addition, it is now becoming apparent that achieving stable protective immunity with recombinant vaccines is only possible with multiple immunizations. This creates significant organizational difficulties, as the patient needs to visit the clinic repeatedly for vaccination. In the case of vaginal vaccination, the patient can do this on his own. Another problem associated with the injectable form of vaccination is that with repeated intramuscular administration of a foreign protein is high the likelihood of anaphylactic shock, which is excluded with the 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 for pathologies that affect the mucous membranes, as in the case of papillomavirus infection.

As a dosage form for mucosal application of vaccines, suppositories or "sprays" can be considered, which allow the vaccine preparation to be applied to the mucosa in the form of lyophilized powder

SUMMARY OF THE INVENTION

The authors of the proposed invention on the basis of long-term studies of papillomatosis came to the conclusion that the optimal vaccine for the treatment of diseases associated with human papilloma viruses should meet the following requirements:

1. The route of administration should include stimulation of local adaptive and innate immunity, namely: in the case of PIIIM, the route of administration is vaginal; in case of colorectal cancer, the rectal route of administration. 2. Given the low immunogenicity of the E7 protein, the recombinant protein, which is the active substance of the vaccine preparation, should contain amino acid sequences of proteins that have an immunomodulatory effect on the formation of cytokines and improve the presentation of the E7 antigen.

3. The dosage form of the vaccine should contain ingredients that increase adhesion on the mucous membranes and the affinity of the protein components of the vaccine for antigen-presenting cells.

In order to solve this problem, in the present work, we developed two dosage forms of a vaccine for BOT immunotherapy - associated pathologies. So, for the treatment of diseases of the anogenital sphere, mucosal vaccines in the form of suppositories have been developed.

For the treatment of cervical cancer, suppositories have been developed that contain the E7 type 16 or type 18 fusion proteins as the main antigens, fused to M.tibercylsis Hsp70. A hybrid protein is a single polypeptide chain consisting of the first protein - HPV type E7, type 16 or 18. As already established, E7 oncoprotein is the main inducer of malignancy of epithelial cells infected with human papilloma viruses (Yip E.K. , Theole O HPV E6 and E7 ESPs are supported by the HPV-Associated Test Server, Sapser Res. Therap., 2005, 37, 6, 319-324) In addition to the oncogenic potential, the E7 protein has pronounced immune properties. It is thanks to the E7 protein that transformed cells manage to "escape" from the body's immune responses (Kapoda S., Faheu L.M. apd Kast WM, Meshapism used bu rillomaviruses so espare teh host immresie reserpse, Serepteregres, 2007, 7, 2007, 89). For these reasons, the E7 protein is an optimal target for immunotherapy. Another part of this protein is represented by the heat shock protein from M.tibercylosis, which has a high affinity for antigen-presenting cells and is a powerful inducer of cytokinrv synthesis (Prozero D. apd Srivastava R., Net Shosk Propeps - repertory commercially available immovable sapser. Moleceloteriptu, Apeltertel, Apeltertel, Apelter, Apolit, Antel. apd urtake ° F Hsp70 - rertide somrlehes, the Joirpal sell Yologu ° F, 2002, 158, 7, 1277-1285; R. Srivastava, Ipterastiop ° F Neat Shosk Rroteips with rertides apd aptigep rreseptipg sells: shareropipg ° F the ippate apd adartive immupe resropses, Appi Rev. Impossible, 2002, 20, 395-425; Lacmapp HJ, Strägewaus L., Väkarmam Apt Evapis G. Rásipgpts bürpulpreptspd app immptsi, siblip 1986, Bac. -27; DeI Giudiсi G. Hsp70: a carrier with built- ip adjuvaptisitu, Expremerptia, 199 '4, 50, 1061-1066).

In the case of colorectal cancer, it is advisable to use type 6 E7 protein as an antigen, which is more common than other types of HPV when the rectal mucosa is damaged.

Our experimental studies of the immunogenicity of hybrid proteins when applied to mucous membranes showed that, in isolation, these proteins are not able to induce pronounced protective immunity against E7 proteins. We made the assumption that the reason for the low immunogenicity of polypeptides is their inability to adsorb on the epithelium of the mucous membranes, as a result of which they can undergo rapid proteolytic degradation. To solve this problem, it was decided to use protein complexes with chitosan. During experimental verification of this assumption, it turned out that the complex of hybrid proteins with chitosan possessing pronounced mucoadhesive property and contributing to the active sorption of antigens on the cell surface, it effectively induces both the humoral and cellular components of immunity against E7 proteins. Based on this, chitosan in the ratio of protein: chitosan 1-1 to 1-10 is included in the composition of vaccines as an adhesive agent that promotes the penetration of antigens through the mucous membranes and a strong stimulator of nonspecific immune reactions. The optimal ratio of protein to chitosan in the dosage form was studied experimentally . It was found that the maximum effect in the induction of immunological reactions against antigenic determinants of proteins was observed when using a ratio in the range of 1-1 to 1-10. With a decrease or increase in the concentration of chitosan in relation to a fixed dosage of protein, a decrease in the efficiency of the induction of immunological reactions was observed.

The following pharmacologically acceptable additives were used for the manufacture of suppositories: Vitepsol, cocoa butter or type A solid fat as a lipophilic base, polyethylene glycols of various molecular weights as a hydrophilic base, nipagin, nipazole or aminoglycosides as antimicrobial agents, succinic acid and sodium benzoate - as stabilizers or preservatives, thiomersal and thiomersal as stabilizers and antimicrobial agents (patent RU 2224542 C2 A61 K39 / 116, 9/02, A 61P 13/00; government decree RF N ° 885 dated August 2, 1999; Patent RU 2150268 A61K9 / 02, A61K35 / 74, A61P15 / 02; “Testing and vaccination against hepatitis B”, “Medicine for All” journal, NsI ₅ 2001; Ratrizi A., Rizzoli L., Viepsezi C. et al. Sepitizatiop to thimerosal ip atoris childrep. Complict Dermatitis, 1999, 40 (2), 974-979).

The present invention also relates to methods of treating diseases associated with human papillomavirus, comprising topical application of a mucosal vaccine. Alternatively, a hybrid protein E7 type 16-Hsp70 or E7 type 18-Hsp70, in combination with chitosan in a ratio of 1: 0.1-10 and pharmacologically acceptable additives, is used to treat cervical dysplasia caused by human papillomavirus. The prepared suppository is administered intravaginally. Depending on the stages of the disease, suppositories are prescribed 2-4 times with an interval of a week with mild cervical dysplasia and 4-8 times with an interval of one week with dysplasia of the 2nd and 3rd degree. For the treatment of anal condyloma, an E7 type 16-Hsp70 or E7type 6-Hsp70 hybrid protein is used, in combination with chitosan in a ratio of 1: 0.1-10 and pharmacologically acceptable additives. The prepared suppository is administered rectally. Suppositories are prescribed for the night with a frequency of once a week. The maximum clinical effect is achieved with the appointment of 4-8 suppositories.

Regional options for vaginal suppositories for the treatment of cervical cancer

Given the peculiarities of the geographical distribution and to achieve maximum efficiency, regional options for the treatment of PIIIM should contain the following combinations: recombinant E7 Hsp70 fusion proteins in the vaginal suppository: 1. Global pentavalent vaccine based on E716, E718, E758, E731, E745 fused to Hsp70;

2. Chinese tetravalent vaccine based on E716, E718, E758, E752, fused with Hsp70; 3. Latin American trivalent vaccine based on E716, E718, E731 fused to Hsp70;

4.African trivalent vaccine based on E716, E718, E745 fused to Hsp70; 5. For the rest of the world (Europe, North America, Australia) a bivalent variant based on E716, E718, merged with Hsp70.

Thus, the proposed invention can significantly increase the effectiveness of the treatment of diseases associated with human papillomavirus. The obtained effect is achieved due to the preferential 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 that ensures their high affinity for epithelial cells of mucous membranes infected with HPV, the active recognition of antigens by the immune system and, as a result, the induction of local immunity against E7 proteins, which play a key role in the development of pathological processes. Using the proposed mucosal vaccine can significantly reduce the cost of treatment compared with the known methods of treating cervical cancer and PKK. The proposed vaccine is intended for local use, which eliminates the unwanted and extremely life-threatening side effects of injectable drugs, for example, anaphylactic shock. The proposed treatment method significantly simplifies the treatment process - the patient can undergo treatment outside the clinic, self-administering the drug.

LIST OF DRAWINGS

Figure 1. The dynamics of the serum IgG immune response in different methods of immunization of RSL mice E76-Hsp70,

Figure 2. Values of mucosal normalized IgA antibody response during intranasal and subcutaneous immunization of RSL mice E76-Hsp70,

Figure 3. The level of secretion of IFN-gamma (l) and IL-4 (2) by lymphocytes from inguinal lymph nodes (A) and spleen (B), stimulated in culture by a mixture of recombinant proteins E716 and E718.

Figure 4. The level of secretion of IFN-gamma (l) and IL-4 (2) lymphocytes from inguinal lymph nodes, stimulated in culture by a mixture of recombinant proteins E716 and E76.

MODES FOR CARRYING OUT THE INVENTION Example N ° l

Preparation of recombinant protein vaccines for mucosal administration with chitosan

Recombinant fusion proteins E711-Hsp70, E716-Hsp70, E718-Hsp70, E76-Hsp70 (RSL) were purified from inclusion bodies by solubilization in 8M urea, followed by metal chelate and ion exchange chromatography to a homogeneous state. The degree of protein purity was monitored by electrophoresis on a 10% polyacrylamide gel under denaturing conditions, the protein concentration was determined by the Lowry method using bovine serum albumin as a standard. Before By preparing the vaccine preparation, the recombinant fusion protein (RSL) was dialyzed to remove preservatives and stabilizers against phosphate-buffered saline (PBS, pH7.2), diluted or concentrated to the required concentration, and sterilized filtration was performed. After these procedures, solutions of recombinant proteins were mixed with a solution of chitosan in various proportions and used to make suppositories. Example M2 Immunization of mice and sampling

Used adult female mice (6-8 weeks old) of the Balb / s line (Nursery RAMS “Stolbovaya”) in the amount of 5 pieces per group. Immunization was performed three times at intervals of 4 weeks (0-28-56 days). During intranasal immunization, mice were injected with 20 μl of the RSL drug with chitosan (10 μl into each nostril) using a micropipette under halotan anesthesia. For vaginal and rectal immunization using a micropipette, 10 μg of protein in complex with chitosan was introduced in a volume of 20 μl. Subcutaneous immunization was carried out at the withers of mice by injection of 200 μl of a suspension of RSL with AlhudroGeC. For subcutaneous (s.c) vaccination, the RSL preparations were adsorbed on 0.5% (w / w) aluminum hydroxide gel (Alhudroel., Bepostag), overnight. In both cases, the dose of RSL for each immunization was 10 μg of protein per mouse. Control groups of mice were immunized in a similar way using 0.5% (w / v) chitosan solution and 0.5% (w / w) Alhudrogel solution for the intranasal and subcutaneous route of administration, respectively. Blood samples for the determination of specific IgG were taken from the retroorbital vein of each mouse using a Pasteur pipette in a volume of 0.1 ml 10 days after the first, second and third immunization. After clot retraction, the serum was separated by low-speed centrifugation and stored at a temperature of -20 ⁰ C until ELISA.

Nasopharyngeal swabs for the determination of specific IgA were obtained 14 days after the third immunization. For this purpose, the mice were sacrificed by cervical dislocation, decapitated, and the nasopharynx was washed from the side of the trachea with 0.5 ml PBS. Nasopharyngeal swabs were stored at -20 ⁰ C until ELISA. To study the T-cell immune response to recombinant proteins and peptide epitopes, mice were sacrificed by cervical dislocation 14 days after the third immunization and the lungs, spleen and mediastinal lymph nodes were aseptically removed. Lymphocyte cultures were obtained by careful mechanical grinding of tissues, lysis of red blood cells, and three-time washing of cells by low-speed centrifugation in RPMI 1640 medium with the addition of 10% fetal serum. 20mM L-Glutamine and antibiotics. Example N ° 3

Comparison of specific IgG and IgA antibody responses in intranasal and subcutaneous immunization with RSL E76-Hsp70

Mice were immunized with the RSL E76-Hsp70 intranasally and subcutaneously in accordance with the protocol and dosage described in Example ZH. Geometric mean titers (CHT) of IgG and IgA antibodies specific for the recombinant E76 protein in serum and nasopharyngeal swabs obtained according to the procedure of Example No. 2 were determined by enzyme-linked immunosorbent assay (ELISA). To determine serum IgG on a 96-well plate, recombinant E76 protein was immobilized from a solution with a concentration of 5 μg / ml in PBS overnight, titrated mouse sera by three-fold serial dilutions, and peroxidase conjugated goat anti-mouse IgG antibodies were introduced. TMB was used as a substrate; optical density (OD) was recorded after 15 min at a wavelength of 450 nm. The titer of specific IgG antibodies for each serum was determined by the endpoint, which corresponded to a dilution of serum with OD> 0.2. The results are shown in figure 1. Figure 1 shows the dynamics of the serum IgG immune response in different ways of immunizing RSL mice E76-Hsp70: Group 1 - E76-Hsp70 + chitosan, intranasally; Group 2 (control) - chitosan, intranasally; Group 3 (control) - aluminum hydroxide, subcutaneously; Group 4 - E76-Hsp70 + aluminum hydroxide, subcutaneous.

The ordinate shows the logarithm of the geometric mean titer of IgG antibodies.

To determine the titer of IgA in nasopharyngeal swabs, an ELISA similar to that described above was used, however, instead of the anti-IgG (H) conjugate, a peroxidase conjugate of goat antibodies against mouse IgA (H) was used. The titration endpoint for each wash was determined as the highest dilution of the sample at which OD> 0.1. To minimize variations associated with the procedure receiving nasopharyngeal swabs, the total IgA content was determined in each sample using a commercial ELISA kit. Ultimately, the magnitude of the mucosal IgA immune response was expressed as the ratio of the titer of E76-specific IgA antibodies to the total IgA content (expressed in micrograms) in the same sample. The results are shown in figure 2. Figure 2 shows the magnitude of the mucosal normalized IgA antibody response during intranasal and subcutaneous immunization of RS7 E711-Hsp70 mice. Group 1- E76-Hsp70 + chitosan, intranasally; Group 2 (control) - chitosan, intranasally; Group 3 (control) - aluminum hydroxide, subcutaneously; Group 4 - E76-Hsp70 + aluminum hydroxide, subcutaneous. Example Ns4 Comparison of a specific T-cell response during intravaginal and subcutaneous immunization with a mixture of RSH E716-Hsp70 and E718-Hsp70

Used adult female mice (age 8-10 weeks) line Balb / s in the amount of 5 pieces per group. Immunization was carried out 3 times with an interval of 10 days according to the scheme: 0-10-20 days. For intravaginal immunization (i.vag.), The mice were injected with 20 μg of a mixture of RSL 10 μg of E716-Hsp70 and E718-Hsp70, respectively, in 16 μl of a 0.5% (w / v) chitosan solution (Chitosap, Celechemia AG, Switzerland) using a micropipette into the vagina. Subcutaneous immunization (s.s.) was carried out at the withers of mice by injection of 200 μl of a suspension of a mixture of RSL (20 μg) with 0.5% (weight / weight) aluminum hydroxide gel (Alhudrogel, Vreptag Biocestor, Denmark). Control groups of mice were immunized similarly, using a 0.5% (w / v) chitosan solution and a 0.5% (w / w) Alhdrogel solution for the intravaginal and subcutaneous route of administration, respectively. 14 days after the third immunization, the mice were sacrificed and the spleen and inguinal lymph nodes were aseptically removed. Lymphocyte cultures were obtained by careful mechanical grinding of tissues, erythrocyte lysis and three-time washing of cells by low-speed centrifugation in RPMI 1640 medium with the addition of 10% fetal serum, 20 mM L-glutamine and antibiotics. Lymphocyte cultures obtained from the spleen and inguinal lymph nodes of mice immunized with a mixture of RSH E716-Hsp70 and E718-Hsp70 and control groups were examined for 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 BIOSECIEPS, Oxford, UK). The data are shown in Figure 3. Figure 3 shows the level of secretion of IFN-gamma (l) and IL-4 (2) by lymphocytes from inguinal lymph nodes (A) and spleen (B), stimulated in culture with a mixture of recombinant proteins E716 and E718. Mice were immunized:

Gray columns - E716-HSP70 + E718-HSP70 + chitosan, intravaginally; Black columns - chitosan, intravaginally; White columns - aluminum hydroxide, subcutaneously;

Shaded columns - E716-HSP70 + E718-HSP-70 + aluminum hydroxide, subcutaneous. The ordinate shows the number of cytokine-secreting lymphocytes per 1 million cells. Example Ns5

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

Used adult female mice (age 8-10 weeks) line Balb / s in the amount of 5 pieces per group. Immunization was carried out 3 times with an interval of 10 days according to the scheme: 0-10-20 days. For rectal immunization (iR), the mice were injected with 20 μg of a mixture of RSL 10 μg of E716-Hsp70 and E76-Hsp70, respectively, in 16 μl of a 0.5% (weight / volume) solution of chitosan (Chitosap, Celechem AG, Switzerland) using a micropipette in the rectum. Subcutaneous immunization (s.s.) was carried out at the withers of mice by injection of 200 μl of a suspension of a mixture of RSL (20 μg) with 0.5% (w / w) aluminum hydroxide gel (Alhudrogel, Vreptag Biocestor, Denmark). The control groups of mice were immunized in a similar way using 0.5% (w / v) chitosan solution and 0.5% (w / w) Alhudrogel solution for rectal and subcutaneous administration, respectively.

14 days after the third immunization, mice were sacrificed and inguinal lymph nodes were aseptically removed. Lymphocyte cultures were obtained by careful mechanical grinding of tissues, erythrocyte lysis and three-time washing of cells by low-speed centrifugation in RPMI 1640 medium with the addition of 10% fetal serum, 20 mM L-glutamine and antibiotics. Lymphocyte cultures obtained from inguinal lymph nodes of mice immunized with a mixture of RSH E716-Hsp70 and E76-Hsp70 and control groups were 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 BIOSECIPSES, Oxford, UK). The data are shown in figure 4.

Figure 4 reflects the level of secretion of IFN-gamma (l) and IL-4 (2) by lymphocytes from inguinal lymph nodes stimulated in culture with a mixture of recombinant proteins E716 and E76. Mice were immunized: Gray columns - E716-HSP70 + E76-HSP70 + chitosan, rectally; Black columns - chitosan, rectally; White columns - aluminum hydroxide, subcutaneously; Shaded columns - E716-HSP70 + E76-HSP-70 + aluminum hydroxide, subcutaneous. The ordinate shows the number of cytokine-secreting lymphocytes per 1 million cells. MB example

The adjuvant effect of Hsp70 M.tibercylosis on cells of the immune system in vitro Immature dendritic cells (DCs) were obtained by perfusion of the hollow bones of the hind limbs of CBA mice using a sterile isotonic solution according to standard methods. Cells were washed three times and cultured in matrices supplemented with thermally inactivated fetal serum KPC in RPMIl 640 medium (150,000 cells / ml) for 48 hours without stimulation, as well as in the presence of Hsp70 at a concentration of 25 μg / ml. Centrifuged culture fluids were analyzed for product mouse cytokines using enzyme immunoassays from R&D Systems (USA). Expression of DC differentiation markers was studied by flow cytometry using monoclonal antibodies manufactured by R&D Systems (USA). The results of determining the production of cytokines (arbitrary units) and differentiation markers of DC (% of cells) are shown in table 1: Table 1

Приведенные данные свидетельствуют о том, что Hsp70 индуцирует в ДК выработку провоспалительных цитокинов: IL-I bеtа, IL-6, IL- 12 и ТNF-аlрhа, что указывает на выраженный адъювантный эффект Hsp70. Исследование изменений фенотипа ДК при стимуляции Hsp70 показало, что доля клеток, экспрессирующих маркеры зрелых ДК (CD38, CD40, CD83, молекулы MHC2), возрастает. Из всего спектра маркеров зрелых ДК не повышается экспрессия костимуляторных молекул CD86 и CDlIc. Остается неясным также изменение уровня TLR2 и TLR4 , более типичных для незрелых ДК. Пример N°7

These data indicate that Hsp70 induces the production of pro-inflammatory cytokines in the DC: IL-I beta, IL-6, IL-12, and TNF-alpha, which indicates a pronounced adjuvant effect of Hsp70. The study of changes in the phenotype of DC during Hsp70 stimulation showed that the proportion of cells expressing markers of mature DC (CD38, CD40, CD83, MHC2 molecules) increases. Of the whole spectrum of markers of mature DCs does not increase the expression of co-stimulatory molecules CD86 and CDlIc. The change in the level of TLR2 and TLR4, which are more typical for immature DCs, remains unclear. Example N ° 7

The manufacture of multicomponent vaginal suppositories for the treatment and prevention of human papillomavirus

1) A solution of recombinant proteins, E716-Hsp70, E718-Hsp70, 2) Chitosan company TAI ZHOU CANDORLY BATCH W 06020301 degree of deacetylation 86%

3) Rawlutheglusol 1500 company APPLI CHEM CAS-NO: 25322-68-3

4) Type A solid fat

5) Vitepsol HW 6) TRIS of the company MP Biomedisals Ips. cat. JChb 195605. Composition per 1 suppository:

A solution of recombinant proteins E716-Hsp70, E718-Hsp70 (c = 1 mg / ml) in O ₅ OlM TRIS 0.1-0.5 ml

Chitosan 0.00165 g-O. OZg Base (Vitepsol / Type A solid fat) up to 3.0 g

The method of obtaining suppositories Suppositories were prepared by pouring. Calculation of the components was carried out per 100 suppositories of 3.0 g each. A solution of recombinant proteins E7 of types 16 and 18 (c = 1 mg / ml) in 0.0 IM TRIS 50 ml

Chitosan 0.165 g

PEG 1500 45.0 g

Cocoa Butter 255.0 g To a solution of recombinant proteins E7 of types 16 and 18 (c = 1 mg / ml) in 50 ml of O ₅ OlM TRIS, 0.165 g of chitosan is added and mixed for 5-10 minutes, then filtered through a paper filter (blue band filter). Pre-molten PEO 1500 is introduced into the solution obtained after filtration and mixed for 5 minutes. The pre-molten hydrophobic base (vitepsol, type A fat) is gradually added to the mixture and mixed for 30 minutes. The resulting suspension is poured into pre-cooled suppository forms and placed in the refrigerator at -4 ⁰ C. Example M 8

Production of multicomponent rectal suppositories for the treatment and prevention of human papillomavirus. Components: 1) Solution of recombinant proteins E7-16-Hsp70,, E7-6-Hsp70

2) Chitosan from TAI ZHOU CANDORLY ATCH JVO 06020301 Deacetylation degree 86%

3) Rawlutheglusol 1500 company APPLI CHEM CAS-NO: 25322-68-3

4) Type A solid fat 5) Witepsol HW

6) Twin-80 to 5%

7) TRIS of the company MP Biomedisals Ips. cat. JNb 195605. Composition per 1 suppository:

A solution of recombinant proteins E716-Hsp70, E76-Hsp70 (c = 1 mg / ml) in 0.01 M TMS 0.1-0.5 ml

Chitosan 0.00165 g-O. OZg.

Base (vitepsol / type A solid fat) up to 3.0 g

The method of obtaining suppositories Suppositories were prepared by pouring. The components were calculated per 100 suppositories of 3.0 g each. Solution of recombinant proteins of type E7 16 and 18 (c = 1 mg / ml) in O ₅ OlM TMS 50 ml Chitosan, 165 g

PEG 1500 45.0 g

Cocoa Butter 255.0 g

To a solution of recombinant proteins E7 of types 16 and 18 (c = 1 mg / ml) in 50 ml of 0.0 IM TRIS, 0.165 g of chitosan is added and mixed for 5-10 minutes, then filtered through a paper filter (filter with a blue band). Pre-molten PEO 1500 is introduced into the solution obtained after filtration and mixed for 5 minutes. The pre-molten hydrophobic base (vitepsol, type A fat) is gradually added to the mixture and mixed for 30 minutes. The resulting suspension is poured into pre-cooled suppository forms and placed in the refrigerator at -4 ⁰ C. Example N ° 9

Study of the clinical efficacy of the developed mucosal vaccine in patients with cervical dysplasia caused by human papilloma viruses type 16 and 18 Three groups of patients with a diagnosis of cervical dysplasia (CIN I) and (CIN II) were selected for clinical studies. Each group consisted of 10 people. The diagnosis of cervical dysplasia was established on the basis of a clinical examination and laboratory analysis of cervical epithelium samples for carriage of HPV types 16 and 18. Each patient was prescribed a course of treatment with vaginal suppositories of various compositions 1 time per week for 4 weeks. In some cases, the course of treatment was carried out for 8-12 weeks, while maintaining the regimen of prescribing the drug for 1 suppository at night once a week. After completing the course of treatment, patients were examined using colposcopy, morphological studies of cervical cells and laboratory analysis of PCR for the presence of HPV DNA of types 16 and 18 were performed. table 2

MlO example

A study of the clinical effectiveness of the developed mucosal vaccine in patients with a diagnosis of rectal condyloma. For the clinical study, 20 patients of the proctology department were diagnosed with recurrent rectal condylomas. Patients complained of pain during the act of deformation, traces of blood in the stool. A visual examination of the rectal mucosa revealed warts 0.2-0.5 cm in size, the mucous membrane is inflamed. Cracks in the anus were found in some patients.

Patients were prescribed monotherapy with rectal suppositories containing the described ingredients, 1 suppository per night, once a week for 4 weeks. In some cases, the course of treatment was carried out for 8-12 weeks, while maintaining the regimen of prescribing the drug 1 candle at night once a week. After the course of treatment, 16 patients experienced a complete clinical recovery, which was accompanied by the disappearance of genital warts and all signs of the inflammatory process. In 4 patients, after treatment, a decrease in the number of condylomas was observed with the absence of a sign of inflammation. The proposed mucosal vaccine in the form of rectal suppositories is very effective in these pathologies.

Claims

Claim 1. A mucosal vaccine against diseases associated with the human papillomavirus, which contains an effective amount of a hybrid protein consisting of the oncoprotein E7 of the human papillomavirus fused to the heat shock protein of mycobacteria Hsp70, chitosan in proportion to the hybrid protein 1: 0.1-10 and pharmacologically acceptable additives for the manufacture of suppositories. 2. The mucosal vaccine according to claim 1, characterized in that for the treatment of cervical dysplasia caused by human papillomavirus, a hybrid protein E7 type 16-Hsp70 or E7 type 18-Hsρ70 is used. 3. The mucosal vaccine according to claim 1, characterized in that the hybrid protein E7 type 16-Hsp70 or E7 type 6-Hsp70 is used to treat anal condyloma. 4. A method of treating a disease associated with human papillomavirus, comprising topical application of the mucosal vaccine according to any one of claims 1-3 in the form of a suppository. 5. A method for the treatment of cervical dysplasia caused by human papilloma virus, comprising intravaginal administration of an effective amount of a mucosal vaccine according to claim 2. 6. A method of treating anal condyloma, comprising rectal administration of an effective amount of a mucosal vaccine according to claim 3. SUBSTITUTE SHEET (RULE 26)