Methods for producing preventive and therapeutic DNA immunological compositions against HPV and cancers associated with the virus, hybrid proteins, expression vectors, immunological compositions, and uses thereof

Abstract

The present invention relates to preventive and therapeutic vaccines against HPV and cancers associated with the virus, which are intended for use in people seeking prevention or those who have developed cancer and have been infected with HPV. The present invention also relates to DNA expression vectors that are capable of effectively producing the capsid protein L2 of the HPV16 virus, and also the viral oncoprotein E6 associated with human papillomavirus tumors. In particular, the present invention relates to recombinant fusion or hybrid proteins obtained by gene cloning into expression vectors, which are produced by gene translation of a fusion gene formed by combining the nucleic acid regulatory sequences of one or more genes with the protein coding sequences of one or more genes, and which are used to generate two different types of responses: a long-lasting humoral immune response that can stimulate the production of specific anti-L2 and anti-E6 antibodies against HPV (preventive effect), and the activation of a cellular immune response to fight tumor cells and induce the expression of the cytokine TNF (tumor necrosis factor) (therapeutic effect).

Description

Field of the Invention

[0001] The present invention relates to prophylactic and therapeutic vaccines against HPV and cancers associated with the virus, intended for use by anyone interested in preventing the disease or those already infected with HPV who have developed cancer. The present invention also relates to DNA expression vectors capable of efficiently producing the HPV16 capsid protein L2, as well as the viral oncoprotein E6, which is associated with the development of tumors caused by human papillomavirus. In particular, the present invention relates to recombinant fusion or hybrid proteins obtained by gene cloning into expression vectors, produced by gene translation of a fusion gene formed by combining nucleic acid regulatory sequences of one or more genes with protein coding sequences of one or more genes, for generating two distinct types of responses: a long-lasting humoral immune response capable of stimulating the production of anti-L2 and anti-E6 antibodies specific to HPV (prophylactic effect), and the activation of a cellular immune response to combat established tumor cells and induce expression of the cytokine TNF (tumor necrosis factor) (therapeutic effect). Background of the Invention

[0003] Cervical cancer is the third most common type of cancer in women worldwide and the second leading cause of cancer-related death in women. The two most prevalent virus types in the population are HPV16 and HPV18, which are detected in the most aggressive cancers. These cancers are primarily caused by persistent infection with high-risk HPV (human papillomavirus) (GUAN P, HOWELL-JONESR, L]N et al., Human papillomavirus types in 115,789 HPV-positive women: a meta-analysis from cervical infection to cancer. Int J. Vol. 131, pp. 2349-2359, 2012).

[0004] Despite the availability of preventive vaccines, HPV infection remains extremely common worldwide. It is estimated that cervical cancer currently affects more than 1.4 million women worldwide and causes the death of more than 300,000 women each year, primarily in developing countries (WHO. Strategic Advisory Group of Experts (SAGE) on immunization WHO. World Health Organization, 2017; DE S,DIAZ M, et al., Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: ameta-analysis. Lancet InfectDis, Vol. 7, pp. 453-459, 2007).

[0005] Among the factors responsible for this high mortality rate, the high cost of the vaccine and the refrigeration requirements are the most limiting factors for implementing large-scale preventive vaccination in developing countries, where approximately 87% of cervical cancer deaths occur (GLOBOCANE. Estimated Incidence, mortality and prevalence worldwide in 2012, v 1.0 [internet, 2012]. In private clinics, the price of the bivalent vaccine against HPV is approximately R$ 200 per dose, while the price of the quadrivalent vaccine is approximately R$ 300 per dose, with 2 to 3 booster doses required every 5 to 8 years (Cianciarullo AM. Profilaxia contra opapilomavirus humano. Rev. Sodebras, Vol. 9, No. 100, pp. 8-15, 2014. Available at: http: / / www.sodebras . com.br / edicoes / N100.pdf ).

[0006] Today, the main forms of prevention for cervical cancer are regular cytology, Pap smears, and preventive vaccinations for prepubertal girls and boys. In Brazil, a quadrivalent preventive vaccine has recently been made available free of charge by the Ministry of Health through the SUS (Unified Health System) for boys and girls aged 9 to 14 years, and for men and women aged 9 to 26 years living with HIV or AIDS, organ and bone marrow transplant recipients, and those undergoing treatment for cancer. (Cianciarullo AM. Profilaxia contra o papilomavirus humano. Rev. Sodebras, Vol. 9, No. 100, pp. 8-15, 2014. Available at: http: / / www.sodebras.com.br / edicoes / N100.pdf INCA. 2017. Available at: http: / / portalsaude.saude.gov.br ).

[0007] The vaccine can also be used by the elderly, but it is only available in private vaccination clinics. It is indicated for girls and women aged 9 to 45 years if it is a quadrivalent vaccine; or girls and women of any age older than 9 years if it is a bivalent vaccine; boys and men aged 9 to 26 years use a quadrivalent vaccine. In addition, the vaccine can be used by people who are in treatment or already have HPV infection because it can protect against other types of HPV viruses and prevent the formation of new genital warts and the risk of cancer (see Cianciarullo AM. Profilaxia contra opapilomavirus humano. Sodebras, Vol. 9, No. 100, pp. 8-15, 2014. Available at: http: / / www.sodebras.com.br / edicoes / N100.pdf ; INCA. 2017. Available at: http: / / portalsaude.saude.gov.br).

[0008] However, such actions are aimed at preventing HPV infection, as there are no specific treatments for established infections and lesions. Among the proteins expressed by HPV, we highlight L2 and E6 as targets for this work. The L2 protein is present in the viral capsid and is conserved between different HPV types, while E6 is an oncoprotein that can induce malignant transformation of infected cells. An intact viral genome is required for malignancy establishment (Herrero R., González P, Markowitz LE. Present status of human papillomavirus vaccine development and implementation. Lancet Oncol, vol. 16, pp. e206-216, 2015; Wang D, Li Z, Xiao J et al. Identification of broad-genotype HPV L2 neutralization site for Pan-HPVvaccine development by a cross-neutralizing antibody. PLOS One, vol. 10, pp. e0123944, 2015; Vande Pol SB, Klingelhutza J. Papillomavirus E6 oncoproteins. Virology, vol. 445, pp. 115-137, 2013; Kims, Chungh, Kongh et al. Identification of novel immunogenic human papillomavirus type 16E7 specific epitopes restricted to HLA-A*b33;03 for cervical cancer immunotherapy. Yonsei Med J, Vol. 58, pp. 43-50, 2017).

[0009] Due to simplicity and low production costs, DNA vaccines have advantages over traditional vaccines because they are heat stable and do not require refrigeration, allow for repeated dosing due to the absence of immune responses to the vector, have the potential to elicit both humoral and cellular immunity, and are highly tolerable in humans [Hancock G, Hellner K, Darrell L. Therapeutic HPV vaccines. Best Practice & Res Clin Obstet Gynecol, 2017. Available at: https: / / doi.org / 10.1016 / j.bpobgyn.2017.09.008 ).

[0010] In 2006, the first preventive vaccine against HPV was approved and released by the FDA (Food and Drug Administration) for sale in the United States.

[0011] Currently, the three vaccines against HPV available on the market and approved by the FDA and ANVISA (National Health Surveillance Agency) are only prophylactic. Both are based on the immunogenicity conferred by the L1 protein when structured in VLPs (virus-like particles).

[0012] The first vaccine was licensed in 2006 and is quadrivalent and is called Produced by MerckSharp Dohme, in which the L1 proteins of HPV 6, 11, 16 and 18 are expressed in yeast (Saccharomyces cerevisae) cells.

[0013] The second vaccine was licensed in 2008 and is bivalent and called Produced by GlaxoSmith Kline, it consists of the L1 proteins of HPV 16 and 18, which constitute the vaccine and are expressed in culture of insect cells of the SF9 line.

[0014] A third vaccine was licensed in 2014; it is nine-valent and is called From the same manufacturer of the vaccine of the same name, but formulated with L1 particles from 9 different types of HPV: 6, 11, 16, 18, 31, 33, 45, 52, and 58 (CDC – Centers for Disease Control and Prevention, available at: http: / / vwvw.cdc.gov / vaccines / hep / vis / vis-statements / hpv-gardasil-9.html ).

[0015] Currently available vaccines are highly effective at inducing the production of specific antibodies, i.e., they are only prophylactic in nature and only directed against the type of virus contained in the vaccine formulation. Some studies have shown that such antibodies can induce a small amount of cross-protection against infection with other types of HPV, but not against the wide range of different types of HPV that cause genital warts and growths.

[0016] A Master's thesis submitted to the Interdepartmental Postgraduate Program at Biotechnology USP / Instituto Butantan / IPT (published in 2009 under the name Bruna Felício MM Portchia; entitled "Development of a therapeutic vaccine against tumors caused by human papillomavirus type 16 (DESENVOLVIMENTO DE UMA VACINA A vaccine strategy against HPV-16-induced tumors using a recombinant form of the E7 protein genetically fused to glycoprotein D (gD) of herpes virus type 1 (HSV-1) is described. The gDE7 protein, produced in a bacterial system, was tested as a vaccine in mice, where it conferred 80% protection against tumor growth. When tested therapeutically, soluble gDE7 was able to protect 30% of the animals.

[0017] The aforementioned master's thesis and the present application both use fusion proteins. However, the fusion proteins described in the aforementioned master's thesis are completely different from the fusion proteins disclosed in the present application.

[0018] University of Sao Paulo PAULO-USP and the São Paulo State Research Support Foundation ( DE AMPARO PESQUISA DO ESTADO DE FAPESP) filed on August 18, 2010 and published on May 2, 2012, and entitled “Hybrid proteins, recombinant nucleic acid sequences, vectors / plasmids, pharmaceutical and / or veterinary preparations and their use in controlling tumors and / or infectious or degenerative diseases induced by human papillomaviruses” (PROTEíNA HíBRIDA, DE RECOMBINANTE,VETORES / PLASMíDEOS, E / OU E SEUS USOS NO CONTROLE DE TUMORES INDUZIDOS PELO VíRUS DO PAPILOMA HUMANO E / OU Patent application PI 10037497 entitled "INFECCIOSAS OU DEGENERATIVAS" describes a non-natural hybrid protein formed by genetic fusion of viral oncoprotein E7 of human papillomavirus type 16 (HPV-16) with a modified form of glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1), having deletions in its signal peptide and in its C-terminal region, including a sequence involved in anchoring to the cytoplasmic membrane (corresponding to amino acid sequence 320 to 344); a recombinant nucleic acid sequence encoding said recombinant protein (SEQ ID NO: 1 and SEQ ID NO: 2). NO:2); a vector / plasmid comprising the recombinant sequence; and a pharmaceutical and / or veterinary preparation comprising the hybrid protein and / or the recombinant sequence encoding the protein, preferably in the form of a vaccine. In addition, the patent application mentioned is directed to the use of the genetic construct as a vaccine adjuvant for other antigens and / or as an active ingredient in the preparation of pharmaceutical and / or veterinary preparations designated for the control of tumors induced by human papillomavirus and herpesvirus infections.

[0019] Patent application PI 10037497 and this patent both disclose vaccines against HPV. However, the genetic construct described in patent application PI 10037497 is completely different from the fusion protein disclosed by this application.

[0020] Patent application filed on July 29, 1996 in the name of CANTAB PHARMACEUTICALS RESEARCH LIMITED and published on July 20, 1999 entitled "POLYPEPTIDE OR POLYPEPTIDE COMPOSITION, IMMUNOGENIC COMPOSITION SUITABLE FOR ADMINISTRATION BY INJECTION, AND USE OF POLYPEPTIDE OR IMMUNOGENIC COMPOSITION" DE POLIPEPTíDEO, ADEQUADA PARA POR E,USO DE POLIPEPTíDEO OU DE Patent application PI 9612675-2 entitled "(I)" describes fusion polypeptides and polypeptide aggregates comprising antigens derived from papillomaviruses, as well as compositions and uses thereof, for example in generating, for example, HPV-specific immune responses together with adjuvants for vaccine and immunogenic purposes. The polypeptides can be purified to result in aggregates that can pass through sterilizing filters when in solution or dispersion, and to result in amorphous aggregates. An example of such a polypeptide is a fusion protein of the L2 and E7 proteins of human papillomavirus.

[0021] In addition to the fusion process and use of L2, patent application PI 9612675-2 also describes a completely different treatment proposal from the present application. Patent application PI 9612675-2 developed an immunotherapy using L2 and E7 of HPV-6 (non-oncogenic) to combat genital warts, while the present application provides L2 and E6 of HPV-16 (oncogenic) for the prevention and treatment of cervical cancer and other cancers associated with oncogenic HPV.

[0022] International patent application WO 2017 / 211886, filed on December 14, 2017 in the name of DEUTSCHES KREBSFORSCHUNGSZENTRUM (DKFZ) and entitled "IMPROVEMENT OF HPV L2 PEPTIDE IMMUNOGENICITY," relates to immunogenic polypeptides comprising a plurality of human papillomavirus (HPV) L2 N-terminal peptides corresponding to amino acids 20 to 50 of the L2 polypeptide of HPV16, wherein the HPV L2 N-terminal peptides are L2-N-terminal peptides from at least two different HPV genotypes. The international patent application WO 2017 / 211886 also relates to the immunogenic polypeptides for use in medicine and for use in vaccination against HPV infection. Furthermore, the international patent application WO 2017 / 211886 relates to polynucleotides encoding the immunogenic polypeptides and host cells comprising the same.

[0023] As can be seen, both international patent application WO 2017 / 211886 and the search target disclose vaccines against HPV. However, the genetic construct described in international patent application WO 2017 / 211886 is completely different from the fusion protein disclosed by the present application.

[0024] TAKE DEUTSCHES KREBSFORSCHUNGSZENTRUM STIFTUNG DES RECHTS and IPK,INSTITUT International patent application WO 2002 / 38769, filed on September 19, 2001 under the name of PFLANZENGENETIK UND KULTURPFLANZENFORSCHUNG and published on May 16, 2002, and entitled "DNA SEQUENCES, WHICH CODE FOR OPTIMISED EUKARYOTIC HPV16-L1 AND HPV 16-L2", relates to a DNA sequence optimized for codon usage, encoding an HPV 16-L1 capsid protein or an HPV 16-L2 capsid protein. The DNA sequence is contained in Figure 5 、 6 or 7 or a fragment or variant thereof, and allows for simple recombinant production of HPV 16-L1 or HPV 16-L2 capsid protein or a fragment thereof in high yield without the use of viral vectors. The capsid protein is preferably used for the production of vaccines.

[0025] International Patent Application WO 2002 / 38769 and the present application both disclose vaccines against HPV. However, the genetic construct described in International Patent Application WO 2002 / 38769 discloses either an HPV 16-L1 capsid protein or an HPV 16-L2 capsid protein, whereas the present application discloses an L2 / E6 fusion protein, which is completely different from the construct disclosed in International Patent Application WO 2002 / 38769.

[0026] As can be seen, none of the prior art documents describe or even suggest a therapeutic vaccine for human use against HPV and cancers associated with the virus. The prior art treatments used for lesions and HPV-positive cancers are not specific and depend on the extent of the lesion. They generally involve treatment with chemicals with cytotoxic effects, chemical / physical / electrical cautery, surgery, radiotherapy, chemotherapy, or a combination of treatments that are highly invasive, aggressive, and often disabling. SUMMARY OF THE INVENTION

[0028] To address the above-mentioned issues, the present invention provides significant advantages over the use of the hybrid recombinant protein L2 / E6, thereby inducing the production of specific cytokines such as TNF and specific antibodies with anti-L2 and anti-E6 effects, thereby effectively combating HPV-transformed cells (cancerous or tumorous) expressing the E6 protein (therapeutic effect) without interfering with the body's healthy cells (tissue-specific tropism), as well as new opportunistic viral infections caused by HPV (broad-spectrum preventive effect). Therefore, it does not produce side effects, thereby enabling improved performance and providing a more favorable cost / benefit ratio.

[0029] The present invention relates to the development of prophylactic and therapeutic vaccines against HPV and cancers associated with the virus, which are intended for HPV-infected humans who have developed cancer. The present invention relates to DNA expression vectors that are capable of efficiently producing the capsid protein L2 of the HPV16 virus and the tumor-associated viral oncoprotein E6 of the human papillomavirus.

[0030] In particular, the present invention relates to recombinant fusion or hybrid proteins obtained by gene cloning into expression vectors, which are produced by gene translation of fusion genes formed by combining nucleic acid regulatory sequences of one or more genes with protein coding sequences of one or more genes, and are used to generate an immune response against HPV.

[0031] In particular, the present invention provides fusions of the amino acid sequences of HPV16 L2 and E6, which contain only immunogenic epitopes (selected peptides) rather than the entire gene, a fact that disables the malignant cell transformation activity (carcinogenesis) of the oncoprotein, thereby conferring safety to L2 / E6 fusions. In addition, these fusions maintain or increase the immunogenic efficacy of L2 and E6. Considering that the production of more complex recombinant proteins should be carried out in eukaryotic expression systems (mammalian cells), as is the case herein, to overcome the disadvantages of prokaryotic systems (bacteria) (which means that certain post-translational modifications (e.g., glycosylation) that are necessary for the functional efficiency of the expressed heterologous protein are not carried out), the biological activity may also be different from that of the native protein; bacteria have a high endotoxin content; lack a secretion mechanism; and the formation of inclusion bodies still occurs, the correction of which means an increase in the final cost of the product.

[0032] The hybrid recombinant protein L2 / E6 produced in this manner of the present invention has the effect of inducing the production of cytokines and specific antibodies with anti-L2 and anti-E6 effects, thereby effectively combating HPV-transformed cells (cancerous or tumorous) that express the E6 protein (therapeutic effect) without interfering with the body's healthy cells (tissue-specific tropism), as well as new opportunistic viral infections caused by HPV (broad-spectrum preventive effect). Therefore, it does not produce side effects.

[0033] The vaccine developed by the present invention, pcDNA3.3 / L2E6, has been shown to be effective in inducing a long-lasting humoral immune response that was able to stimulate the production of anti-L2 and anti-E6 specific antibodies in an incremental manner and throughout the experimental period (49 days), as well as activating a cellular immune response against tumor cells and inducing the expression of the cytokine TNF.

[0034] Therefore, it is noteworthy that the vaccine of the present invention not only induces the production of specific antibodies against HPV (preventive effect), but also induces the production of cytokines, as demonstrated by experiments. Cytokines are important for demonstrating the induction of cellular responses that are responsible for antagonizing tumor cells (therapeutic effect). This is the main difference between this work and other work presented herein, as those works only induce responses via antibodies.

[0035] So far, there is no therapeutic vaccine for HPV and cancers associated with the virus that has been approved for human use in the world. DNA vaccines are stable and easy to produce, transport and store because they are heat-stable and do not require refrigeration. They can maintain the expression of the target gene, promote antigen presentation through class I MHC, and allow repeated doses to be administered without causing the production of neutralizing antibodies against the vaccine. Compared to other vaccines, the cost is also very low. They do not require adjuvants to ensure their functional efficiency, and they are highly tolerable in humans.

[0036] The vaccine of the present invention is obtained through genetic engineering techniques: recombinant DNA technology, gene cloning, and gene expression of recombinant proteins. A vaccine vector containing peptides selected from the HPV16 L2 and E6 proteins was constructed and expressed in mammalian cells. The vaccine vector was amplified in Escherichia coli DH5α bacterial cultures. Expression of the vector was tested in human epithelial cell lines and in murine models, in which the vaccine's efficacy was evaluated in either prophylactic or therapeutic models, in which mice received the vaccine before or after challenge with TC-1 lineage tumor cells.

[0037] Expression assays of L2E6 protein in HEK 293T and 293F cells demonstrated the efficacy of the vector in producing recombinant protein as demonstrated in indirect immunofluorescence assays and Western blotting.

[0038] Tests in animal models have demonstrated that the vaccine is able to induce the production of anti-L2 and anti-E6 specific antibodies, as tested in ELISA, and the production of the cytokine TNF, as demonstrated by assays in flow cytometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The structure and operation of the present invention, as well as further advantages thereof, may be better understood by referring to the accompanying drawings and the following description:

[0041] Figure 1 The construction diagram of the expression vector is shown. The L2E6 insert was inserted into the vector pcDNA3.3, flanked by the Hind III and Nhe I endonuclease sites, and thus designated pcDNA3.3 / L2E6. The constructed plasmid contains the bacterial replication origin pUC, the kanamycin resistance gene Amp(R), and the CMV (cytomegalovirus) promoter. For vector analysis, electrophoresis was performed on 0.8% (m / v) agarose gels in 0.5xTAE buffer (40 mM Tris, 20 mM acetate, 1 mM EDTA) and subjected to 80 V electrophoresis in the presence of Blue Green loading dye (LGC Biotecnologia, Cotia, SP, Brazil).

[0042] Figure 2 A photographic file of an agarose gel subjected to electrophoresis to select colonies containing the vector pcDNA3.3 / L2E6 is shown.

[0043] Figure 3 Shown are cells transfected with the vector pcDNA3.3 / L2E6 expressing recombinant L2 protein (green-b), cell nuclei were visualized by PI (red-c), and in (a), the images (b) and (c) are superimposed.

[0044] Figure 4 E6 expression in 293F cells transfected with pcDNA3.3 / L2E6 is shown in an indirect immunofluorescence assay (green - a). In (b), cell nuclei visualized with PI reagent (red) can be observed. In (c), images (a) and (b) are superimposed.

[0045] Figure 5Photographic documentation of the results obtained from a Western blot assay for the detection and characterization of the recombinant protein L2E6 is shown, wherein samples (1) and (5) are aliquots of lysates from untransfected cells; samples (2) and (6) are lysates from cells transfected and expressing L2E6; samples (3) and (7) are eluted intermediates obtained from a gel filtration chromatography assay and interacting with anti-L2 (3) and anti-E6 (7) specific monoclonal antibodies, demonstrating the presence of low molecular weight proteins recognized by these two commercial antibodies; and sample (4) is the final elution sample from the gel filtration process. (MM) Color Burst TM Molecular weight standard reference.

[0046] Figure 6 A graph showing animal weights in a DNA vaccine prophylactic evaluation assay is shown.

[0047] Figure 7 A graph showing the tumor volume of TC-1 cells in animals previously immunized with DNA vaccine.

[0048] Figure 8 Shown is a graph of animal body weights from a treatment study with a DNA vaccine of the present invention.

[0049] Figure 9 A graph showing tumor volume after treatment with the DNA vaccine of the present invention is shown.

[0050] Figure 10 Shown is an anti-L2 antibody detection assay by ELISA, including a graph of the reactivity of serum samples obtained from animals on the last experimental day (day 49), which demonstrates that all animals immunized with the vaccine pcDNA3.3 / L2E6 produced specific anti-L2 antibodies as detected by the ELISA assay (cutoff = 0.0636).

[0051] Figure 11 Shown is an anti-E6 antibody detection assay by ELISA, including a graph of serum samples obtained from animals on the last experimental day (day 49), which demonstrates the reactivity of anti-E6 antibodies produced by all immunized animals at the end of the experiment (cutoff = 0.0675). Detailed Description of the Invention

[0053] While the invention may be susceptible to different embodiments, a preferred embodiment is shown in the drawings and in the detailed discussion that follows, it should be understood that this embodiment should be considered as an example of the principles of the invention and is not intended to limit the invention to those illustrated or described in this report.

[0054] The present invention relates to vaccines that can provide broad-spectrum protection against HPV infection, as well as against cells modified by the virus. In particular, the present invention relates to recombinant fusion or hybrid proteins obtained by gene cloning into an expression vector, which are produced by gene translation of a fusion gene formed by combining the nucleic acid regulatory sequences of one or more genes with the protein coding sequences of one or more genes, for generating an immune response against HPV. In particular, the present invention provides fusions of the amino acid sequences of HPV16 L2 and E6, which contain only immunogenic epitopes, a fact that disables the transforming activity of the oncogenic protein, thereby conferring safety to L2 / E6 fusions. Furthermore, these fusions maintain or increase the immunogenic efficacy of L2 and E6.

[0055] Although humoral immune responses have important preventive effects, several lines of evidence suggest that cell-mediated immune responses play a key role in the regression of precancerous lesions and the elimination of infection, and are of high therapeutic importance. First, persistent infection and preinvasive lesions with high-risk HPV are significantly more frequent in immunosuppressive states (e.g., untreated HIV infection or receiving post-transplant anti-rejection therapy).

[0056] Spontaneous regression of high-grade CIN (cervical intraepithelial neoplasia) has been reported to be associated with a cell-mediated immune response. The regressing lesions are typically infiltrated by CD8+ T cells specific for the viral oncoproteins E6 and E7.

[0057] In addition, CD4+ T cell responses to E2 (a key transcriptional regulator) have been detected in individuals after CIN has resolved. An increase in the CD4:CD8 ratio in the stroma has also been reported. Conversely, there is a decrease in the number of CD4+ T cells in persistent and / or progressive CIN lesions. Regulatory T cells (Tregs) have also been found in persistent HPV infections, with their frequency increasing with the size of genital warts.

[0058] After administration, the DNA vaccine encoding the hybrid L2 / E6 protein will express it in the patient's tissues. The resulting hybrid recombinant L2 / E6 protein has the effect of inducing the production of cytokines and specific antibodies with anti-L2 and anti-E6 effects, effectively combating HPV-transformed cells (cancerous or tumorous) that express the E6 protein (a therapeutic effect) without interfering with the body's healthy cells (tissue-specific tropism) or new opportunistic viral infections caused by HPV (a broad-spectrum preventive effect). Therefore, it produces no side effects.

[0059] 1. Epitope Selection and Vector Construction

[0060] The sequences of the HPV16 L2 and E6 genes were obtained from the international database GenBank(r), using the accession code AAD33252 for the E6 gene and the accession reference number NC_001526.2 for the L2 gene. Starting from the complete gene sequence, peptide sequences were selected based on their ability to induce an immune response, as described elsewhere.

[0061] The peptide selected from the E6 gene is between amino acid residues 50 and 57 (YDFAFRDL) (SEQ ID NO: 5), which is responsible for inducing cellular immune responses by activating cytotoxic T lymphocytes.

[0062] For greater success in its expression in human cells, the codons were optimized by using the codon optimization tool of IDT-Integrated DNA Technology, and the final sequence obtained was TATGATTTCGCCTTCAGGGACCTC (SEQ ID NO: 4).

[0063] For the L2 gene, the selected peptide was located between amino acid residues 17 to 36 (QLYKTCKQAGTCPPDIIPKV) (SEQ ID NO: 2), wherein the sequence was optimized as described above, resulting in: CAGCTGTATAAAACTTGTAAGCAGGCCGGTACGTGCCCCCCAGATATCATTCCAAAGGTC (SEQ ID NO: 3).

[0064] These two sequences were joined via three "linker" sequences, resulting in a 96 bp (base pair) sequence, to which start and stop sequences were added, resulting in the following sequence of 102 bp: ATG CAG CTG TAT AAA ACC TGT AAACAG GCA GGT ACA TGT CCG CCG GAT ATT ATT CCG AAA GTT GGT GGT AGC GGC TAT GATTTT GCC TTT CGC GAT CTG TAA (SEQ ID NO: 1). The final sequence was provided by GeneArt. TM The gene was synthesized by ThermoFisher Scientific, resulting in a synthetic gene called L2E6.

[0065] 2. Vector Construction

[0066] Two vectors containing the synthetic gene L2E6 were designed and manufactured by Gene Art TMDeveloped by Thermo Fisher Scientific, they are cloning vectors based on the commercial vector pMAT (Thermo Fisher Scientific), and expression vectors or vaccine vectors in pcDNA (Thermo Fisher Scientific), which will be better described below.

[0067] To construct the cloning vector, the synthetic gene L2E6 was inserted into pMA-T, wherein both ends of the synthetic gene and the vector were flanked by sequences for the endonuclease Sfi I (Thermo Fisher Scientific).

[0068] The vector, designated pMA-T / L2E6, has the bacterial origin of replication, Col E1, and the kanamycin resistance gene, Amp R. The entire vector has 2494 bp.

[0069] To construct the expression vector, the L2E6 insert was inserted into the vector pcDNA3.3, flanked by the endonuclease sites Hind III and Nhe I, and thus designated pcDNA3.3 / L2E6. The constructed plasmid has the bacterial replication origin pUC, the kanamycin resistance gene Amp(R), and the CMV (cytomegalovirus) promoter.

[0070] For carrier analysis, electrophoresis assays were performed on 0.8% (m / v) agarose gels in 0.5x TAE buffer (40 mM Tris, 20 mM acetate, 1 mM EDTA) and subjected to 80 V electrophoresis in the presence of Blue Green loading dye (LGC Biotecnologia, Cotia, SP, Brazil).

[0071] 3. Chemical competence of bacteria

[0072] For amplification of the vector, E. coli bacterial strains DH5a and TOP 10 were used. The bacterial strains were cultured in 3 mL of Luria-Bertani (LB) medium (2% tryptone, 1% yeast extract, 1% NaCl) at 37° C. with shaking at 250 rpm for approximately 18 hours.

[0073] Aliquots of this culture were then streaked onto petri dishes containing LB medium supplemented with 1.5% agar and incubated at 37°C for approximately 18 hours.

[0074] The next day, one colony was selected to inoculate 3 mL of LB medium and cultured under the same conditions as described above. After this period, 1 mL of the culture was used to inoculate 100 mL of LB medium and cultured at 37°C with shaking until the optical density (OD600) of the culture at 600 nm reached 0.4, indicating that the cultured bacteria were in the middle of the exponential growth phase.

[0075] The induction of competence was carried out according to the experimental protocol established by Sambrook et al. (2001), wherein after reaching the desired cell density, the bacterial culture was transferred to a conical tube and centrifuged at 5000 x g for 10 minutes and incubated on ice for 1 hour in a 0.1 M CaCl solution. The bacteria were then centrifuged again and resuspended in a 0.1 M CaCl solution with 10% glycerol (w / v).

[0076] Aliquots of 200 μL were frozen at -80°C until use.

[0077] 4. Bacterial Transformation

[0078] Start bacterial transformation from competent bacteria described above, wherein by the mixture of the aliquot of the plasmid DNA of about 15g of 10 μ L and chemical competent bacteria incubated on ice for 30 minutes, experience the heat shock at 42 ℃ for 2 minutes, and again incubated on ice for 5 minutes.Then, add the SOC culture medium (2% tryptone, 0.5% yeast extract, 10mM NaCl, 2.5mM KCl, 10mM MgCl , 10mM MgSO , 20mM glucose) of 350 μ L, by mixture incubated 90 minutes at 37 ℃ under the shaking of 250rpm.Then, will be inoculated in the LB-agar medium flat board that comprises or does not comprise 100 μ g / ml kanamycin through transformed bacteria and incubated 18 hours at 37 ℃.

[0079] Positive clones selected by colony PCR were identified from colonies grown on LB-agar medium plates containing kanamycin, inoculated into tubes containing 3 mL of LB medium, and incubated at 37° C. with shaking for approximately 18 hours.

[0080] 5. Selection of Bacterial Colonies

[0081] To select bacterial colonies after transformation, the colony PCR technique was used, wherein two primers, EAK03 and EAK02, were designed using specific primers synthesized starting from the L2E6 gene sequence and synthesized by EXXTEND Biotecnologia Ltda (Campinas, SP, Brazil), which were able to generate a 144 bp product. The sequences of the primers can be seen below.

[0082] EAK03-CCCYYAAGCTTGCACCATGCAGC(SEQ ID NO:6)

[0083] EAK02-GAGTGTCTAGATGCCACGCT(SEQ ID NO:7)

[0084] The primers were resuspended in TE buffer (10 mM Tris / HCl, 1 mM EDTA) at a stock concentration of 100 μM.

[0085] PCR products were prepared separately in microtubes containing 25 μL of PCR-Mix solution (1.5 mM MgCl , 0.5 U Taq, 5 M dNTPs 200-LGC Biotecnologia, Cotia, SP, Brazil), 0.5 μL of 25 μM of each primer and ultrapure water to a final volume of 50 μL. As template DNA, each transformed bacterial colony sample was selected and grown in LB culture medium with kanamycin. Amplification was performed in a Mastercycler Ep Gradient thermal cycler (Eppendorf, Germany) using a cycle of 95°C for 5 minutes, 1 minute at 95°C, 30 seconds at 56°C, and 1 minute at 72°C for 25 cycles, followed by a final extension at 72°C for 7 minutes.

[0086] The detection of PCR product is carried out by electrophoresis in 0.8% agarose gel in 0.5X TAE buffer.Prepare sample by adding the Blue Green loading dye I of 0.5 μ L and make it experience the sample running 30 minutes under 80A.As the parameter of fragment size, use 2 kinds of molecular weight standards with reference to thing, the universal standard reference (LGC Biotecnologia) that promptly comprises 8 10000bp to the fragment of 100bp and the 2nd 50bp DNA Ladder standard reference (LGC Biotecnologia) that comprises 12 1000bp to the fragment of 50bp.Described gel is analyzed in high-performance ultraviolet transilluminator (HighPerfomance Ultraviolet Translluminator) (UVP Ultraviolet Products, Upland, USA), and in photo file processor Alliance 9.7 (UVITEC Cambridge, Cambridge, England), capture image.

[0087] 6. Extraction of Plasmid DNA

[0088] The positive clones selected by colony PCR were cultured in 5 mL of LB medium at 37 ° C under shaking in the presence of selective antibiotics for approximately 18 hours. The next day, complete bacteria were precipitated at 12,000 x g for 1 minute at ambient temperature. In order to purify the plasmid DNA, a GeneJet Plasmid Miniprep kit (Thermo Fisher Scientific) was used, which is based on an alkaline lysis procedure combined with a selective binding column for circular DNA, according to the manufacturer's instructions. The purified DNA was analyzed in 0.8% agarose gel in 0.5X TAE buffer and subjected to sequencing technology to confirm that the target gene was inserted into the vector, quantified in a spectrophotometer (ACTGene, New Jersey, USA), and stored at -20 ° C.

[0089] 7. DNA Sequencing

[0090] Plasmid DNA samples were sequenced to confirm the correct insertion and integrity of the L2E6 gene. To this end, DNA samples purified from the gel as previously described were quantified by spectrophotometry. A 5 μL volume of plasmid was aliquoted and 2.5 μL of 5 μM EAK03 primer was added. The samples were sent to the Human Genome Center at the University of São Paulo, where sequencing reactions were performed on an ABI 3730 DNA Analyzer (Thermo Fisher Scientific).

[0091] 8. Cell Lines and Culture Conditions

[0092] In principle, to verify the expression of the vector pcDNA3.3 / L2E6, the transfection process was performed on HEK293T cells, which were cultured in Dulbecco's modified Eagle's medium (DMEM, Cultilab, Campinas, SP, Brazil) D10 supplemented with 10% fetal bovine serum (FBS, Cultilab, Campinas, SP, Brazil) and maintained in a humidified atmosphere containing 5% CO2 until they reached 50-80% confluence. Replantation was performed every 3 or 4 days.

[0093] One cell line that has been selected for large-scale expression of recombinant proteins is 293F, FreeStyle TM 293Expression System(Life Technologies TM ), which was developed to allow large-scale transfection of cells suspended in defined and serum-free culture medium.

[0094] 293F cells were cultured in 30 mL of FreeStyle in a sterile glass bottle. TM 293 Expression Medium (Life Technologies TM ) were cultured at 37°C and 5% CO2 in a humidified atmosphere with shaking at approximately 135 rpm. Cells were reseeded every 3 or 4 days at a rate of approximately 2-3 × 10 6 The cells were transferred to a conical tube and centrifuged at 800 rpm for 5 minutes. Cell viability was then analyzed and the cells were diluted to 3×10 cells / mL in fresh pre-warmed culture medium. 5 The final cell density was 10 viable cells / mL.

[0095] To determine cell viability, exclusion by trypan blue (0.4% in PBS) and counting in a Neubauer chamber were used.

[0096] 9. Cell Transfection

[0097] Use transfection reagent according to the manufacturer's instructions. Transfection Reagents (EMDBiosciences / Merck, Darmstadt, Germany) is used to perform transient transfection assays on HEK 293T cells using lipid transfection. In short, the transfection reagent is mixed with an aliquot of DMEM culture medium by vortexing and incubated at ambient temperature for 5 minutes. The target plasmid DNA is added to the mixture in a suitable ratio, homogenized gently, and incubated at ambient temperature for 15 minutes. The mixture of culture medium / transfection reagent / DNA is then added to the cells and complete culture medium and incubated in an incubator at 37°C and 5% CO2. The culture medium is replaced after 8 hours, now with complete culture medium containing antibiotics and 10% FBS. When the cells are collected and analyzed (which will be described in the following topics) with immunofluorescence and in SDS-PAGE electrophoresis gel, the incubation lasted for approximately 48 hours.

[0098] For 293F cells, transfection assays were performed by using the transfection reagent 293fectin according to the manufacturer's instructions.

[0099] One day before transfection, the cell density of live 293F cells was determined and adjusted. TM Expression Medium was supplemented with 7×10 5 cells and cultured under the conditions described above.

[0100] The next day, transfer a small aliquot of the cell culture to a microtube to determine cell viability, which should be around 90%. 7 For each living cell, prepare two solutions in separate sterile conical tubes:

[0101] Tube 1-In having 30 μg of pcDNA3.3 / L2E6 plasmid DNA in a final volume of 1 mL of culture medium was gently homogenized;

[0102] Tube 2- has 60 μL of 293fectin in a final volume of 1 mL of culture medium TM , mix gently and incubate at ambient temperature for 5 minutes.

[0103] The contents of tubes 1 and 2 described above were then gently mixed, resulting in a 2 mL solution, which was incubated at ambient temperature for 30 minutes to allow the plasmid DNA to react with the reagents. Form a complex.

[0104] In incubation While the complex is in progress, the cells in culture are centrifuged at 800 rpm for 5 minutes in a conical tube with 28 mL of fresh, pre-warmed FreeStyle TM Expression Medium was used to replace the culture medium.

[0105] Finally, 2 mL of The complexes, a total of 30 mL of solution for transfection, were incubated at 37° C. and 5% CO 2 in a humidified atmosphere in an orbital shaker with shaking at approximately 125 rpm for 48 hours.

[0106] 10. Expression and Characterization of Recombinant Proteins

[0107] 10.1. Intracellular Expression of Proteins

[0108] After transfection, aliquots of the cells were isolated for analysis of intracellular recombinant protein expression by immunofluorescence assay.

[0109] HEK 293T cells were cultured in 12-well plates. Cultures were performed on 13 mm diameter circular coverslips. Strains that readily adhered were cultured directly on the coverslips in D10 medium in a humidified atmosphere containing 5% CO₂ for approximately 18 hours. The following day, cell transfection assays were performed as described in the previous entry.

[0110] For assays using 293F cells that had been transfected as described above, the cells were centrifuged in microtubes and the supernatant discarded.

[0111] After transfection, both cell lines were washed twice in PBS and fixed in a 1% paraformaldehyde solution in PBS for 1 hour at 4°C. After continuous washing to remove all fixatives, the cells were incubated in a PBS+5% BSA solution to block nonspecific binding and incubated at ambient temperature for 1 hour. The cells were then washed 3 times in PBS and subsequently incubated with specific anti-HPV16 L2 or anti-HPV16 E6 primary antibodies diluted in 0.5% BSA and 0.05% Tween 20 in PBS for 1 hour at ambient temperature, with frequent shaking of the tubes. After rewashing, the cells were incubated with the respective specific secondary antibodies diluted in 1.5% BSA and 0.01% Tween 20 in PBS for 1 hour at ambient temperature, with frequent shaking of the tubes containing the cells and the solution with the antibodies. The secondary antibody used was anti-mouse IgG raised in goats. 488(Molecular OR) and anti-rabbit IgG raised in goat 633. After the final wash in PBS, 293F cells were spread on glass coverslips coated with 0.5% poly-L-lysine in PBS.

[0112] Finally, coverslips containing HEK 293T or 293F cells were incubated with a PI-Molecular Detector (MDR) containing propidium iodide (PI-Molecular Detector) to detect the presence and location of nucleic acids. The cells were mounted on glass slides with Mowiol (Calbiochem, CA, USA) in a 5% PBS solution.

[0113] The preparations were kept at 4°C protected from light until analyzed on a Zeiss LSM 510 Meta laser scanning confocal microscope.

[0114] 10.2. Analysis of Recombinant Protein Expression

[0115] After each transfection reaction, cells were lysed in buffer (0.5 M Tris pH 7.4, 0.5 M NaCl, 0.5 M EDTA pH 8.0, 0.2 M EGTA, Triton X-100, protease inhibitor cocktail), centrifuged at 10,000 x g for 5 minutes, the pellet discarded, and the supernatant stored at -20°C until use.

[0116] To confirm transfection, a sample of the cell lysate was analyzed by dot blot assay in a faster and simpler way. Therefore, 10 μL of the sample was exposed to a nitrocellulose membrane strip (Bio-Rad Laboratories, Inc.). After drying, the strip was incubated in a solution of 5% skim milk powder in PBST for 1 hour at ambient temperature under shaking. The strip was then washed 4 times for 10 minutes in PBST and incubated with anti-HPV16 L2 (Santa Cruz Biotechnology, Inc.) or anti-HPV16 / 18E6 diluted in 1% skim milk powder in PBST. The membrane strips were then washed again in PBST for 10 minutes, four times, and incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody diluted in PBST and 1% skim milk powder. The samples were then washed four times (10 min) in PBST and revealed with a substrate solution of DAB (diaminobenzidine)-peroxidase (0.05% DAB, 0.015% H2O2, 0.01 M PBS, pH 7.2), which was incubated at ambient temperature for 10 min with shaking and in the dark.

[0117] The positive samples in dot blot assay are subsequently used for the expression analysis of the recombinant protein carried out by Western blotting assay. For this reason, 16% Tricine-SDS-PAGE gel (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is prepared and the electrophoresis separation (30V, 300 minutes) under denaturing conditions is experienced, which is included in the lysate sample of the transfected cell diluted in the running buffer. Then, the gel is dyed in Coomassie blue solution (7% acetic acid, 50% methanol, 0.1% Coomassie brilliant blue R-250), or the protein is transferred to nitrocellulose membrane for carrying out Western blotting assay. After electrophoretic transfer (30 V and 15 mA for 18 hours or 70 V and 35 mA for 2 hours) at 4 ° C. in transfer buffer (0.025 M Tris, 0.192 M glycine, 20% methanol), the membrane was stained in a solution containing Ponceau temporary dye (0.1% Ponceau S, 5% acetic acid) to confirm the transfer and labeling of the molecular weight indicator. The membrane was then washed in distilled water to remove the temporary stain and incubated in a solution containing 5% skim milk powder diluted in PBST (0.05% Tween 20, PBS) at ambient temperature for 1 hour with shaking. After this period, the membranes were washed four times in PBST and incubated with a 1% skim milk powder solution in PBST containing a monoclonal anti-HPV16 L2 primary antibody, a polyclonal anti-HPV16 L2 primary antibody raised in rabbit (kindly provided by Prof. Dr. RBS Roden, John Hopkins School of Medicine, USA) and / or an anti-HPV16 E6 primary antibody for 2 hours at ambient temperature with shaking. The membranes were then washed four times again in PBST and incubated with a solution containing the respective anti-mouse IgG or anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase in a 1% skim milk powder solution in PBST for 2 hours at ambient temperature with shaking. The reaction was detected using an ECL HRP chemiluminescent substrate reagent kit (Invitrogen) on green light-sensitive film (Kodak).

[0118] 11. Protein Analysis

[0119] Cell lysate samples were analyzed on 16% Tricine-SDS-PAGE electrophoresis gels (SCHAGGER, 2006), which are best suited for separating proteins between 1 and 100 kD.

[0120] To this end, a 16% separating gel was first prepared using AB-6 buffer (46.5 g acrylamide, 3 g bisacrylamide) diluted in gel buffer (3 M Tris, 1 M HCl, 0.3% SDS, pH 8.45) plus 10% PA [(NH4)2S2O8] and TEMED [(CH3)2NCH2CH2N(CH3)2]. The gel was quickly placed in the instrument to polymerize. Next, a new 10% separating gel was prepared using AB-3 buffer (48 g acrylamide, 1.5 g bisacrylamide) diluted in the same gel buffer, water, 10% PA, and TEMED. After preparation, the 10% gel was carefully placed on top of the 16% gel. Finally, a stacking gel was prepared using AB-3 buffer diluted in gel buffer, water, 10% PA, and TEMED to a final concentration of 4%.

[0121] Protein samples are quantitatively analyzed, and their concentration is adjusted to allow each sample to contain approximately 15 μg of protein. Protein content is determined by using the Bradford method (Bio-Rad Laboratories Inc.) according to the manufacturer's instructions. Samples are diluted in sample buffer (1 M Tris-Cl pH 6.8, 0.8% SDS, 0.1% bromophenol blue, 40% glycerol, 14.4 M β-mercaptoethanol) and subjected to electrophoretic separation (30 V, 300 minutes) in tris-tricine buffer (0.1 M Tris; 0.1 M Tricine; 0.1% SDS, pH 8.5). After separation, the gel is stained with Coomassie blue solution.

[0122] 12. Peptide Synthesis

[0123] To serve as a positive control for the peptides designed in this study, Preto, SP) requested the synthesis of peptides L2 (PEP2) and E6 (PEP1).

[0124] PEP1 refers to a peptide selected from the E6 protein of HPV16, whose sequence is YDFAFRDL, contains 8 amino acids and has a purity of >85%.

[0125] PEP2, selected from the L2 protein of HPV16, has the sequence LYKTCKQAGTCPPDIIPKV, has 19 amino acids and is >85% pure.

[0126] The peptide was received in lyophilized form and was resuspended in ultrapure water at a concentration of 5 mg / mL.

[0127] 13. Culture of TC-1 tumor cells

[0128] Primary cells from the lungs of C57BL / 6 mice were transduced with the E6 and E7 genes of HPV16, and the immortalized cells were then re-transduced with the activated human c-Has-ras gene to generate the stable transformed cell line TC-1 expressing the E6 and E7 proteins of HPV16, which is able to generate solid tumors in mice with the same genetic background (LIN, 1996).

[0129] TC-1 cells were cultured at 75 cm 2 The cells were cultured in RPMI medium (Cultilab, Campinas, SP, Brazil) supplemented with 10% fetal bovine serum (FBS) (Cultilab, Campinas, SP, Brazil) at 37°C and 5% CO2 in polystyrene bottles (TPP-TPP Techno Plastic Products AG, Switzerland). The cells were replanted or the culture medium was changed every 3 or 4 days. For replantation, 250mg% trypsin / EDTA solution (Cultilab) was used to remove cells attached to the bottom of the culture flask and incubated at 37°C for 15 minutes. The cells were then centrifuged at 800 × g for 15 minutes at 4°C. The supernatant was discarded and the cells were resuspended in new culture medium. For long-term storage of the cell line, aliquots of the cells were stored in a solution of RPMI medium containing 10% FBS plus 10% DMSO in liquid nitrogen.

[0130] 14. In vivo experiments

[0131] In order to evaluate the possibility of using the constructed vector as a vaccine vector (as the chosen plasmid pcDNA also allows such use), in vivo assays were performed.

[0132] For the assays in animals, the present invention was analyzed and approved by the Animal Use Ethics Committee of the Butantan Institute under experimental protocol number 923 / 12.

[0133] Male C57BL / 6 mice aged 4 to 8 weeks with an average weight of 20 g and provided by the Butantan Institute's animal husbandry were divided into 4 groups (each containing 10 animals) and maintained in the animal house with free access to food and water and a controlled light cycle.

[0134] Animals were weighed weekly before and throughout the experiment.

[0135] 14.1. Prevention Evaluation

[0136] Two groups of 4 or 6 animals were selected for the prophylactic evaluation of the proposed DNA vaccine.

[0137] The first group (referred to as Group I, containing 4 animals) received 50 μL of intramuscular PBS solution in 3 doses with an interval of 14 days between the first dose (Day 1) and the second dose (Day 14), and with an interval of 7 days between the second and third doses (Day 21).

[0138] The other 6 animals constituting Group II received 3 doses of the vaccine construct pcDNA3.3 / L2E6 in PBS solution at decreasing concentrations. The first dose was administered on day 1, and the animals received 50 μg of the DNA vaccine. The second dose was administered 14 days later at a concentration of 20 μg, while the third and final dose had a concentration of 5 μg of the vaccine vector and was applied 7 days after the previous dose.

[0139] The vaccine was administered intramuscularly in the tibialis anterior muscle of the hind leg, and the doses were alternating between the left and right hind legs. The volume inoculated never exceeded 50 μL of solution.

[0140] Seven days after the last dose (day 28), each animal was treated subcutaneously with 10 mg of 5 All animals in the two groups were challenged with TC-1 cells. Mice were observed daily and the formation of palpable masses could be detected 8 days after the administration of tumor cells. Starting from the detection of tumors, the CT scans were performed with the help of digital calipers ( Tumors were measured every 2 days at the China Medical University Hospital. Tumors grew in an irregular shape and were measured about the length, vertical axis, and width in the same plane. From these data, the circumference in cm was calculated. 3 The tumor volume of each mouse is shown. The data were grouped and statistically analyzed using Student's t-test (p = 5.00%) and Graphic-Pad Prism 6 software version 3.0.

[0141] Small blood aliquots of approximately 100 μL were collected from all animals on days 0, 14, 28, and 42. Following ethical standards in animal experimentation (TAMBOURGI et al., 2010), mice were euthanized approximately 3 weeks after tumor implantation (day 49), and all blood was collected.

[0142] Starting from the blood samples, the animals' serum was collected by centrifugation at 800 rpm, where approximately 30 μL of serum was obtained and stored at -20°C until the time of analysis.

[0143] 14.2. Treatment Evaluation

[0144] To evaluate the efficacy of the constructed vaccine vector pcDNA3.3 / L2E6 in treating HPV-associated cancers, three animal groups were selected for the assay.

[0145] The groups consisted of 10 animals and were fed with 7×10 4 The challenge was performed with TC-1 cells and inoculated subcutaneously in the left dorsolateral region of each animal. The area had been prepared in advance by shaving to facilitate the application of tumor cells through the correct route.

[0146] The day after tumor cell inoculation, day 1, animals in the control group (Group III) received 5 μg of the empty vector pcDNA3.3, and on day 10, a second dose containing 5 μg of the same vector was administered.

[0147] Animals were monitored daily and the area was palpated to verify tumor implantation. Once detected, tumors were measured manually with the aid of a digital caliper every 2 days or until the tumors reached approximately 20 mm in diameter, at which time the animals were euthanized.

[0148] For the experimental assay, animals were divided into two new groups, with the first group (Group IV) receiving the first dose of the DNA vaccine, pcDNA3.3 / L2E6, on the first day of the experiment and the second dose on day 15. The first dose contained 5 μg of the vector pcDNA3.3 / L2E6, while the second dose consisted of 5 μg of the vaccine vector. On day 28 of the experiment, the animals also received a third vaccine dose of 5 μg. Both doses were diluted in PBS solution.

[0149] The animals in the second experimental group (Group V) received a first dose of DNA vaccine containing 5 μg of pcDNA3.3 / L2E6 on the first experimental day, 1 day after receiving tumor cells, and a second dose containing 5 μg pcDNA3.3 / L2E6 10 days after the first dose.

[0150] Animals were monitored daily and the area was palpated to verify tumor growth. Once detected, tumors were measured manually with the aid of a digital caliper every 2 days or until the tumors reached approximately 20 mm in diameter, at which time the animals were euthanized.

[0151] Small blood aliquots were drawn on days 0, 21, 28 and 49 to obtain serum and stored at -20°C until use.

[0152] Spleens from animals that were part of the groups III, IV and V tested for the therapeutic effects of the DNA vaccine were collected in a sterile environment, washed twice in sterile PBS solution and cultured as described below.

[0153] 15. Culture of Spleen Cells

[0154] The spleen was removed from each animal, washed twice in sterile PBS, and then macerated against a sterile gauze pad in a petri dish using a syringe plunger. 3 mL of RPMI medium (R10) supplemented with 10% fetal bovine serum was added to the macerate. The cells were collected in a sterile conical tube and centrifuged at 12,000 rpm for 5 minutes.

[0155] After centrifugation, the supernatant was discarded and the erythrocytes were lysed by adding 500 μL of sterile distilled water, quickly homogenizing, and adding another 500 μL of twice concentrated and sterile PBS solution, homogenizing thoroughly. After centrifugation again at 1000 rpm for 10 minutes, the supernatant was discarded again and the pellet was resuspended with 1 mL of R10 medium.

[0156] Viable splenocytes were counted in a Neubauer chamber with the aid of a 0.4% trypan blue dye solution in PBS.

[0157] The cells were 10 6 Splenocytes were plated in triplicate at a concentration of 10 cells / mL in 12-well culture plates, with the volume filled with 1 mL of R10 medium. 10 μL of the previously described synthetic peptide PEP1 (E6) was added to each well. The plates containing splenocytes and PEP1 were then incubated at 37°C and 5% CO2 in a humidified atmosphere for 48 hours. After this period, the culture supernatant was recovered and stored at -80°C until analysis of cytokine levels as described below.

[0158] 16. Cytokine Detection Assay

[0159] Starting from serum aliquots and spleen cell culture supernatants collected from the experimental animals, soluble cytokine content was determined with the aid of the BD CBA Mouse Th1 / Th2 Cytokine Kit (BD™ Biosciences, USA) according to the manufacturer's instructions.

[0160] Briefly, samples were separated by experimental group and day, with serum samples collected only on days 0, 28, and 49, and spleen cell culture supernatant samples analyzed as pools. The samples were incubated with beads coated with specific antibodies against five different types of cytokines (IL-2, IL-4, IL-5, INF-g, and TNF), along with a detection reagent conjugated to the fluorescent protein PE (phycoerythrin), and incubated for 2 hours at ambient temperature in the dark.

[0161] The beads were then washed with wash buffer (component of the kit) and resuspended in 300 μL of wash buffer, which were then analyzed on a flow cytometer BD FACSCanto II (BD TM The data were analyzed using FCAP Array software version 3.0 (BD Biosciences). TM Biosciences) for analysis.

[0162] 17. Indirect ELISA

[0163] To analyze the induction of humoral immune responses by the vaccine pcDNA3.3 / L2E6, an ELISA assay (enzyme-linked immunosorbent assay) was performed, wherein a flat-bottomed plate (Sarstedt, Numbrecht, Germany) with high adhesion capacity was sensitized with 0.6 mg / mL PEP1 or 0.3 mg / mL PEP2 in carbonate-bicarbonate buffer (0.2 M Na CO , 0.2 M NaHCO , pH 9.6) and incubated at 4° C. for 18 hours. The next day, the antigen was recovered and the plate was washed 3 times with PBST. Then, 150 μL of PBST solution + 5% skim milk powder was added to block nonspecific binding and incubated at ambient temperature for 1 hour. The plate was washed again 3 times with PBST, and serum samples collected from animals at days 0, 28, and 49, diluted 1:100 in PBST solution + 1% skim milk powder, were distributed in each well. The primary antibody was incubated for 2 hours at ambient temperature. After this period of time, discard sample and flat board is washed 3 times again with PBST solution.Then, in hole, add 100 μ L of anti-mouse IgG secondary antibody (Sigma-Aldrich) that carries out 1:250 dilution, that is conjugated with peroxidase in sheep, and incubation 1 hour 30 minutes at ambient temperature.Flat board is washed 3 times again with PBST solution and washed 1 time with PBS.Finally, each hole accepts the disclosure solution (0.05M C6H8O7, 0.05M Na2HPO4, pH 5) of 100 μ L, dilutes OPD (o-phenylenediamine dihydrochloride, Invitrogen) tablet comprising 5mg therein and adds the H2O2 of 12 μ L to 30%.At ambient temperature, incubation 15 minutes in the position avoiding direct light.Then, each hole accepts the 2.5MH2SO4 solution of 50 μ L with termination reaction.Then, on MultiSkan EX reader (LabSystems), read flat board at 492nm place. Samples were tested in triplicate.

[0164] The results were analyzed by calculating the cutoff point by taking the mean of the negative samples, adding 3 times the standard deviation of these samples, and adding 10% of the final value. The number of standard deviations used in the formula ensures the confidence level of the results.

[0165] Cutoff point = mean + 3 x s + 10%

[0166] All points obtained above this cut-off point were considered positive samples.

[0167] 18. Statistical Analysis

[0168] Statistical analysis was performed with the help of GraphPad Software (GraphPad Software, Inc.) was used for statistical analysis of the data obtained in the animal experiments (animal body weight and tumor volume). Student's t test (p = 0.05%) was used. In the cytokine detection assay, ANOVA parametric test (p < 0.05) was used.

[0169] 19. Bioinformatics Analysis of L2E6 Sequence

[0170] Starting with the DNA sequence encoding the defined L2E6 protein, computer analysis was performed to better understand the product to be generated and the characteristics of the protein in order to assist in the development of this study.

[0171] Table 1 shows the bioinformatics analysis of the L2E6 sequence. Starting from the DNA sequence encoding the defined L2E6 protein, computer analysis was performed to better understand the products to be produced and the characteristics of the protein, in order to assist in the development of this research. Several software and online websites for proteome analysis have emerged in the past few years based on algorithms that predict the characteristics of proteins starting from their amino acid sequences. One of the most commonly used and described websites in the literature that can predict the physicochemical characteristics of proteins is http: / / protcale.sourceforge.net / , which estimates molecular weight, pH, pI and performs amino acid residue counting. Several reference websites were consulted, the data analyzed and compiled in the table below, where the defined sequence QLYKTCKQAGTCPPDIIPKVGGSGYDFAFRDL with 32 amino acid residues shows additional characteristics:

[0172] Table 1 – Physicochemical properties of the designed L2E6 protein in this study, according to the Protein Calculator website ( http: / / protcale.sourceforge.net / )

[0173] Physical and chemical properties Molecular weight About 3.5 kDa pI 7.98 Estimated charge at pH 7.0 0.9

[0174] Use other predicted sites of possible post-translational changes by linking http: / / www.cbs.dtu.dk / index.shtml Several parameters were analyzed via the website of the "Center for Biological Sequence Analysis (CBS)" of the Technical University of Denmark.

[0175] The analysis demonstrated that the developed L2E6 peptide possesses predicted sites for acetylation and phosphorylation, a leucine-rich site indicating signaling export to the nucleus, and a cleavage site between amino acid residues 23 and 24, thereby generating a signal peptide with potential signaling for export of the protein to the transmembrane region. In addition, the presence of two amino acid residues, cysteine, can lead to disulfide bond formation, which is a post-translational modification, thereby potentially altering the conformation and stability of the protein.

[0176] Figure 2 Photographic file showing an electrophoresis gel of 0.8% agarose used to select bacterial colonies after transformation with the vector pcDNA3.3 / L2E6. Following the bacterial transformation assay for amplification of the vector, the bacterial colonies were subjected to PCR to select positive clones. The PCR products were analyzed on an agarose gel and subjected to electrophoresis ( Figure 2 ), where (MM1) represents a universal standard reference, and (MM2) represents a 50 bp DNA ladder standard reference. In (1) is a sample of the complete plasmid pcDNA3.3 / L2E6, in (2) and (3) are PCR products from bacterial colonies transformed with pcDNA3.3 / L2E6, and in (4) is a PCR product from an untransformed colony.

[0177] Therefore, select the third column ( Figure 2 ) were used for bacterial colony amplification and subsequent purification of plasmid DNA. The recovered plasmid DNA was quantified spectrophotometrically, resulting in an average value of 150 ng / μL per sample.

[0178] Sequencing of the plasmid DNA recovered and quantified as described above confirmed the correct formation and sequence of the L2E6 gene developed in this work.

[0179] Figure 3 and 4 Shown in HEK293T cells ( Figure 3 ) and 293F cells ( Figure 4 ) was used to evaluate the intracellular expression of the fusion recombinant protein L2E6.

[0180] By indirect immunofluorescence assay, the expression of the recombinant protein was demonstrated, which was recognized by a commercial anti-L2 specific monoclonal antibody. The protein was visualized as dots distributed throughout the cell ( Figure 3 and 4 ).

[0181] Figure 5The expression analysis of the recombinant protein L2E6 is shown. After transfection of 293F cells with the vector pcDNA3.3 / L2E6, the cells were harvested and lysed as described above. The cell lysates were analyzed on 12% SDS-PAGE as shown in Figure 5 The theoretical molecular weight of the recombinant protein L2E6 produced would be approximately 3.5 kDa.

[0182] Comparison of the morphology of cell lysate samples from untransfected and transfected cells revealed the presence of a very low molecular weight protein below 8 kDa, which was present only in the transfected samples, suggesting that it was the recombinant protein L2E6. In addition, Western blot assays were performed to confirm the expression of the recombinant proteins and the recognition of these proteins by commercial specific anti-L2 and anti-E6 monoclonal antibodies in order to confirm the identity of the expressed proteins. The results obtained revealed that the two monoclonal antibodies, anti-L2 and anti-E6, recognized a protein of approximately 3.5 kDa, suggesting confirmation of the identity of the protein of interest ( Figure 5 ).

[0183] Photographic documentation of the results obtained from a Western blot assay for the detection and characterization of the recombinant protein L2E6, wherein samples (1) and (5) are aliquots of lysates from untransfected cells; samples (2) and (6) are lysates from cells transfected and expressing L2E6; samples (3) and (7) are eluted intermediates obtained from the gel filtration chromatography assay and interacting with anti-L2 (3) and anti-E6 (7) specific monoclonal antibodies, demonstrating the presence of low molecular weight proteins recognized by these two commercial antibodies; sample (4) is the final elution sample from the gel filtration process. (MM) Color Burst TM Molecular weight standard reference.

[0184] Figure 6 The present invention shows the protective evaluation in an in vivo assay performed in this work, in experiments for analyzing the use of the vector as a DNA vaccine in a murine model. The animals were divided into four experimental groups and evaluated for their potential to induce prophylactic and therapeutic protection against challenge with TC-1 tumor cells.

[0185] The animals were weighed weekly and it was observed that the body weight remained constant throughout the experiment. A small difference in body weight was observed between the experimental group animals and the control group animals (which received only PBS from day 0 (zero) of the experiment), but such a difference was not statistically significant according to the Student's t-test, suggesting that the DNA vaccine did not interfere with the animals' weight gain or loss ( Figure 6 ).

[0186] Figure 7 It is shown that one week after the administration of 3 doses of the DNA vaccine proposed in this study, animals were challenged with TC-1 tumor cells to evaluate whether a protective immune response was induced and its preventive protective ability in the tumor development of HPV-positive cells. Starting from the moment the tumors were established and became palpable, they were measured every 2 days and the data were analyzed. The tumor volume increased throughout the experimental period, which proves the presence of tumor implantation with tumor development. There were no statistically significant differences between animals in the same group, as it was not possible to observe differences in the tumor volume of animals between the experimental and control groups ( Figure 7 ), suggesting that the DNA vaccine, when used prophylactically, does not induce an immune response capable of preventing or controlling tumor development. However, such a result is expected, as the preventive power of the vaccine under development will be limited to its ability to induce specific antibodies capable of preventing cells from being infected by HPV.

[0187] Figure 8 The therapeutic evaluation of the DNA vaccine is shown. During the entire experimental procedure, animals were weighed weekly to monitor their development and possible changes induced by applying the DNA vaccine. When applied subcutaneously, TC-1 cells have the ability to establish and form solid and non-invasive tumors, but their growth is rapid and can induce the formation of external wounds in the skin of animals. Therefore, after applying tumor cells, animals were monitored for tumor development and euthanized when they induced the formation of exposed wounds or when the maximum diameter of the tumor mass reached 200 mm. In this way, all animals in the control group, i.e., Group II, were euthanized on the 28th experimental day. They received tumor cells and did not receive the DNA vaccine, but only received the empty vector. Therefore, in the animal group that received tumor cells and the treatment with the empty vector, it was observed that tumor implantation induced weight loss in the animals from the 7th day after inoculation of the neoplastic cells, but this weight difference was not statistically significant. Only on the 28th experimental day was a huge weight loss observed in the animals (Groups III and IV, Figure 8 ), which was statistically significant when compared to the previous weighing (p>0.0001). Within the same group, there were no significant differences in animal body weight ( Figure 8 Animals that received tumor cells and the vaccine pcDNA3.3 / L2E6 did not show significant weight loss throughout the experiment, including the group that received the vaccine with a shorter time period between doses, Group V, and the growth of the animals appeared to be continuous throughout the experiment.

[0188] Figure 9The results show that, starting from the 10th day after inoculation of TC-1 cells, palpable (but not yet measurable) tumor masses could be detected in all animals of Group III that had received the vaccine containing the empty vector. However, with the rapid growth of the tumor, tumor diameter measurements were started after 2 days and then measured every 2 days. The data obtained demonstrated rapid tumor growth, with similar tumor volumes between animals in the same group. In the animals of Group IV that had received 3 doses of the vaccine pcDNA3.3 / L2E6 at 15-day intervals, 3 animals in this group developed solid tumors starting from the 12th experimental day. With the rapid development of the tumor, these 3 animals were euthanized on the 28th experimental day. The remaining 7 animals in this group did not develop tumors during the entire analysis period.

[0189] Of the animals in group V, which received only two doses of the vaccine pcDNA3.3 / L2E6 at 10-day intervals, eight did not develop any tumors associated with the administration of TC-1 cells, which remained throughout the experimental period. Two animals in this group developed tumors, but of much smaller size than those in the control group, group III.

[0190] Immediately after the second dose of DNA vaccine, the difference in tumor volume between the groups was statistically significant (p < 0.0001), suggesting that it induced an immune response sufficient to suppress tumor growth in animals of Group IV. As time passed, this difference in tumor volume between Groups III and IV became more pronounced, as can be seen in the Figure 9 The tumor volumes of animals in the group that received the DNA vaccine, Group IV, continued to increase, albeit only slightly, suggesting stability until 14 days after the last dose, when measurements again showed a small, but not statistically significant, increase. On the other hand, animals in Group V, those that exhibited tumor mass formation, grew at a much slower pace when compared to the other animals exhibiting tumor progression. Furthermore, the final volume reached by these tumors was much smaller when compared to the other animals.

[0191] Table 2 shows the analysis of cytokine production.

[0192] Starting from sera obtained from the animals used in the experiments, cytokine detection and content assays were performed in an attempt to characterize the characteristic profile of the immune response induced by the DNA vaccine.

[0193] The serum sample pools from days 0, 28 and 49 were separated into groups using the BD CBA mouse Th1 / Th2 cytokine kit. The presence of the analyzed cytokines could be detected in all samples tested, but the results obtained for the cytokines IFN, IL-2, IL-4 and IL-5 were low and no differences in concentrations could be observed between the samples collected before and after treatment, as can be seen in Table 2 below (Table 2).

[0194] Statistical analysis using the Mann & Whitney nonparametric test revealed that there was no significant difference in the detected TNF concentrations between the control group and the experimental group (p<0.05).

[0195] Table 2 – Cytokine levels detected in serum samples

[0196]

[0197] However, in the analysis of TNF (Tumor Necrosis Factor) in serum samples, a significant increase in the concentration of this cytokine could be observed on the last experimental day (day 49), as can be observed in the table below (Table 3).

[0198] Table 3 - TNF levels detected in the serum of the animals studied

[0199]

[0200] The same cytokine detection analysis was also performed on samples of spleen cell culture supernatant when incubated with aliquots of the synthetic peptide PEP1 (E6). IFN, IL-2, IL-4, and IL-5 levels were not detected, as the concentrations found were outside the standard concentration curve. However, high levels of TNF were detected in samples from animals in groups IV and V, as can be seen in Table 4 (below).

[0201] Table 4 - TNF levels detected in splenocyte culture supernatants

[0202] Group TNF content (pg / mL) III – Treatment evaluation, control group 42.02 IV-treatment evaluation, experimental group, at a dose of 15 days 85.31* V – Treatment evaluation, experimental group, dosed for 10 days 97.40*

[0203] Figure 10 and 11Analysis of specific antibody production is shown. Serum samples collected from all animals and groups were analyzed in an indirect ELISA assay to demonstrate the presence of an induction of a humoral immune response induced by the vaccine pcDNA3.3 / L2E6 with the production of anti-L2 or anti-E6 specific antibodies. The samples were tested in triplicate and the results analyzed as previously described. The data obtained demonstrated that no anti-L2 or anti-E6 antibodies were detected in any of the animals in the samples at day 0, as expected. However, the presence of high levels of specific antibodies could already be detected in the samples at day 28, but a peak of reactivity was found at day 49 and then demonstrated anti-L2 ( Figure 10 ) and anti-E6 ( Figure 11 )-specific antibodies. The animals in Group IV that developed tumors were the same animals that showed lesser production of anti-L2 and anti-E6 antibodies, numbered 5, 6, 7, and 9. The two animals in Group V that developed solid tumors also had the lowest levels of anti-L2 and anti-E6 antibody production, numbered 4 and 5. Data for animals not shown in the following figures represent those with larger tumor development that did not persist to day 49 and were euthanized when the aforementioned parameters were reached.

[0204] Thus, although only a few embodiments of the present invention are shown, it will be understood that various omissions, substitutions and changes can be made by those skilled in the art in the preventive and therapeutic DNA vaccines against HPV and cancers associated with the virus without departing from the spirit and scope of the invention.

[0205] It is expressly intended that all combinations of elements which perform the same function in substantially the same way to achieve the same results are within the scope of the invention. Substitutions of elements from one described embodiment to another are also fully intended and contemplated.

[0206] It is also to be understood that the drawings are not necessarily to scale, but that they are merely conceptual in nature. The intention is, therefore, to be limited as indicated by the scope of the appended claims. Sequence Listing <110> INSTITUTO BUTANTAN <120> Methods for producing preventive and therapeutic DNA immunological compositions against HPV and cancers associated with the virus, hybrid proteins, expression vectors, immunological compositions, and uses thereof <130> PN007351 <150> BR 10 2019 025802 0 <151> 05-12-2019 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 102 <212> DNA <213> Sequence of the L2 / E6 gene <400> 1 atgcagctgt ataaaacctg taaacaggca ggtacatgtc cgccggatat tattccgaaa 60 gttggtggta gcggctatga ttttgccttt cgcgatctgt aa 102 <210> 2 <211> 30 <212> PRT <213> Peptide encoded by L2 gene sequence <400> 2 Gln Leu Tyr Lys Thr Cys Lys Gln Ala Gly Thr Cys Pro Pro Asp Ile 1 5 10 15 Ile Pro Lys Val Gly Ser Gly Tyr Asp Phe Ala Phe Asp Leu 20 25 30 <210> 3 <211> 60 <212> DNA <213> Optimized L2 gene sequence <400> 3 cagctgtata aaacttgtaa gcaggccggt acgtgccccc cagatatcat tccaaaggtc 60 <210> 4 <211> twenty four <212> DNA <213> Optimized E6 gene sequence <400> 4 tatgatttcg ccttcaggga cctc 24 <210> 5 <211> 8 <212> PRT <213> Peptide encoded by the E6 gene sequence <400> 5 Tyr Asp Phe Ala Phe Arg Asp Leu 1 5 <210> 6 <211> twenty four <212> DNA <213> Sense primer <400> 6 cccttaagct tgccaccatg cagc 24 <210> 7 <211> 20 <212> DNA <213> antisense primer <400> 7 gagtgtctag atgccacgct 20

Claims

1. A nucleic acid sequence, characterized in that The nucleic acid sequence comprises the optimized L2 gene sequence shown in SEQ ID NO: 3 and the optimized E6 gene sequence shown in SEQ ID NO: 4, which are connected by three "linker" sequences, and the nucleic acid sequence is shown by SEQ ID NO:

1.

2. A hybrid protein, characterized in that The hybrid protein is encoded by the nucleic acid sequence defined by claim 1.

3. An expression vector, characterized in that The expression vector comprises the nucleic acid sequence defined by claim 1.

4. The expression vector according to claim 3, characterized in that To construct an expression vector, the L2E6 nucleic acid sequence was inserted into the vector pcDNA3.3, flanked by the endonuclease sites Hind III and Nhe I, and thus named pcDNA3.3 / L2E6, wherein the constructed plasmid has the bacterial replication origin pUC, the ampicillin resistance gene Amp and the CMV promoter.

5. An immunological composition, characterized in that The immunological composition comprises the hybrid protein defined in claim 2 or the carrier defined in claims 3 and 4, and a pharmaceutically acceptable excipient.

6. The immunological composition according to claim 5, characterized in that The immunological composition optionally comprises a vaccine adjuvant, or is free of an adjuvant.

7. The immunological composition according to claim 6, characterized in that The vaccine adjuvant is a cationic lipid or HPV virus-like particles.

8. The immunological composition according to any one of claims 5 to 7, characterized in that The immunological composition includes dosage forms for intramuscular and intradermal administration, administration via DNA tattooing, or oral administration.

9. The immunological composition according to any one of claims 5 to 7, characterized in that The immunological compositions include dosage forms for administration with a syringe and needle, by electroporation, or with a biological syringe without a needle.

10. Use of the hybrid protein defined in claim 2 for preparing preventive and therapeutic vaccines against HPV and cervical cancer.

11. Use of the expression vector defined in claim 3 or 4 for preparing preventive and therapeutic vaccines against HPV and cervical cancer.

12. Use according to claim 10 or 11, characterized in that It generates a long-lasting humoral immune response that stimulates the production of anti-L2 and anti-E6 antibodies specific for HPV infection, as well as activates a cellular immune response against tumor cells and induces the expression of the cytokine TNF.

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