PROCESS FOR PRODUCING A PROPHYLACTIC AND THERAPEUTIC DNA IMMUNOLOGICAL COMPOSITION FOR HPV AND CANCERS ASSOCIATED WITH THE VIRUS, HYBRID PROTEIN, EXPRESSION VECTOR, IMMUNOLOGICAL COMPOSITION AND USES THEREOF

Abstract

The present invention relates to the prophylactic and therapeutic vaccine against HPV and cancers associated with the virus, aimed at people seeking prevention or those already infected with HPV who have developed cancer. It also refers to DNA expression vectors that can efficiently produce the L2 protein of the HPV16 virus capsid, in addition to the viral E6 oncoprotein associated with human papillomavirus tumors. In particular, this invention relates to obtaining recombinant fusion or hybrid proteins, through gene cloning into expression vectors, produced by the genetic translation of fused genes, formed by the combination of nucleic acid regulatory sequences of one or more genes, with the protein coding sequences of one or more genes, used to generate two distinct types of responses: lasting humoral immune response, capable of stimulating the production of specific anti-L2 and anti-E6 antibodies against HPV (prophylactic action), as well as activate the cellular immune response, to fight tumor cells and induce the expression of TNF cytokines (Tumor Necrosis Factor) or Tumor Necrosis Factor (therapeutic action).

Claims

1-47. (canceled)

48. A Process for producing prophylactic and therapeutic vaccine against HPV and cancers associated with the virus characterized by the fact that it consists of the following steps: a) selection of epitopes and construction of a vaccine vector containing peptides selected from HPV16 L2 and E6 proteins, wherein there were used HPV16 L2 and E6 gene sequences which were obtained from the international GenBank(r) database, wherein the access code of the E6 gene used was AAD33252 and the reference for access to L2 was NC_001526.2, preferentially, the peptides selected from the E6 gene are among the amino acid residues 50 to 57 as established in SEQ ID NO: 5; b) amplification of the vaccine vector, wherein amplification of the vaccine vector occurs in bacterial cultures of Escherichia coli DH5α; c) bacterial transformation, wherein was carried out from the competent bacteria, where a mixture of 10 .Math.L of plasmideal DNA, approximately 15 g, and an aliquot of chemo-competent bacteria was incubated for 30 minutes on ice, subjected to a thermal shock for 2 minutes at 42° C. and incubated again on ice for 5 minutes, then 350 .Math.L of S.O.C medium (Tryptone 2%, Yeast extract 0.5%, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM Glucose), the mixture being incubated for 90 minutes at 37° C., under agitation of 250 rpm, then the transformed bacteria were seeded in plates of LB-agar medium containing or not 100 .Math.g/ml ampicillin and incubated at 37° C. for 18 hours, preferentially, colonies grown on LB-agar plates containing ampicillin identify the positive clones that were selected by colony PCR and seeded in tubes containing 3 mL of LB medium, incubated at 37° C., under stirring for approximately 18 hours; d) selection of bacterial colonies, wherein for the selection of bacterial colonies after transformation, the colony PCR technique was used, where 2 specific primers synthesized from the L2E6 gene sequence were designed, EAK03 and EAK02, and capable of generating a 144 bp product, preferentially, the initiators are as established by SEQ ID NO 6 and SEQ ID NO: 7, and the primers were resuspended in TE buffer (10 mM Tris / HCl, 1 mM EDTA) at a stock concentration of 100 .Math.M, in which for PCR, each solution was prepared separately in microtubes containing 25 .Math.L of PCR-Mix solution (1.5 mM MgCl.sub.2, 0.5U Taq, 200 5 M dNTPs – LGC Biotecnologia, Cotia, SP, Brazil), 0.5 .Math.L of each primer at 25 .Math.M and ultrapure water for a final solution volume of 50 .Math.L, and as template DNA, a sample of each transformed bacterial colony was used, selected and grown in LB medium with ampicillin, wherein the amplification was performed in a thermocycler occurring as the protocol: 95° C. for 5 minutes, 25 cycles of 1 minute at 95° C., 30 seconds at 56° C. and 1 minute at 72° C., followed by final extension at 72° C. for 7 minutes, which the detection of PCR products occurred through electrophoresis in 0.8% agarose gel in 0.5X TAE buffer, The samples were prepared with the addition of 0.5 .Math.L of Blue Green Loading Dye I and subjected to a run at 80 A for 30 minutes; e) extraction of plasmideal DNA; wherein the positive clones selected by the colony PCR were cultured in 5 mL of LB medium, at 37° C., for approximately 18 hours, under agitation, in the presence of the antibiotic of selection, then they were centrifuged at room temperature, for 1 minute at 12000xg to sediment the intact bacteria, and for the purification of plasmideal DNA, the GeneJet Plasmid Miniprep Kit (Thermo Fisher Scientific) was used, where the purified DNA was analyzed in 0.8% agarose gel in 0.5X TAE buffer and submitted to the sequencing technique to confirm the insertion of the gene of interest to the vector, quantified in a spectrophotometer and stored at -20° C.; f) DNA sequencing; wherein plasmideal DNA samples were sequenced to confirm the correct insertion of the L2E6 gene and its integrity, where the DNA samples purified from the gel were quantified by spectrophotometry, and a volume of 5 .Math.L of the plasmid was aliquoted and 2.5 .Math.L of the EAK03 primer was added at a concentration of 5 .Math.M; g) cell transfection; wherein the transient transfection of HEK 293T cells was performed by lipofection using the GeneJuice® Transfection Reagents, where the transfection reagent was mixed by vortex to an aliquot of DMEM medium, being incubated for 5 minutes at room temperature; to the mixture, the plasmideal DNA of interest was added, in the appropriate proportion, gently homogenized and incubated for 15 minutes at room temperature, then the mixture Medium/transfection reagent/DNA was added to the cells with complete culture medium, and incubated in oven at 37° C. and 5% CO.sub.2, where after 8 hours the culture medium was changed, now with a complete medium containing antibiotics and 10% SBF, wherein the incubation continued for approximately 48 hours when the cells were collected and analyzed in immunofluorescence and in SDS-PAGE electrophoresis gel, and in step (g) for 293F cells, the transfection assays were carried out using the 293fectin transfection reagent; h) expression and characterization of recombinant proteins; wherein HEK 293T cells were grown on 13 mm diameter round coverslips in 12-well culture plates (TPP®), where the easily adherent strains were grown directly on the coverslips in D10 medium, kept in a humid atmosphere, containing 5% CO.sub.2, for approximately 18 hours, and in step (h), for the assays using the 293F cells already transfected, said cells were centrifuged in microtubes and the supernatant discarded; and i) Protein synthesis.

49. Process, according to claim 48, characterized in that the codons have been optimized for greater success in their expression in human cells, using the Codon Optimization tool from IDT Integrated DNA Technology, and the final sequence obtained was as set out in SEQ ID NO: 4 and for the L2 gene, the selected peptide was found among amino acid residues 17 to 36 as established in SEQ ID NO: 2, with the optimized sequence resulting in: SEQ ID NO: 3; and wherein both sequences were linked by 3 “Linker” sequences, resulting in the 96 bp sequence (base pairs), to which the initiator and termination sequences were added, giving rise to the 102 bp sequence as established by SEQ ID NO: 1, and the final sequence was synthesized giving rise to the synthetic gene called L2E6.

50. Process, according to claim 48, characterized in that in step (a), 2 vectors were designed containing the synthetic gene L2E6, being a cloning vector based on the commercial vector pMAT and an expression vector or vaccine vector in pcDNA, preferentially, the construction of the cloning vector, the synthetic gene L2E6 was inserted in pMA-T, with both ends of the synthetic gene and the vector flanked by endonuclease sequences Sfi I, preferentially, the vector called pMA-T/L2E6 has bacterial Col E1 replication origin and ampicillin resistance gene, AmpR, and the complete vector has 2494 bp; and wherein for the construction of the expression vector, the L2E6 insert was inserted in the vector pcDNA3.3 flanked by endonuclease sites Hind III and Nhe I, being named pcDNA3.3/L2E6, in which the constructed plasmid originates from bacterial replication, pUC, Ampicillin resistance gene, Amp (R) and CMV promoter (Cytomegalovirus), and vector analysis, electrophoresis assays were performed on 0.8% (m/v) agarose gel in 0.5x TAE buffer (Tris 40 mM, 20 mM acetate, 1 mM EDTA) and subjected to an 80 V electrophoresis run in the presence of Blue Green Loading Dye.

51. Process, according to claim 48, characterized in that in step (b) for the amplification of the vectors, the bacterial strains Escherichia coli DH5a and TOP10 were used, wherein the bacterial strains were grown in 3 mL of Luria medium-Bertani (LB) (2% tryptone, 1% yeast extract, 1% NaCl) at 37° C., under stirring at 250 rpm for approximately 18 hours, then an aliquot of this culture was streaked into a Petri dish containing LB medium plus 1.5% agar, incubated at 37° C., for approximately 18 hours, where a colony was chosen to sow 3 mL of LB medium and grown under the same conditions described above, after that period, 1 ml of this culture was used to sow 100 mL of LB medium and cultivated at 37° C., under agitation until the optical density of the culture at 600 nm (DO600) reaches 0.4, wherein the cultured bacteria were in the middle of the exponential growth phase, preferentially, the competence induction was performed, wherein after reaching the desired cell density, the culture of bacteria was transferred to a conical tube and centrifuged at 5000xg for 10 minutes and incubated in a solution of 0.1 M CaCl.sub.2 for 1 hour on ice, then the bacteria were again centrifuged and resuspended in a solution of 0.1 M CaCl.sub.2 plus 10% glycerol (w/v), where aliquots of 200 .Math.L were frozen at -80° C. until use.

52. Process, according to claim 48, characterized in that after transfections, both cell lines were washed 2 times in PBS and fixed in 1% paraformaldehyde solution in PBS, for 1 hour at 4° C., where after successive washes to remove all fixative, said cells were incubated in 5% PBS+BSA solution to block nonspecific bonds and incubated for 1 hour at room temperature, then the cells were washed in PBS for 3 times and then incubated with specific primary anti-HPV16 L2 or anti-HPV16 E6 antibodies diluted in 0.5% BSA and 0.05% Tween 20 in PBS for 1 hour at room temperature, shaking the tube frequently, where after re-washing, 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 room temperature, with frequent shaking in the tube containing the cells and the solution with the antibodies, wherein the secondary antibodies used were ram AlexaFluor® 488 anti-mouse IgG produced in goat (Molecular Probes®, OR) and AlexaFluor® 633 anti-IgG rabbit produced in goat, where after a last wash in PBS, 293F cells were distributed over coated glass coverslips with 0.5% poly-L-lysine in PBS; and wherein after each transfection reaction, the cells were lysed in buffer (0.5 M Tris pH 7.4, 0.5 M NaCl, EDTA 0.5 M pH 8.0, 0.2 M EGTA, Triton X-100, protease inhibitor cocktail), centrifuged at 10,000xg for 5 minutes, with the precipitate discarded and the supernatant stored at 20° C. until the time of use; and wherein the positive samples in the dot blot assays proceeded to analyze the expression of recombinant proteins, carried out through Western blotting assays.

53. Process, according to claim 52, characterized in that a 16% Tricina-SDS-PAGE gel (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) was prepared and submitted to electrophoretic separation (30 V for 300 minutes), under denaturing conditions, containing samples of transfected cell lysates diluted in running buffer, then the gel was stained in Coomassie Blue solution (7% acetic acid, 50% methanol, 0.1% Coomassie Bright Blue R-250) or proteins were transferred to a nitrocellulose membrane for Western blotting assays, after electrophoretic transfer (30 V and 15 mA for 18 hours, or 70 V and 35 mA for 2 hours) in transfer buffer (Tris 0.025 M, Glycine 0.192 M, 20% Methanol) at 4° C., the membrane was stained in a solution containing Ponceau temporary dye (Ponceau S 0.1%, acetic acid 5%) to confirm the transfer and marking of the molecular weight indicators, then the membranes were washed in distilled water to remove temporary staining and incubated in a solution containing 5% skimmed-milk powder diluted in PBST (Tween 20 0.05%, PBS), for 1 hour, at room temperature, under agitation After that period, the membranes were washed 4 times in PBST and incubated with 1% skimmed-milk powder solutions in PBST containing the primary antibodies anti-HPV16 L2 monoclonal, anti-HPV16 L2 polyclonal produced in rabbit and/or anti-HPV16 E6 for 2 hours at room temperature, under agitation, then the membranes were washed again 4 times in PBST and incubated in a solution containing the respective secondary antibodies anti-mouse IgG or anti-rabbit IgG conjugated with horseradish peroxidase in solution of 1% skimmed-milk powder in PBST, incubated for 2 hours at room temperature, under agitation, and wherein the samples of cell lysate were analyzed in 16% Tricina-SDS-PAGE electrophoresis gel, the most suitable for the separation of proteins from 1 to 100 KDa; and wherein the protein samples were quantified and their concentrations adjusted so that each sample contains approximately 15 .Math.g of proteins, where the protein dosage occurred using the Bradford method (Bio-Rad Laboratories Inc.), where the samples were diluted in sample buffer (Tris-Cl 1 M pH 6.8, SDS 0.8%, bromophenol blue 0.1%, glycerol 40%, b-mercaptoethanol 14, 4 M) and finally submitted to electrophoretic separation (30 V for 300 minutes) in tris-tricine buffer (0.1 M Tris; 0.1 M Tricine; 0.1% SDS, pH 8.5), after separation, the gel was stained with Coomassie blue solution.

54. Nucleic acid sequence characterized by comprising the optimized sequence of the L2 gene as established in SEQ ID NO: 3 and the optimized sequence of the E6 gene as established in SEQ ID NO: 4 linked by 3 “Linker” sequences.

55. Nucleic acid sequence, according to claim 54, characterized in that it is as established by SEQ ID NO: 1.

56. Hybrid protein characterized in that it is encoded by the nucleic acid sequence as defined by claim 54.

57. Expression vector characterized by comprising the nucleic acid sequence as defined by claims 54, wherein for the construction of the expression vector, the L2E6 nucleic acid sequence was inserted into the pcDNA3.3 vector flanked by Hind III and Nhe I endonuclease sites, receiving the name pcDNA3.3/L2E6, in which the constructed plasmid originates from bacterial replication, pUC, Ampicillin resistance gene, Amp (R) and CMV promoter (Cytomegalovirus).

58. Immune composition characterized by comprising the hybrid protein as defined by claim 56 and pharmaceutically acceptable vehicles, excipients and carriers, and optionally comprises vaccine adjuvants, particularly cationic lipids and HPV Virus-like particles (VLPs), wherein the composition comprises dosage forms administered intramuscularly and intradermally, preferably administered with syringes and needles, by electroporation, bioinjectors without needles, or by techniques like DNA tattooing, or orally.

59. Use of the hybrid protein, as defined by claim 56, characterized in that it is for the preparation of a prophylactic and therapeutic vaccine against HPV and cancers associated with the virus.

60. Use of the expression vector, as defined by claim 57, characterized in that it is for the preparation of a prophylactic and therapeutic vaccine against HPV and cancers associated with the virus.

61. Use, according to claims 59, characterized in that it produces a lasting humoral immune response, capable of stimulating the production of specific anti-L2 and anti-E6 antibodies against HPV infection (prophylactic action), as well as activating the cellular immune response, to fight tumor cells and induced by the expression of cytokines TNF (Tumor Necrosis Factor) or Tumor Necrosis Factor (therapeutic action).

62. Immune composition characterized by comprising the vector, as defined by claim 57 and pharmaceutically acceptable vehicles, excipients and carriers, and optionally comprises vaccine adjuvants, particularly cationic lipids and HPV Virus-like particles (VLPs), wherein the composition comprises dosage forms administered intramuscularly and intradermally, preferably administered with syringes and needles, by electroporation, bioinjectors without needles, or by techniques like DNA tattooing, or orally.

63. Use, according to claims 60, characterized in that it produces a lasting humoral immune response, capable of stimulating the production of specific anti-L2 and anti-E6 antibodies against HPV infection (prophylactic action), as well as activating the cellular immune response, to fight tumor cells and induced by the expression of cytokines TNF (Tumor Necrosis Factor) or Tumor Necrosis Factor (therapeutic action).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The structure and operation of the present invention, together with additional advantages of the same can be better understood by reference to the attached drawings and the following description:

[0039] FIG. 1 shows the construction map of the expression vector. The L2E6 insert was inserted into the pcDNA3.3 vector flanked by Hind III and Nhe I endonuclease sites, named pcDNA3.3/L2E6. The constructed plasmid originates from bacterial replication, pUC, kanamycin resistance gene, Amp (R), and CMV promoter (Cytomegalovirus). For vector analysis, electrophoresis assays were performed on 0.8% (m/v) agarose gel in 0.5x TAE buffer (40 mM Tris, 20 mM Acetate, 1 mM EDTA) and subjected to an 80 V electrophoresis run in the presence of Blue Green Loading Dye (LGC Biotecnologia, Cotia, SP, Brazil).

[0040] FIG. 2 shows photodocumentation of the agarose gel submitted to electrophoresis for selection of colonies containing the pcDNA3.3/L2E6 vector.

[0041] FIG. 3 shows Cells transfected with the pcDNA3.3/L2E6 vector expressing the recombinant L2 protein (green – b), the cell nucleus was evidenced by PI (red – c) and in (a) the overlay of the images (b) and (c).

[0042] FIG. 4 shows E6 Expression (green - a) in 293F cells transfected by pcDNA3.3/L2E6 in an indirect immunofluorescence assay. In (b) it is possible to observe the cell nucleus evidenced by the PI reagent (red). In (c) the overlay of the image (a) and (b).

[0043] FIG. 5 shows the Photodocumentation of the result obtained from the Western blotting assay used for detection and characterization of the recombinant protein L2E6, where the samples (1) and (5) are aliquots of non-transfected cells lysate, (2) and (6) transfected cells lysate and expressing L2E6, (3) and (7) eluted intermediates obtained from the filtration gel chromatography assay and interacting with specific anti-L2 (3) and anti-E6 monoclonal antibodies, demonstrating the presence of a low molecular weight protein being recognized by both commercial antibodies, (4) final elution sample from the gel filtration process. (MM) Color Burst™ molecular marker.

[0044] FIG. 6 shows a graphic of the animals’ weighing in the DNA vaccine prophylactic evaluation test.

[0045] FIG. 7 shows a graph of the volume of TC-1 cell tumors in animals previously immunized with the DNA vaccine.

[0046] FIG. 8 shows a graph of the animals’ weight in the therapeutic study of the DNA vaccine of the present invention.

[0047] FIG. 9 shows a graph of tumor volumes upon treatment with the DNA vaccine of the present invention.

[0048] FIG. 10 shows the anti-L2 antibody detection assay by ELISA, where the graph of the reactivity of serum samples obtained from the animals on the last experimental day (49 days), demonstrating that all animals immunized with the pcDNA3.3/L2E6 vaccine produced specific anti-L2 antibodies detected by ELISA assay (Cut off = 0.0636).

[0049] FIG. 11 shows the anti-E6 antibody detection assay by ELISA, where the graph of serum samples obtained from animals on the last experimental day (49 days), is demonstrating the reactivity of anti-E6 antibodies that all immunized animals produced at the end of the experiment (Cut off = 0.0675).

DETAILED DESCRIPTION OF THE INVENTION

[0050] Although the present invention may be susceptible to different embodiments, it is shown in the drawings and in the following detailed discussion, a preferred embodiment with the understanding that the present embodiment should be considered an example of the principles of the invention and is not intended to limit the present invention to what was illustrated and described in this report.

[0051] The present invention relates to a vaccine that is capable of protecting against infection by the HPV virus in a broad spectrum of protection, as well as fighting cells already modified thereby. In particular, this invention relates to obtaining recombinant fusion or hybrid proteins, through gene cloning into expression vectors, produced by the genetic translation of fused genes formed by the combination of nucleic acid regulatory sequences of one or more genes, with the protein coding sequences of one or more genes, used to generate immune responses against HPV. Specifically, this invention provides fusions of HPV16 L2 and E6 amino acid sequences, containing only immunogenic epitopes, a fact that disables the transformation activity of this oncogenic protein, providing safety to L2/E6 fusions. In addition, these fusions maintain or increase the immunogenic efficacy of L2 and E6.

[0052] Although humoral immune responses have important preventive utility, several lines of evidence suggest that cell-mediated immune responses play a crucial role in the regression of precancerous lesions and the elimination of infection, of high therapeutic importance. Above all, persistent high-risk HPV infections and pre-invasive lesions are significantly more frequent in immunosuppressed states, such as untreated HIV infection or receiving post-transplant anti-rejection therapy.

[0053] Spontaneous regression of high-grade CIN (Cervical Intraepithelial Neoplasm) has been reported to correlate with cell-mediated immune responses. Regression lesions are typically infiltrated by CD8 + T cells specific for viral oncoproteins, E6 and E7.

[0054] In addition, CD4 + T cell responses to E2, a key transcriptional regulator, were detected in individuals after CIN regression. Increased proportions of CD4:CD8 in the stroma have also been reported. In contrast, reduced numbers of CD4 + T cells are present in persistent and/or progressive CIN lesions. Regulatory T cells (Tregs) are also found in persistent HPV infections, with increasing frequency with the size of genital warts.

[0055] After administration, the DNA vaccine encoding the hybrid L2/E6 proteins will express them in the patient’s tissues. The hybrid recombinant protein L2/E6 thus produced has the effect of inducing the production of cytokines and specific antibodies with anti-L2 and anti-E6 action, effectively combating cells transformed by HPV (cancerous or tumor) that express the E6 protein (effect therapeutic), without interfering with healthy body cells (tissue-specific tropism), and new opportunistic viral infections by HPV (broad spectrum prophylactic effect). Thus, it does not produce side effects.

1. Selection of Epitopes and Construction of Vectors

[0056] The sequences of the HPV16 L2 and E6 genes were obtained from the international database GenBank (r), and the access code of the E6 gene used was AAD33252 and the reference for L2 access was NC_001526.2. From the sequences of the complete genes, peptide sequences were selected according to their ability to induce an immune response described in other studies.

[0057] The peptides selected from the E6 gene are among the amino acid residues 50 to 57 (YDFAFRDL) (SEQ ID NO: 5), responsible for inducing cellular immune response through the activation of cytotoxic T lymphocytes.

[0058] The codons were optimized for greater success in their expression in human cells, using the Codon Optimization tool from IDT – Integrated DNA Technology, and the final sequence obtained was

TABLE-US-00001 TATGATTTCGCCTTCAGGGACCTC (SEQ ID NO: 4).

[0059] For the L2 gene, the selected peptide was found among amino acid residues 17 to 36 (QLYKTCKQAGTCPPDIIPKV) (SEQ ID NO: 2), the sequence being optimized as described above, resulting in:

TABLE-US-00002 CAGCTGTATAAAACTTGTAAGCAGGCCGGTACGTGCCCCCCAGATATCAT TCCAAAGGTC (SEQ ID NO: 3).

[0060] Both sequences were linked by 3 “Linker” sequences, resulting in the 96 bp sequence (base pairs), to which the initiator and termination sequences were added, giving rise to the following 102 bp sequence:

TABLE-US-00003 ATG CAG CTG TAT AAA ACC TGT AAA CAG GCA GGT ACA TGT CCG CCG GAT ATT ATT CCG AAA GTT GGT GGT AGC GGC TAT GAT TTT GCC TTT CGC GAT CTG TAA. (SEQ ID NO: 1).

The final sequence was synthesized by the company Gene Art™ (Thermo Fisher Scientific) giving rise to the synthetic gene called L2E6.

2. Construction of Vectors

[0061] 2 vectors were designed containing the synthetic gene L2E6 and developed by the company Gene Art™ (Thermo Fisher Scientific), being a cloning vector based on the commercial vector pMAT (Thermo Fisher Scientific) and an expression vector or vaccine vector in pcDNA (Thermo Fisher Scientific) which will be better described below.

[0062] For the construction of the cloning vector, the synthetic gene L2E6 was inserted in pMA-T, with both ends of the synthetic gene and the vector flanked by endonuclease Sfi I sequences (Thermo Fisher Scientific).

[0063] The vector called pMA-T/L2E6 has bacterial Col E1 replication origin and kanamycin resistance gene, AmpR. The complete vector has 2494 bp.

[0064] For the construction of the expression vector, the L2E6 insert was inserted into the pcDNA3.3 vector flanked by Hind III and Nhe I endonuclease sites, receiving the name pcDNA3.3 / L2E6. The constructed plasmid originates from bacterial replication, pUC, kanamycin resistance gene, Amp (R), and CMV promoter (Cytomegalovirus).

[0065] For vector analysis, electrophoresis assays were performed on 0.8% (m/v) agarose gel in 0.5x TAE buffer (40 mM Tris, 20 mM acetate, 1 mM EDTA) and subjected to running 80 V electrophoresis in the presence of Blue Green Loading Dye (LGC Biotecnologia, Cotia, SP, Brazil).

3. Chemiocompetence of Bacteria

[0066] For the amplification of the vectors, bacterial strains Escherichia coli DH5a and TOP10 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., under agitation at 250 rpm for approximately 18 hours.

[0067] Then, an aliquot of this culture was streaked in a Petri plate containing LB medium plus 1.5% agar, incubated at 37° C., for approximately 18 hours.

[0068] The next day, a colony was chosen to sow 3 mL of LB medium and grown under the same conditions as previously described. After that period, 1 mL of this culture was used to sow 100 mL of LB medium and grown at 37° C., under agitation until the optical density of the culture at 600 nm (DO600) reached 0.4, wherein the cultured bacteria were in half exponential growth phase.

[0069] Competence induction was performed according to a protocol established by Sambrook et al. (2001), where after reaching the desired cell density, the bacterial culture was transferred to a conical tube and centrifuged at 5000xg for 10 minutes and incubated in a 0.1 M CaCl.sub.2 solution for 1 hour on ice. Then, the bacteria were again centrifuged and resuspended in a 0.1 M CaCl.sub.2 solution plus 10% glycerol (w/v).

[0070] 200 .Math.L aliquots were frozen at -80° C. until use.

4. Bacterial Transformation

[0071] From the competent bacteria described above, bacterial transformation was carried out, wherein a mixture of 10 .Math.L of plasmideal DNA, approximately 15 g, and an aliquot of chemocompetent bacteria was incubated for 30 minutes on ice, subjected to a thermal shock for 2 minutes at 42° C. and incubated again on ice for 5 minutes. Then, 350 .Math.L of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) were added, and the mixture was incubated for 90 minutes at 37° C., under agitation of 250 rpm. Then, the transformed bacteria were seeded on plates of LB-agar medium containing or not 100 .Math.g/ml kanamycin and incubated at 37° C. for 18 hours.

[0072] Colonies grown on LB-agar plates containing kanamycin identify the positive clones that were selected by colony PCR and seeded in tubes containing 3 mL of LB medium, incubated at 37° C., under agitation for approximately 18 hours.

5. Selection of Bacterial Colonies

[0073] For selection of bacterial colonies after transformation, the colony PCR technique was used, where using specific primers synthesized from the L2E6 gene sequence, 2 primers were designed, EAK03 and EAK02, and synthesized by the company EXXTEND Biotecnologia Ltda. (Campinas, SP, Brazil), capable of generating a 144 bp product. The sequence of the primers can be seen below.

TABLE-US-00004 EAK03 - CCCYYAAGCTTGCCACCATGCAGC (SEQ ID NO: 6)

TABLE-US-00005 EAK02 - GAGTGTCTAGATGCCACGCT (SEQ ID NO: 7)

[0074] The primers were resuspended in TE buffer (10 mM Tris/HCl, 1 mM EDTA) at a stock concentration of 100 .Math.M.

[0075] For PCR, each solution was prepared separately in microtubes containing 25 .Math.L of PCR-Mix solution (1.5 mM MgCl.sub.2, 0.5U Taq, 200 5 M dNTPs - LGC Biotecnologia, Cotia, SP, Brazil), 0.5 .Math.L of each initiator at 25 .Math.M and ultrapure water for a final solution volume of 50 .Math.L. As a template DNA, a sample of each colony of transformed bacteria was used, selected and grown in LB medium with kanamycin. Amplification in a Mastercycler EpGradient thermocycler (Eppendorf, Germany) occurred following the protocol: 95° C. for 5 minutes, 25 cycles of 1 minute at 95° C., 30 seconds at 56° C. and 1 minute at 72° C., followed by final extension at 72° C. for 7 minutes.

[0076] The detection of PCR products occurred through electrophoresis in 0.8% agarose gel in 0.5X TAE buffer. The samples were prepared with the addition of 0.5 .Math.L of Blue Green Loading Dye I and subjected to a run at 80 A for 30 minutes. As a parameter for fragment size, 2 molecular weight markers were used, the Universal Marker containing 8 fragments from 10000 bp to 100 bp (LGC Biotechnology) and the second 50 bp DNA Ladder marker containing 12 fragments from 1000 bp to 50 bp (LGC Biotechnology). The gel was analyzed in the High Perfomance Ultraviolet Translluminator (UVP Ultraviolet Products, Upland, USA) and the images captured in Alliance 9.7 photo-documenter (UVITEC Cambridget, Cambridget, England).

6. Plasmid DNA Extraction

[0077] The positive clones selected by the colony PCR were cultured in 5 mL of LB medium, at 37° C., for approximately 18 hours, under agitation, in the presence of the antibiotic of selection. The next day, they were centrifuged at room temperature, for 1 minute at 12000xg to sediment the intact bacteria. For the purification of plasmideal DNA, the GeneJet Plasmid Miniprep Kit (Thermo Fisher Scientific) was used, which is based on the alkaline lysis procedure combined with a selective binding column to the circular DNA, performed according to the manufacturer’s instructions. The purified DNA was analyzed on 0.8% agarose gel in 0.5X TAE buffer and subjected to the sequencing technique to confirm the insertion of the gene of interest to the vector, quantified in a spectrophotometer (ACTGene, New Jersey, USA) and stored at -20° C.

7. DNA Sequencing

[0078] Plasmid DNA samples were sequenced to confirm the correct insertion of the L2E6 gene and its integrity. For this purpose, the DNA samples purified from the gel as described in the previous session were quantified by spectrophotometry. A volume of 5 .Math.L of the plasmid was aliquoted and 2.5 .Math.L of the EAK03 primer was added at a concentration of 5 .Math.M. The samples were sent to the Human Genome Center, at the University of São Paulo, where the sequencing reaction took place on the ABI 3730 DNA Analyzer (Thermo Fisher Scientific).

8. Cell Lines and Cultivation Conditions

[0079] At first, to check the expression of the pcDNA3.3/L2E6 vector, the transfection process was performed on HEK293T cells cultured in Eagle medium modified by Dulbecco (DMEM, Cultilab, Campinas, SP, Brazil) supplemented with 10% fetal bovine serum (SBF, Cultilab, Campinas, SP, Brazil), D10, kept in a humid atmosphere, containing 5% CO.sub.2 until reaching 50-80% confluence. The raising occurred every 3 or 4 days.

[0080] The cell line chosen for the expression of recombinant proteins on a larger scale was the 293F, FreeStyle™ 293 Expression System (Life Techonologies™), which was developed to allow large-scale transfection of cells suspended in a defined and serum-free medium.

[0081] 293F cells were cultured in 30 mL of FreeStyle™ 293 Expression medium (Life Technologies™) in sterile glass flasks at 37° C. and 5% CO.sub.2, in a humid atmosphere, under agitation with rotation at approximately 135 rpm. Cell replays were performed every 3 or 4 days, with cell density of approximately 2 to 3x10.sup.6 viable cells/mL, where the cells were transferred to conical tubes and centrifuged at 800 rpm for 5 minutes. Then, cell viability was analyzed and the cells diluted to a final cell density of 3x10.sup.5 viable cells/mL in fresh preheated medium.

[0082] Trypan blue exclusion method (0.4% in PBS) and counting in a Neubauer chamber were used to determine cell viability.

9. Cell Transfection

[0083] The transient transfection assay of HEK 293T cells was carried out by lipofection using the GeneJuice® Transfection Reagents (EMD Biosciences/Merck, Darmstadt, Germany), according to the manufacturer’s specifications. Briefly, the transfection reagent was mixed by vortex to an aliquot of DMEM medium, being incubated for 5 minutes at room temperature. The plasmideal DNA of interest was added to the mixture, in the appropriate proportion, gently homogenized and incubated for 15 minutes at room temperature. Then, the Medium/transfection reagent/DNA mixture was added to the cells with complete culture medium, and incubated in an oven at 37° C. and 5% CO.sub.2. After the 8-hour period, the culture medium was changed, now with a complete medium containing antibiotics and 10% SBF. The incubation continued for approximately 48 hours when the cells were collected and analyzed in immunofluorescence and in SDS-PAGE electrophoresis gel, which will be described in later topics.

[0084] For 293F cells, transfection assays were performed according to the manufacturer’s instructions, using 293fectin transfection reagent.

[0085] One day before transfection, the cell density of viable 293F cells was determined and adjusted. In 28 mL of FreeStyle™ Expression Medium, 7x10.sup.5 cells were added, cultured under conditions described above.

[0086] The next day, a small aliquot of the cell culture was transferred to a microtube to determine the cell viability rate, which should remain around 90%. For the transfection of 3x107 viable cells, two solutions were prepared in different sterile conical tubes: [0087] Tube 1 - 30 .Math.g of plasmideal pcDNA3.3 / L2E6 DNA in 1 mL of final volume with Opti-MEM® medium, gently homogenized; [0088] Tube 2 - 60 .Math.L of 293fectin™ in 1 mL of final volume with Opti-MEM.sup.® medium, mixed gently and incubated for 5 minutes at room temperature.

[0089] Then, the contents of tubes 1 and 2, described above, were mixed gently resulting in a 2 mL solution, which was incubated for 30 minutes at room temperature, to allow plasmideal DNA to form complexes with 293fectin® reagent.

[0090] While the DNA-293fectin® complex was incubated, the cells in culture were centrifuged in conical tubes at 800 rpm for 5 minutes, with the medium in culture being replaced by 28 ml of fresh, preheated FreeStyle™ Expression Medium.

[0091] Finally, the 2 mL of the DNA-293fectin® complex was added to 28 mL of medium and cells in suspension, totaling 30 mL of solution for transfection, incubated 48 hours at 37° C. and 5% CO.sub.2, in a humid atmosphere, under shaking in an orbital shaker at approximately 125 rpm.

10. Expression and Characterization of Recombinant Proteins

10.1. Intracellular Expression of Proteins

[0092] After transfection, an aliquot of the cells was separated for analysis of the expression of the intracellular recombinant proteins through immunofluorescence assays.

[0093] HEK 293T cells were cultured on 13 mm diameter round coverslips in 12 well culture plates (TPP®). The easily adherent strains were grown directly on the coverslips in D10 medium, kept in a humid atmosphere, containing 5% CO.sub.2, for approximately 18 hours. In the following day, the cell transfection assay was performed as described in the previous item.

[0094] For assays using 293F cells already transfected, as previously described, they were centrifuged in microtubes and the supernatant discarded.

[0095] After the transfections, both cell lines were washed 2 times in PBS and fixed in a 1% paraformaldehyde solution in PBS, for 1 hour at 4° C. After successive washes to remove all fixative, the cells were incubated in a PBS+BSA 5% solution to block nonspecific bonds and incubated for 1 hour at room temperature. Then, the cells were washed in PBS for 3 times and then incubated with specific primary anti-HPV16 L2 or anti-HPV16 E6 antibodies diluted in 0.5% BSA and 0.05% Tween 20 in PBS for 1 hour at room temperature, shaking the tube frequently. After washing again, 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 room temperature, with frequent shaking in the tube containing the cells and the solution with the antibodies. The secondary antibodies used were AlexaFluor® 488 anti-mouse IgG produced in goat (Molecular Probes®, OR) and AlexaFluor® 633 anti-rabbit IgG produced in goat. After a final wash in PBS, 293F cells were distributed on glass coverslips coated with 0.5% poly-L-lysine in PBS.

[0096] Finally, the coverslips containing HEK 293T or 293F cells were mounted on slides with Mowiol (Calbiochem, CA, USA), containing propidium iodide (PI - Molecular Probes®, OR) that evidence the presence and location of nucleic acids.

[0097] The preparations were kept at 4° C., protected from light until the moment of their analysis in the Zeiss LSM 510 Meta laser-scanning confocal microscope.

10.2. Analysis of the Expression of Recombinant proteins

[0098] 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 10,000xg for 5 minutes, with the precipitate discarded and the supernatant stored at -20° C. until the time of use.

[0099] In order to confirm the transfection, the samples of cell lysates were analyzed in a faster and simpler way through dot blotting assays. Thus, 10 .Math.L of samples were exposed to nitrocellulose membrane strips (Bio-Rad Laboratories, Inc.). After drying, the strips were incubated in a solution containing 5% skimmed-milk powder in PBST for 1 hour, under agitation at room temperature. Then the membrane strips were washed 4 times in PBST, for 10 minutes, and incubated with anti-HPV16 L2 (Santa Cruz Biotechnology, Inc.) or anti-HPV16/18 E6 (Abcam®) monoclonal antibodies diluted in 1% skimmed-milk powder in PBST for 1 hour at room temperature, under stirring. Then, the strips were washed again for 10 minutes in PBST, 4 times, and incubated in secondary anti-mouse IgG antibody conjugated to horseradish peroxidase (Sigma-Aldrich®) diluted in PBST and 1% skimmed milk powder, for 1 more hour, stirring at room temperature. Then, the samples were washed for another 4 times of 10 minutes in PBST and the development was done by a DAB substrate solution (diaminobenzidine) - peroxidase (0.05% DAB, 0.015% H.sub.2O.sub.2, 0.01 M PBS, pH 7.2), incubated at room temperature for up to 10 minutes, under agitation and protected from light.

[0100] The positive samples in the dot blot assays were taken for analysis of the expression of recombinant proteins, carried out through Western blotting assays. For this purpose, a 16% Tricina-SDS-PAGE gel (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) was prepared and submitted to electrophoretic separation (30 V for 300 minutes), under denaturing conditions, containing samples of transfected cell lysates diluted in buffer of racing. Then, the gel was stained in Coomassie Blue solution (7% acetic acid, 50% Methanol, 0.1% Coomassie Brilliant Blue R-250) or the proteins were transferred to a nitrocellulose membrane for Western blotting assays. After electrophoretic transfer (30 V and 15 mA for 18 hours, or 70 V and 35 mA for 2 hours) in transfer buffer (0.025 M Tris, 0.192 M Glycine, 20% Methanol) at 4° C., the membrane was stained in a solution containing Ponceau temporary dye (Ponceau S 0.1%, acetic acid 5%) to confirm the transfer and marking of molecular weight indicators. Then, the membranes were washed in distilled water to remove temporary staining and incubated in a solution containing 5% skimmed-milk powder diluted in PBST (Tween 20 0.05%, PBS), for 1 hour, at room temperature, under agitation. After that period, the membranes were washed 4 times in PBST and incubated with 1% skimmed-milk powder solutions in PBST containing the primary antibodies anti-HPV16 L2 monoclonal, anti-HPV16 L2 produced in rabbit (kindly provided by Prof Dr RBS Roden, John Hopkins School of Medicine, USA) and/or anti-HPV16 E6 for 2 hours at room temperature, under stirring. Then, the membranes were again washed 4 times in PBST and incubated in a solution containing the respective secondary anti-mouse IgG or anti-rabbit IgG antibodies conjugated with horseradish peroxidase in 1% skimmed-milk powder solution in PBST, incubated for 2 hours at room temperature, under agitation. The reaction was detected using the Novex® ECL HRP Chemiluminescent Substrate Reagent (Invitrogen) kit on green light sensitive films (Kodak).

11. Protein Analysis

[0101] The samples of cell lysate were analyzed on 16% Tricina-SDS-PAGE electrophoresis gel, the most suitable for the separation of proteins from 1 to 100 KDa (SCHAGGER, 2006).

[0102] For this purpose, the 16% separation gel was first prepared, where the AB-6 buffer (acrylamide 46.5 g, bis-acrylamide 3 g) was diluted in gel buffer (Tris 3 M, HCl 1 M, 0.3% SDS, pH 8.45), plus 10% PA [(NH.sub.4) 2S.sub.2O.sub.8] and TEMED [ (CH.sub.3) 2NCH.sub.2CH.sub.2N (CH.sub.3) .sub.2]. The gel was quickly placed in the apparatus to polymerize. Then, a new 10% separation gel was prepared, using AB-3 buffer (48 g acrylamide, 1.5 g bis-acrylamide), diluted in the same gel buffer, water, 10% PA and TEMED. After preparation, the 10% gel was gently placed on the 16% gel. Finally, a stacking gel was prepared, where the AB-3 buffer was diluted in gel buffer, water, 10% PA and TEMED to a final concentration of 4%.

[0103] Protein samples were quantified and the concentrations adjusted so that each sample contains approximately 15 .Math.g of proteins. Protein dosage was performed using the Bradford method (Bio-Rad Laboratories Inc.), according to the manufacturer’s instructions. Thus, the samples were diluted in sample buffer (Tris-Cl 1 M pH 6.8, SDS 0.8%, bromophenol blue 0.1%, glycerol 40%, b-mercaptoethanol 14.4 M) and finally submitted to electrophoretic separation (30 V for 300 minutes) in tris-tricine buffer (0.1 M Tris; 0.1 M Tricine; 0.1% SDS, pH 8.5). After separation, the gel was stained with Coomassie blue solution.

12. Peptide Synthesis

[0104] To be used as positive controls of the peptides designed in the present study, synthetic peptides L2 (PEP2) and E6 (PEP1) were requested from FastBio Ltda. (Ribeirão Preto, SP).

[0105] PEP1, refers to the selected peptide of the HPV16 E6 protein, whose sequence YDFAFRDL, containing 8 amino acids and purity > 85%.

[0106] PEP2, selected from the HPV16 L2 protein, has the sequence LYKTCKQAGTCPPDIIPKV, with 19 amino acids and purity > 85%.

[0107] The peptides were received lyophilized and were resuspended in ultrapure water at a concentration of 5 mg/mL.

13. Cultivation of TC-1 Tumor Cells

[0108] Primary lung cells from C57BL/6 mice were transduced with the HPV16 E6 and E7 genes, then the immortalized cells were again transduced with the activated human c-Has-ras gene, generating the stable transformed cell line TC-1 that express the HPV16 protein E6 and E7, being able to generate solid tumors in mice with the same genetic background (LIN, 1996).

[0109] TC-1 cells were cultured in 75 cm.sup.2 polystyrene flasks (TPP – TPP Techno Plastic Products AG, Switzerland) in RPMI medium (Cultilab, Campinas, SP, Brazil), supplemented with 10% fetal bovine serum (SBF) (Cultilab, Campinas, SP, Brazil) at 37° C. and 5% CO.sub.2. The cells were replanted or the culture media changed every 3 or 4 days. For raising, trypsin / EDTA 250 mg% solution (Cultilab) was used to remove the cells adhered to the bottom of the culture flasks, incubated for up to 15 minutes, at 37° C. Then, the cells were centrifuged at 800xg for 15 minutes at 4° C. The supernatant is discarded and the cells resuspended in new culture medium. For cell line storage for long periods, aliquots of the cells were stored in a solution of RPMI culture medium containing 10% SBF, plus 10% DMSO, in liquid nitrogen.

14. In Vivo Experiments

[0110] In order to evaluate the possibility of using the vectors constructed as vaccine vectors, since the chosen plasmid, pcDNA, also allows such use, in vivo tests were performed.

[0111] For animal tests, the present invention was analyzed and approved by the Animal Use Ethics Committee of the Butantan Institute, under protocol number 923/12.

[0112] C57BL/6 male mice of 4 to 8 weeks provided by the Central Animal Hospital of the Butantan Institute, with an average weight of 20 g, were separated into 4 groups containing 10 animals in each group, kept in a vivarium with food and water ad libitum and controlled light cycle.

[0113] The animals were weighed weekly before and throughout the experiment.

14.1. Prophylactic Evaluation

[0114] Two groups of 4 or 6 animals were selected for prophylactic evaluation of the proposed DNA vaccine.

[0115] The first group, called group I, containing 4 animals received 50 .Math.L of intramuscular PBS solution in 3 doses in the interval of 14 days between the first (day 1) and second dose (day 14), and 7 days (day 21) between the second and third doses.

[0116] The other 6 animals that made up group II, received 3 doses of the vaccine construct pcDNA3.3/L2E6 in PBS solution, in decreasing concentrations. The first dose occurred on day 1 and the animals received 50 .Math.g of the DNA vaccine. The second dose was administered 14 days later, at a concentration of 20 .Math.g, while the third and last dose presented a concentration of 5 .Math.g of the vaccine vector, applied 7 days after the previous dose.

[0117] The vaccines were administered intramuscularly in the anterior tibial muscle of the hind leg, and the doses were administered intercalating between the right and left hind legs. The inoculated volume never exceeded 50 .Math.L of solution.

[0118] Seven days after the last dose (day 28), all animals in both groups were challenged with 10.sup.5 TC-1 cells in PBS solution via the subcutaneous route in the left dorsolateral region of each animal. The mice were observed daily and 8 days after the implementation of the tumor cells it was possible to detect the formation of a palpable mass. From the detection of the tumors, they were measured every 2 days with the aid of a digital caliper (Western®, China). The tumors grew in irregular shapes and were measured for length and the perpendicular axis, width, in the same plane. From these data, the tumor volume of each mouse in cm.sup.3 was calculated, the data were grouped and analyzed statistically using Student’s t-tests (p = 5.00%), and the Graphic-Pad Prism 6 software, version 3.0.

[0119] Small blood aliquots, approximately 100 .Math.L, were collected from all animals on days 0, 14, 28, 42. The mice were euthanized about 3 weeks after the implantation of the tumors (day 49), following the instructions ethical standards in animal experimentation (TAMBOURGI et al., 2010) and all blood was collected.

[0120] From the blood samples, the animals’ serum was collected by centrifugation at 800 rpm, where approximately 30 .Math.L of serum were obtained and stored at -20° C. until the time of analysis.

14.2. Therapeutic Evaluation

[0121] To evaluate the efficiency of the use of the constructed vaccine vector, pcDNA3.3/L2E6, in the treatment of cancers associated with HPV, 3 animal groups were selected for the trials.

[0122] The groups were formed by 10 animals and challenged with 7x10.sup.4 TC-1 cells in DMEM culture medium and inoculated subcutaneously in the left dorsolateral region of each animal. Previously, the area was prepared by shaving the hair to facilitate the application of tumor cells by the correct route.

[0123] The day after tumor cell inoculation, day 1, the animals in the control group, group III, received 5 .Math.g of the empty vector, pcDNA3.3, and on day 10, a second dose containing 5 .Math.g of the same vector was administered.

[0124] The animals were monitored daily and the region was palpated to check the tumor implantation. As soon as the tumors were detected, they were measured manually with the aid of a digital caliper every 2 days, or until the tumors reached approximately 20 mm in diameter, when the animals were euthanized.

[0125] For the experimental tests, the animals were divided into 2 new groups, where the first group, group IV, received the first dose of the DNA vaccine, pcDNA3.3/L2E6, on the first day of experimentation, and the second dose, at 15 days. The first dose with 5 .Math.g of vector pcDNA3.3/L2E6, while the second dose was composed of 5 .Math.g of the vaccine vector. These animals also received a third vaccine dose of 5 .Math.g at 28 experimental days. Both doses were diluted in PBS solution.

[0126] Animals from the second experimental group, group V, received the first dose of the DNA vaccine containing 5 .Math.g of pcDNA3.3/L2E6 on the first experimental day, that is, 1 day after receiving the tumor cells, and the second dose 10 days after the first one containing 5 .Math.g.

[0127] The animals were monitored daily and the region was palpated to check tumor growth. If detected, the tumors would be measured manually with the aid of a digital caliper every 2 days, or until the tumors reached approximately 20 mm in diameter, when the animals would be euthanized.

[0128] Small aliquots of blood were taken on days 0, 21, 28 and 49 to obtain serum and stored at -20° C. until the time of use.

[0129] The spleens of the animals that were part of the groups tested for the therapeutic action of the DNA vaccine, groups III, IV and V, were collected in a sterile environment, washed twice in sterile PBS solution and cultured as described below.

15. Splenocyte Culture

[0130] The spleen removed from each animal, after double washing in sterile PBS solution, was macerated against a piece of sterile gauze with the aid of a syringe plunger in a Petri plate. 3 ml of RPMI culture medium supplemented with 10% fetal bovine serum (R10) was added to the macerate. The cells were collected in a sterile conical tube and centrifuged at 12000 rpm for 5 minutes.

[0131] After centrifugation, the supernatant was discarded and the red blood cells were lysed by adding 500 .Math.L of sterile distilled water, homogenized quickly and adding another 500 .Math.L of PBS solution twice concentrated and sterile, homogenizing well. After another centrifugation at 1000 rpm for 10 minutes, the supernatant was again discarded and the precipitate was resuspended with 1 mL of R10 medium.

[0132] Viable spleen cells were counted with the help of trypan blue dye in 0.4% solution in PBS in a Neubauer chamber.

[0133] The cells were distributed in triplicate in a 12-well culture plate at a concentration of 10.sup.6 cells/ml, the volume being made up to 1 ml of R10 medium. 10 .Math.L of the synthetic peptide PEP1 (E6), described above, was added to each well. Then, the plates containing the splenocytes and PEP1 were incubated for 48 hours at 37° C. and 5% CO.sub.2, in a humid environment. After this period, the culture supernatant was recovered and stored at -80° C. until the moment of cytokine dosage analyzes, described below.

16. Cytokine Detection Assay

[0134] From serum aliquots collected from the animals under experiment and from the splenocyte culture supernatant, dosages of soluble cytokines were performed with the aid of the BD CBA Mouse Th1/Th2 Cytokine kit (BDTM Biosciences, USA) according to manufacturer specifications.

[0135] Briefly, the samples were separated by groups and experimental dates, being analyzed in pools only the serum samples collected on days 0, 28 and 49, as well as the samples of the splenocyte culture supernatant. The samples were incubated with beads coated with specific antibodies against 5 different types of cytokines (IL-2, IL-4, IL-5, INF-g and TNF), together with the detection reagent conjugated to fluorescent protein PE (Ficoeritrina) and incubated for 2 hours at room temperature, protected from light.

[0136] Then, the beads were washed with washing buffer (kit component) and resuspended in 300 .Math.L of washing buffer, being then analyzed in a flow cytometer, BD FACSCanto II (BD™ Biosciences). The data were analyzed using FCAP Array software version 3.0 (BD™ Biosciences).

17. Indirect ELISA

[0137] For analysis of the induction of humoral immune response by the pcDNA3.3/L2E6 vaccine, ELISA assays (Enzymed-Linked ImmunoSorbent Assay) were performed, where the flat-bottom plates with high adhesion capacity (Sarstedt, Numbrecht, Germany) were sensitized with 0.6 mg/ml PEP1 or 0.3 mg/ml PEP2 in carbonate-bicarbonate buffer (0.2 M Na.sub.2CO.sub.3, 0.2 M NaHCO.sub.3, pH 9.6) and incubated for 18 hours at 4° C. The next day, the antigen was recovered and the plate was washed 3 times with PBST. Then, 150 .Math.L of PBST solution + 5% skimmed-milk powder were added to block nonspecific bonds and incubated for 1 hour at room temperature. The plate was washed again 3 times with PBST and in each well the serum samples collected from the animals were distributed on days 0, 28 and 49, diluted 1:100 in PBST solution + 1% skimmed milk powder. The primary antibodies were incubated for 2 hours at room temperature. After this period, the samples were discarded and the plate was washed 3 more times with PBST solution. Then, 100 .Math.L of secondary antibody, anti-mouse IgG produced in sheep conjugated to peroxidase (Sigma-Aldrich), diluted 1:250 in PBST solution + 1% skimmed milk powder and incubated for 1 hour and 30 minutes at room temperature. The plate was washed again 3 times with PBST solution and 1 time with PBS. Finally, each well received 100 .Math.L of developer solution (0.05 M CeH.sub.8O.sub.7, 0.05 M Na.sub.2HPO.sub.4, pH 5) in which an OPD tablet (o-phenylenediamine dihydrochloride, Invitrogen) containing 5 mg and 12 .Math.L of H.sub.2O.sub.2 was added to 30%. Incubated for 15 minutes at room temperature in a place protected from direct light. Then, each well received 50 .Math.L of 2.5 M H.sub.2SO.sub.4 solution to stop the reaction. The plate was then read on a MultiSkan EX Reader (LabSystems) at 492 nm. The samples were tested in triplicate.

[0138] The analysis of the results was performed by calculating the cut-off point (“cut off”) by the average of the negative samples plus 3 times the value of the standard deviation(s) of these samples, with an additional 10% of the final value. The number of standard deviations used in the formula guarantees the confidence level of the result.

[0139] Cutoff point = average + 3x s + 10%

[0140] All points obtained above this cutoff point were considered to be positive samples.

18. Statistical Analysis

[0141] Statistical analyzes were performed with the aid of the GraphPad Prism.sup.® software (GraphPad Software, Inc.). Student’s t tests (p = 0.05%) were used for statistical analysis on data obtained in animal experiments, animal weight and tumor volume. In the cytokine detection tests, the ANOVA parametric test (p <0.05) was used.

19 Bioinformatics Analysis of the L2E6 Sequence

[0142] From the DNA sequence coding for the defined L2E6 protein, in silico analyzes were performed to better understand the product to be generated and the characteristics of the protein to assist the development of the present study.

[0143] Table 1 shows the bioinformatics analyzes of the L2E6 sequence. From the DNA sequence coding for the defined L2E6 protein, in silico analyzes were performed to better understand the product to be generated and the characteristics of the protein to assist in the development of the present study. Several software and online sites for proteomic analysis have emerged in recent years, based on algorithms that predict protein characteristics from their amino acid sequences. One of the most used and described sites in the literature capable of predicting the physicochemical characteristics of proteins is http: //protcalc.sourceforge.net/, which estimates molecular mass, pH, pI and performs the counting of amino acid residues. Several reference sites were consulted and the data were analyzed and compiled in the table below, where the defined sequence QLYKTCKQAGTCPPDIIPKVGGSGYDFAFRDL with 32 amino acid residues showing the other characteristics:

TABLE-US-00006 Physico-chemical properties of the L2E6protein drawn in the present study, according to the Protein Calculator website (http://protcalc.sourceforge.net/) Physico-chemical properties Molecular mass Approximately 3.5 KDa pI 7.98 Estimated load at pH 7.0 0.9

[0144] Using other prediction sites for possible post-translational changes, several parameters were analyzed through the website of the Center for Biological Sequence Analysis – CBS – of the Technical University of Denmark through the link http://www.cbs.dtu.dk/index.shtml.

[0145] The analyzes demonstrated that the developed L2E6 peptide presents prediction sites for acetylation, phosphorylation, leucine-rich sites indicating signs of export to the nucleus and a cleavage site between amino acid residues 23 and 24, generating a signal peptide with possible signaling of export of this protein to the transmembrane region. In addition to the presence of 2 cysteine amino acid residues, which can lead to the formation of disulfide bonds with post-translational modifications generating possible changes in the conformation and stability of the protein.

[0146] FIG. 2 shows the photodocumentation of electrophoresis gel in 0.8% agarose for selection of bacterial colonies after transformation with the pcDNA3.3/L2E6 vector. After the bacterial transformation assays to amplify the vectors, the bacterial colonies were submitted to PCR to select the positive clones. The PCR products were analyzed on an agarose gel and subjected to electrophoresis (FIG. 2), where (MM1) represents the universal marker and (MM2) the 50 bp DNA Ladder marker. In (1) we have a sample of the intact plasmid pcDNA3.3/L2E6, in (2) and (3) PCR products from bacterial colonies transformed by pcDNA3.3/L2E6 and (4) PCR product from an untransformed colony.

[0147] Thus, the colony presented in column 3 (FIG. 2) was selected for amplification of the bacterial colony and then for the purification of plasmideal DNA. The recovered plasmideal DNA was quantified by spectrophotometry, resulting in an average of 150 ng/pL per sample.

[0148] The sequencing of the plasmideal DNA recovered and quantified as previously described confirmed the correct formation and sequence of the L2E6 gene developed in the present work.

[0149] FIGS. 3 and 4 show the evaluation of the intracellular expression of the recombinant fused protein L2E6, performed in HEK293T (FIG. 3) and 293F cells (FIG. 4).

[0150] Through indirect immunofluorescence assays it was possible to evidence the expression of the recombinant protein, being recognized by the specific anti-L2 commercial monoclonal antibody. The protein was visualized as dots distributed throughout the cell (FIGS. 3 and 4).

[0151] FIG. 5 shows the analysis of the expression of the recombinant protein L2E6. After transfection of 293F cells with the pcDNA3.3/L2E6 vector, the cells were collected and lysed as previously described. The cell lysate was analyzed on SDS-PAGE 12%, as seen in FIG. 5. The theoretical molecular mass of the recombinant L2E6 protein produced would be approximately 3.5 KDa.

[0152] The comparison of the running profile of the cell lysate samples from non-transfected and transfected cells revealed the presence of a very low molecular weight protein, below 8 KDa, present only in the transfected samples, suggesting that it is the recombinant protein L2E6. Additionally, Western blotting assays were performed to confirm the expression of the recombinant proteins and the recognition of these proteins by specific commercial monoclonal antibodies anti-L2 and anti-E6, to confirm the identity of the expressed protein. The results obtained revealed that both 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 (FIG. 5).

[0153] The photodocumentation of the result obtained from the Western blotting assay used for detection and characterization of the recombinant protein L2E6, where the samples (1) and (5) are aliquots of non-transfected cells lysate, (2) and (6) lysate of transfected cells expressing L2E6, (3) and (7) eluted intermediates obtained from the filtration gel chromatography assay and interacting with the specific monoclonal antibodies anti-L2 (3) and anti-E6 (7), demonstrating the presence of a low molecular weight protein being recognized by both commercial antibodies, (4) final elution sample from the gel filtration process. (MM) Color Burst™ molecular marker.

[0154] FIG. 6 shows the prophylactic evaluation in in vivo tests performed in the present work, in experiments to analyze the use of the vector as a DNA vaccine in a murine model. The animals were separated into 4 experimental groups, being evaluated for the possibility of inducing prophylactic and therapeutic protection to the challenge with TC-1 tumor cells.

[0155] From the weekly weighing of the animals it was possible to observe that the weight remained constant throughout the experiment. There was a small difference in weight between the animals of the experimental and control group, which received only PBS, since day 0 (zero) experimental, however such difference is not statistically significant, according to Student’s t test, suggesting that the vaccine of DNA did not interfere in the animals’ weight gain or loss (FIG. 6) .

[0156] FIG. 7 shows that one week after the application of the 3 doses of the DNA vaccine proposed in the present study, the animals were challenged with tumor cells TC-1 to assess whether there was an induction of protective immune response and its prophylactic protective capacity, in the tumor development of HPV-positive cells. From the moment the tumors were established and became palpable, they were measured every 2 days and the data analyzed. The tumor volume increased throughout the experiment, demonstrating that there was a tumor implantation with tumor development. There was no statistically significant difference between animals in the same group, just as it was not possible to observe a difference in the tumor volume of animals between experimental and control groups (FIG. 7), suggesting that the DNA vaccine used prophylactically did not induce an immune response capable of prevent or control tumor development. However, such a result was expected, since the prophylactic capacity of the developing vaccine would be limited in its ability to induce the production of specific antibodies capable of preventing the infection of cells by HPV.

[0157] FIG. 8 shows the therapeutic evaluation of the DNA vaccine. Throughout the experimental procedure, the animals were weighed weekly to monitor their development and possible changes induced by the application of DNA vaccines. TC-1 cells, when applied subcutaneously, have the ability to establish and form solid and non-invasive tumors, however their growth is fast and capable of inducing the formation of external wounds on the animals’ skin. Thus, after application of the tumor cells, the animals were monitored for tumor development, and when they induced formation of exposed wounds or when the largest diameter of the tumor mass reached 200 mm, the animals were euthanized. Thus, all animals in the control group, group II, which received tumor cells and did not receive the DNA vaccine, receiving only the empty vector, were euthanized on the twenty-eighth experimental day. Thus, in the group of animals that received the tumor cells and the treatment with the empty vector, it was possible to observe that the tumor implantation induced the weight loss of the animals since the seventh day after the inoculation of the neoplastic cells, however this weight difference is not statistically significant. Only on the twenty-eighth experimental day it was possible to observe a great decrease in weight in the animals (Groups III and IV, FIG. 8), being statistically significant when compared to the previous weighings (p>0.0001). Within the same group there was no significant difference in the animals’ weight (FIG. 8). The animals that received tumor cells and the pcDNA3.3/L2E6 vaccine did not demonstrate significant weight loss during the entire experiment, including the group that received the vaccines in shorter periods between doses, group V, the growth of the animals showed to be continuous throughout the experiment.

[0158] FIG. 9 shows that from the tenth day after inoculation of TC-1 cells it was already possible to detect the presence of a palpable tumor mass, but not yet measurable in all animals in group III, which received the vaccine containing the vector empty. However, with the rapid growth of tumors, measurements of tumor diameters were started after 2 days, followed by measurements every 2 days. The data obtained demonstrate rapid tumor growth, with similar tumor volumes among animals in the same group. Regarding animals of group IV, which received 3 doses of pcDNA3.3/L2E6 vaccine at 15-day intervals, 3 animals in the group developed solid tumors from the 12.sup.th experimental day on. With the rapid development of the tumors, these 3 animals were euthanized on the twenty-eighth experimental day. The remaining 7 animals in the group did not develop tumors throughout the analyzed period.

[0159] Regarding animals of group V, which received only 2 doses of the pcDNA3.3/L2E6 vaccine at 10-day intervals, 8 of them did not develop any tumor associated with the application of TC-1 cells, being maintained throughout the experimental period. Two animals in this group developed a tumor, but with much smaller volumes when compared to animals in the control group, III.

[0160] Among groups, the difference in tumor volumes was statistically significant (p<0.0001), immediately after the immunization of the second dose of the DNA vaccine, suggesting that it induced an immune response sufficiently capable of containing tumor growth in animals of group IV. Over time, this difference in tumor volume between groups III and IV became more evident, as can be seen in FIG. 9. The tumor volumes of animals in the group that received the DNA vaccine, group IV, remained increased, although only slightly, suggesting stability up to 14 days after the last dose, when the measurements again showed a small increase, but not statistically significant. The animals in group V, on the other hand, those that presented the formation of tumor masses, were growing at a very slow pace, when compared to the other animals that presented tumor development. And the final volume reached by these tumors was much smaller when compared to the other animals.

[0161] Table 2 shows the analysis of cytokine production.

[0162] From the sera obtained from the animals used in the experiments, cytokine detection and dosage tests were performed in an attempt to trace an immune response profile induced by the DNA vaccine.

[0163] A pool of serum samples from days 0, 28 and 49 were separated by groups and with the aid of the BD CBA Mouse Th1/Th2 Cytokine kit. In all tested samples it was possible to detect the presence of the analyzed cytokines, however the results obtained for the IFN, IL-2, IL-4 and IL-5 cytokines, the concentrations found were low and it was not possible to observe differences in the concentrations of the samples collected before and after treatments, as can be seen in Table 2, below (Table 2).

[0164] The statistical analysis using the Mann & Whitney non-parametric test revealed that there was no significant difference in the TNF concentrations detected between the control and experimental groups (p<0.05) .

TABLE-US-00007 Levels of cytokines detected in serum samples Groups IFN (pg/mL) IL-2 (pg/mL) IL-4 (pg/mL) IL-5 (pg/mL) Day 0 Day 49 Day 0 Day 49 Day 0 Day 49 Day 0 Day 49 Group I (Prophylactic – Control) N/D.sup.# N/D.sup.# N/D.sup.# N/D.sup.# N/D.sup.# N/D.sup.# N/D.sup.# N/D.sup.# Group II (Prophylactic – Experimental) N/D.sup.# N/D.sup.# N/D.sup.# N/D.sup.# N/D.sup.# N/D.sup.# N/D.sup.# N/D.sup.# Group III (Therapeutic – Control) 16.53 11.02 10.65 9.10 11.61 8.00 13.77 9.11 Group IV (Therapeutic – Experimental 1) 15.13 16.23 9.96 10.81 0.79 11.45 3.85 13.70 Group V (Therapeutic – Experimental 2) 17.03 17.64 1.14 11.18 1.77 11.12 3.92 15.35

[0165] However, in the TNF (tumor necrosis factor) analyzes of the serum samples, it was possible to observe a significant increase in the concentrations of this cytokine on the last experimental day, 49, as can be seen in the table below (Table 3).

TABLE-US-00008 Levels of TNF detected in the serum of the animals studied Groups TNF dosage (pg/mL) Day 0 Day 28 Day 49 I – Prophylactic evaluation, control group 7.06 4.44 5.80 II – Prophylactic evaluation, vaccinated group 4.80 5.02 4.80 III – Therapeutic evaluation, control group 4.44 6.02 — IV – Therapeutic evaluation, vaccinated group, doses in 15 days 5.95 7.52 12.33* V – Therapeutic evaluation, vaccinated group, doses in 10 days 5.08 7.17 5.12

[0166] The same cytokine detection analyzes were also performed with the samples of the splenocyte culture supernatant when incubated with aliquots of the synthetic peptide PEP1 (E6). The levels of IFN, IL-2, IL-4 and IL-5 were not detected, as the concentrations found were all 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).

TABLE-US-00009 TNF levels detected in the supernatant of the splenocyte culture Groups TNF dosage (pg/mL) III - Therapeutic evaluation, control group 42.02 IV - Therapeutic evaluation, experimental group, doses in 15 days 85.31* V - Therapeutic evaluation, experimental group, doses in 10 days 97.40*

[0167] FIGS. 10 and 11 show the analysis of the production of specific antibodies. Serum samples collected from all animals and groups were analyzed in indirect ELISA assays, to demonstrate whether there was an induction of humoral immune response induced by the pcDNA3.3/L2E6 vaccine with the production of specific anti-L2 or anti-E6 antibodies. The samples were tested in triplicate and their results were analyzed as previously described. The data obtained demonstrated that in the samples of day 0 no anti-L2 or anti-E6 antibodies were detected in any of the animals, as expected. However, in samples on the 28th it was already possible to detect the presence of specific antibodies at high levels, however, the peak of reactivity was found on day 49 and the presence of specific anti-L2 (FIG. 10) and anti-E6 antibodies (FIG. 11) was then demonstrated. The animals in group IV that developed tumors were the same ones that showed less capacity to produce anti-L2 and anti-E6 antibodies, receiving the numbers 5, 6, 7 and 9. The 2 animals in group V that developed solid tumors also had the lowest levels of production of anti-L2 and anti-E6 antibodies, being 4 and 5. The data of the animals that do not appear in the figures below represent those that had a greater tumor development, not resisting until the 49 experimental days, being euthanized when reaching the previously mentioned parameters.

[0168] Thus, although only some embodiments of the present invention have been shown, it will be understood that various omissions, substitutions and changes in the prophylactic and therapeutic DNA vaccine against HPV and cancers associated with the virus can be made by a person skilled in the art, without depart from the spirit and scope of the present invention.

[0169] It is expressly provided that all combinations of elements that 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 embodiment described to another are also fully intended and contemplated.

[0170] It is also necessary to understand that the drawings are not necessarily to scale, but that they are only of a conceptual nature. The intention is, therefore, to be limited, as indicated by the scope of the attached claims.