DNA vaccine against human papillomavirus and method of use thereof

Abstract

The present disclosure provides a DNA vaccine for subjects with human papillomavirus (HPV)-associated disease. The DNA vaccine may comprise a DNA construct comprising a fusion gene. The fusion gene may be a subsegment of the DNA construct comprising an optimized HPV partial sequence encoding at least one HPV antigen. The optimized HPV partial sequence may comprise one or more of the HPV-16 E6 expression gene set forth in SEQ ID NO:1, the HPV-16 E7 expression gene set forth in SEQ ID NO:2, the HPV-18 E6 expression gene set forth in SEQ ID NO:3, and the HPV-18 E7 expression gene set forth in SEQ ID NO:4.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 117,473, entitled "HUMAN PAPILLOMAVIRUS DNA VACCINE," filed November 24, 2020, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing Reference In this application: January 7, 2022 Created on the name " 220107-137111-sequence-listing-v1F ",size 19,844 Accompanying is a sequence listing as an American Standard Code for Information Interchange (ASCII) text file of 10 bytes, which is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates generally to DNA vaccines, particularly DNA vaccines against HPV-associated diseases, and more particularly to improving DNA vaccines through codon optimization. [Background technology]

[0004] Human papillomavirus (HPV) is the causative agent in several human cancers, including cervical cancer, anal cancer, penile cancer, vulvar cancer, vaginal cancer, and head and neck cancer. Current vaccines against HPV, such as Gardasil® and Cervarix®, have shown clinical efficacy in preventing HPV infection, but are ineffective in treating patients who already have HPV infection or HPV-related cancer. Therefore, there is a strong need to develop a therapeutic vaccine for patients infected with HPV or even suffering from HPV-related diseases.

[0005] Deoxyribonucleic acid (DNA) vaccination is a technique for protecting against infection or treating disease by injecting genetically engineered plasmids containing DNA sequences encoding one or more antigens. DNA vaccines have theoretical advantages over conventional vaccines, including safety, speed, and predictability of production, temperature stability, flexibility in design, and the ability to induce a broader range of immune response types. Despite the above advantages, DNA vaccines generally have obstacles to inducing strong antigen-specific immune responses in animals. For example, DNA vaccination with HPV E6 and E7 genes may be poorly immunogenic because the virus has evolved mechanisms to evade host recognition, including low levels of E6 / E7 expression. Thus, a number of different strategies are being tested to enhance DNA vaccine immunogenicity, including improved vector design, antigen codon optimization, the use of conventional and molecular adjuvants, electroporation (EP), co-expression of molecular adjuvants, and prime-boost strategies (L. Li and N. Petrovsky, Expert Rev Vaccines. 2016;15 (3): 313-29).

[0006] Codon optimization refers to the technique of using synonymous mutations to increase protein expression of a gene of interest, such as an antigen-expressing gene in a DNA vaccine. However, codon optimization does not necessarily correlate positively with DNA vaccine efficacy. Many studies have shown that rare codons may not necessarily be the speed-limiting step, and frequently used codons do not guarantee increased protein production. Furthermore, coding regions not only specify amino acid sequences, but also often contain redundant genetic information, including RNA secondary structures that may affect protein folding. It has been shown that synonymous codons potentially alter protein conformation and function (L. Li and N. Petrovsky, Expert Rev Vaccines. 2016;15 (3):313-29; VP Mauro and SA Chappell, Methods Mol Biol. 2018;1850:275-288). Therefore, the use of codon optimization to enhance the expression of an encoded antigen in a DNA vaccine may cause unexpected adverse consequences and requires careful consideration. Furthermore, it is more difficult to predict whether a DNA vaccine containing a codon-optimized sequence will achieve the desired immunogenicity. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] L. Li and N. Petrovsky, Expert Rev Vaccines. 2016;15 (3): 313-29 [Non-Patent Document 2] VP Mauro and SA Chappell, Methods Mol Biol. 2018;1850:275-288 Summary of the Invention [Problem to be solved by the invention]

[0008] Considering the above reasons, there is an unmet need to provide DNA vaccine with better efficacy in treating HPV-related disease.However, the efficacy of DNA vaccine against HPV-related disease is still highly unpredictable by codon optimization, and therefore there is still a problem to be solved in this field. [Means for solving the problem]

[0009] In some embodiments of the present disclosure, a DNA vaccine for a subject with a human papillomavirus (HPV)-associated disease may be provided. The DNA vaccine may include a DNA construct comprising a fusion gene. The fusion gene may be a subsegment of the DNA construct comprising an optimized HPV subsequence encoding at least one HPV antigen. The optimized HPV subsequence may include one or more of the HPV-16 E6 expression gene set forth in SEQ ID NO:1, the HPV-16 E7 expression gene set forth in SEQ ID NO:2, the HPV-18 E6 expression gene set forth in SEQ ID NO:3, and the HPV-18 E7 expression gene set forth in SEQ ID NO:4.

[0010] In some embodiments, the fusion gene may further comprise a subsequence encoding an immunostimulant.

[0011] In some embodiments, the immunostimulant may be 70 kilodalton (kDa) heat shock protein (HSP70) or human calreticulin (CRT) protein.

[0012] In some embodiments, the optimized HPV partial sequence may include HPV-16 E6, HPV-16 E7, HPV-18 E6, and HPV-18 E7 expression genes, and the fusion gene may include SEQ ID NO:5.

[0013] In some embodiments, the fusion gene may further comprise an optimized signal sequence, and the fusion gene may comprise SEQ ID NO:6.

[0014] Another aspect of the present disclosure may provide a method for treating HPV-associated disease in a subject in need thereof. The method may include administering to the subject the DNA vaccine described above.

[0015] In some embodiments, the method may further include administering to the subject a recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18, where the DNA vaccine is administered as a priming vaccine and the recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 is administered as a boosting vaccine.

[0016] In some embodiments, the recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 can be a TA-HPV.

[0017] In some embodiments, the TA-HPV is 1×10 4 2 × 10 plaque-forming units (pfu) 8 The vaccine may be administered in doses ranging from up to pfu.

[0018] In some embodiments, the DNA vaccine may be administered at doses ranging from 100 micrograms per subject to 20 milligrams per subject.

[0019] So that the features of the present disclosure described above can be understood in detail, a more particular description of the present disclosure, briefly summarized above, can be had by reference to the embodiments, some of which are illustrated in the accompanying drawings, in which it should be noted, however, that the accompanying drawings illustrate only typical embodiments of the present disclosure and therefore should not be considered as limiting its scope, as other equally effective embodiments may be recognized in the present disclosure. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a schematic diagram illustrating a DNA construct according to some embodiments of the present disclosure. [Diagram 2] 1 is a western blot image showing expression levels of HPV antigens in human embryonic kidney (HEK) 293 cells transfected with either pBI-1, pBI-10.1, pBI-11, or pBI-12, according to certain embodiments of the present disclosure. [Figure 3A] FIG. 3B is a bar graph comparing the percentage of IFN-γ+ CD8+ T cells / total CD8+ cells in control, pBI-10.1, pBI-11, and pBI-12 groups in an in vitro T cell activation assay using mouse H-2Db-restricted HPV-16 E7 peptide (amino acids 49-57)-specific CD8+ T cells ( FIG. 3A ) to compare the ability of HPV antigen presentation by cells transfected with each of the DNA constructs according to an embodiment of the present disclosure. [Figure 3B] FIG. 3B is a bar graph comparing the percentage of IFN-γ+ CD8+ T cells / total CD8+ cells in control, pBI-10.1, pBI-11, and pBI-12 groups in an in vitro T cell activation assay using mouse H-2Kb-restricted HPV-18 E6 peptide (amino acids 67-75)-specific CD8+ T cells ( FIG. 3B ) to compare the ability of HPV antigen presentation by cells transfected with each of the DNA constructs according to an embodiment of the present disclosure. [Figure 4A] FIG. 1 is a schematic diagram of the experimental design for comparing HPV-16 E7-specific CD8+ T cell responses generated by various DNA constructs, according to certain embodiments of the present disclosure. [Figure 4B] 1 is a bar graph comparing the number of HPV-16 E7-specific IFN-γ+ CD8+ T cells per 3×105 splenocytes after preparation of splenocytes from mice vaccinated with either pBI-10.1, pBI-11, or pBI-12 stimulated with HPV-16 E7 (amino acids 49-57) peptide, according to an embodiment of the present disclosure. [Figure 5A]FIG. 1 is a schematic diagram of the experimental design of an in vivo tumor treatment experiment using the HPV-16 E6 / E7+ TC-1 tumor model, according to certain embodiments of the present disclosure. [Figure 5B] 1 is a bar graph showing the percentage of HPV-16 E7-specific CD8+ T cells / total CD8+ cells in peripheral blood mononuclear cells (PBMCs) prepared from untreated mice or mice treated with pBI-10.1, pBI-11, or pBI-12 using HPV-16 E7 (amino acids 49-57) peptide-loaded tetramer staining using a two-tailed Student's t-test, according to an embodiment of the present disclosure. [Figure 5C] 1 is a bar graph showing the percentage of HPV-18 E6-specific CD8+ T cells / total CD8+ cells in PBMCs prepared from untreated mice or mice treated with pBI-10.1, pBI-11, or pBI-12 using HPV-18 E6 (amino acids 67-75) peptide-loaded tetramer staining using a two-tailed Student's t-test, according to certain embodiments of the present disclosure. [Figure 5D] 1 is a curve chart showing TC-1 tumor volume in untreated mice and mice treated with pBI-10.1, pBI-11, or pBI-12 in an in vivo tumor treatment experiment according to an embodiment of the present disclosure. [Figure 5E] 1 is a Kaplan-Meier survival curve showing the survival probability of untreated mice and mice treated with pBI-10.1, pBI-11, or pBI-12 in an in vivo tumor treatment experiment according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] According to common practice, the various description features are not drawn to scale but rather to emphasize features relevant to the present disclosure. Like reference characters refer to like elements throughout the drawings and text.

[0022] The present disclosure will now be described in more detail below with reference to the accompanying drawings, in which exemplary embodiments of the present disclosure are shown. However, the present disclosure may be embodied in many different forms and should not be construed as being limited to the exemplary embodiments set forth herein. Rather, these exemplary embodiments are presented so that this disclosure will be thorough and complete, and so that the scope of the disclosure will be fully conveyed to those skilled in the art. Like reference numerals refer to like elements throughout.

[0023] The terminology used herein is merely for the purpose of describing certain exemplary embodiments and is not intended to limit the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural unless the context clearly indicates otherwise. It will further be understood that the terms "comprises" and / or "comprising" or "includes" and / or "including" or "has" and / or "having" as used herein specify the presence of the stated features, regions, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0024] Unless otherwise defined, all terms used herein (including scientific and technical terms) have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Furthermore, terms defined in commonly used dictionaries, etc. should be interpreted to have a meaning consistent with their meaning in the context of the relevant technical field and this disclosure, and will not be interpreted in an ideal or overly formal sense unless expressly defined in this specification.

[0025] The DNA vaccine of the present disclosure is provided to a subject with a human papillomavirus (HPV)-associated disease. The subject with an HPV-associated disease may be a mammal (e.g., a human, a mouse, etc.) suffering from, but not limited to, warts, papillomas, intraepithelial neoplasia, penile cancer, vaginal cancer, vulvar cancer, anal cancer, oropharyngeal cancer, non-melanoma skin cancer, conjunctival cancer, or cervical cancer.

[0026] The DNA vaccine may comprise a DNA construct comprising a fusion gene, wherein the fusion gene comprises a codon-optimized human papillomavirus (HPV) subsequence. The DNA vaccine may also be referred to as an optimized DNA vaccine in the context described herein.

[0027] A synthetic fusion gene may be provided that includes a codon-optimized subsequence (e.g., an optimized HPV subsequence) encoding at least one HPV antigen. Specifically, the optimized HPV subsequence may include one or more of an HPV-16 E6 expression gene encoding the HPV-16 E6 antigen set forth in SEQ ID NO:1, an HPV-16 E7 expression gene encoding the HPV-16 E7 antigen set forth in SEQ ID NO:2, an HPV-18 E6 expression gene encoding the HPV-18 E6 antigen set forth in SEQ ID NO:3, and an HPV-18 E7 expression gene encoding the HPV-18 E7 antigen set forth in SEQ ID NO:4.

[0028] In some embodiments, the fusion gene can be cloned into the pNGVL4a plasmid, which functions as a vector, to provide a DNA construct, thus making the fusion gene a subsegment of the DNA construct. In further embodiments, alternative and / or additional plasmids can be used to provide the vectors described herein.

[0029] In some embodiments, the DNA vaccine may further include components such as adjuvants to generate a stronger immune response in the subject receiving the vaccine.

[0030] In some embodiments, to provide a combination therapy used in a heterologous prime boost regimen to enhance a subject's immune response to HPV-associated disease, a DNA vaccine may be administered as a priming vaccine, and a recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 may be subsequently administered as a boosting vaccine. For example, the DNA vaccine may be administered at a dose ranging from 100 micrograms per subject to 20 milligrams per subject, and a recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 may be administered at a dose ranging from 1×10 4 pfu to 2 x 10 8 The vaccine may be administered in doses ranging from up to pfu.

[0031] In some embodiments, the recombinant vaccinia virus expressing both HPV-16 and HPV-18 E6 and E7 can be TA-HPV. TA-HPV is a recombinant vaccinia virus vaccine expressing HPV 16 and 18 oncogenes E6 and E7. The HPV-16 and HPV-18 oncogenes E6 and E7 can be inserted in a head-to-head orientation under the control of p7.5 and H6 promoters at a neutral site in the genome of the Wyeth strain of vaccinia virus (L. K, Borysiewicz et al., Lancet., 1996 Jun 1;347 (9014):1523-7). For both the HPV-16 and HPV-18 genes, the E6 stop codon can be altered to create an E6 / E7 fusion open reading frame, and a defined mutation can be introduced to inactivate the Rb binding site of E7. TA-HPV is a 1×10 4 pfu to 2 x 10 8 pfu. Preferably, the TA-HPV is administered in a dose ranging from 2×10 4 5×10 from pfu 7 The vaccine may be administered in doses ranging from up to pfu.

[0032] Examples of DNA constructs for DNA vaccines are described as follows. Four different DNA constructs (pBI-1, pBI-10.1, pBI-11, and pBI-12) are illustrated in FIG. 1. Each DNA construct contains a partial sequence encoding a signal peptide (indicated as "S" in FIG. 1), a partial sequence encoding an immunostimulant (e.g., HSP70 in FIG. 1), and one or more HPV antigens (e.g., HPV-16 E7 (or E7 in FIG. 1)). (16) ), HPV-18 E7 (or E7 in Figure 1) (18) ), HPV-16 E6 (or E6 in Fig. 1) (16) ), and / or HPV-18 E6 (or E6 in FIG. 1) (18) In some embodiments, the immunostimulant may include, but is not limited to, a 70 kilodalton heat shock protein (HSP70) or a human calreticulin (CRT) protein.

[0033] In some embodiments, pBI-1 is a DNA construct having a nucleotide sequence as set forth in SEQ ID NO:8, and comprising a fusion gene comprising a partial sequence encoding a signal peptide, an HPV partial sequence encoding a "detox" form of the HPV-16 E7 expression gene, and a partial sequence encoding HSP70. The term "detox" means that the HPV oncogene (e.g., E6 or E7) DNA has been altered by mutation to express a protein that cannot be oncogenic. In some embodiments, pBI-10.1 is a DNA construct having a nucleotide sequence as set forth in SEQ ID NO:7, and comprising a fusion gene comprising a partial sequence encoding a signal peptide, an HPV partial sequence encoding a "detox" form of the HPV-16 E7 expression gene, an "detox" form of the HPV-18 E7 expression gene, an "detox" form of the HPV-16 E6 expression gene, and a partial sequence encoding HSP70.

[0034] In order to increase the expression of HPV antigens and further realize a DNA vaccine against HPV-associated diseases with safety and improved immunogenicity, optimized HPV partial sequences encoding HPV-16 E6, HPV-16 E7, HPV-18 E6, and HPV-18 E7, respectively, can be designed by mutating to express proteins that cannot be oncogenic and by codon optimization. The optimized HPV-16 E6 expression gene can include the nucleotide sequence set forth in SEQ ID NO:1. The optimized HPV-16 E7 expression gene can include the nucleotide sequence set forth in SEQ ID NO:2. The optimized HPV-18 E6 expression gene can include the nucleotide sequence set forth in SEQ ID NO:3. The optimized HPV-18 E7 expression gene can include the nucleotide sequence set forth in SEQ ID NO:4.

[0035] The DNA construct can be designed to include a fusion gene that may include an optimized HPV partial sequence encoding at least one HPV antigen selected from the group consisting of the HPV-16 E6 expression gene set forth in SEQ ID NO: 1, the HPV-16 E7 expression gene set forth in SEQ ID NO: 2, the HPV-18 E6 expression gene set forth in SEQ ID NO: 3, and the HPV-18 E7 expression gene set forth in SEQ ID NO: 4. In some embodiments, pBI-11 is a DNA construct having a nucleotide sequence set forth in SEQ ID NO: 5, and including a fusion gene including an optimized HPV partial sequence encoding a signal peptide, the HPV-16 E7 expression gene set forth in SEQ ID NO: 2, the HPV-18 E7 expression gene set forth in SEQ ID NO: 4, the HPV-16 E6 expression gene set forth in SEQ ID NO: 1, and the HPV-18 E6 expression gene set forth in SEQ ID NO: 3, and a partial sequence encoding HSP70. In some embodiments, pBI-12 is a DNA construct having a nucleotide sequence set forth in SEQ ID NO:6 and comprising a fusion gene comprising an optimized HPV partial sequence encoding an optimized signal sequence encoding a signal peptide, an HPV-16 E7 expression gene set forth in SEQ ID NO:2, an HPV-18 E7 expression gene set forth in SEQ ID NO:4, an HPV-16 E6 expression gene set forth in SEQ ID NO:1, and an HPV-18 E6 expression gene set forth in SEQ ID NO:3, and a partial sequence encoding HSP70. EXAMPLES

[0036] Design and synthesis of HPV DNA vaccine constructs The pBI-1 DNA construct was previously described as pNGVL4a-SigE7(antitoxin)HSP70 (C. Trimple et al. Vaccine 2003, 21:4036-4042). In one embodiment, the pBI-10.1 DNA construct was derived by Gibson assembly of DNA fragments encoding fusion proteins of signal peptide, HPV-16 E7(antitoxin), HPV-18 E7(antitoxin), HPV-16 E6(antitoxin), and HPV-18 E6(antitoxin), flanked by EcoRI and Kozak sites on the 5' side and Tth111I site on the 3' side, and the 5' portion of HSP70 (up to the Tth111I site), synthesized by Biobasic (Markham, ON, Canada). The synthesized DNA fragments were cloned into pBI-1 to replace the fragment between EcoRI and Tth111I in frame with HSP70.

[0037] Similar to pBI-10.1, the pBI-11 DNA construct also contained synthesized DNA fragments encoding the signal peptide, HPV-16 E7, HPV-18 E7, HPV-16 E6, and HPV-18 E6, and the fusion protein of HSP70, but the expression genes of HPV-16 E7, HPV-18 E7, HPV-16 E6, and HPV-18 E6 in pBI-11 were further codon-optimized. The pBI-12 DNA construct also contained synthesized DNA fragments encoding the signal peptide, HPV-16 E7, HPV-18 E7, HPV-16 E6, and HPV-18 E6, and the fusion protein of HSP70, but the expression genes of HPV-16 E7, HPV-18 E7, HPV-16 E6, and HPV-18 E6 in pBI-12 were further codon-optimized. The synthesized DNA fragment of pBI-11 or pBI-12 was cloned into pBI-1 to replace the fragment between EcoRI and Tth111I in frame with HSP70.

[0038] The genes within the synthesized DNA fragments of each DNA construct, either optimized for gene expression (Figure 1, fragment 101), mutated for detoxification (Figure 1, fragment 102), or based on the native papillomavirus sequence (Figure 1, fragment 103), are shown in Figure 1. EXAMPLES

[0039] Codon optimization enhances expression levels of HPV antigens Western blot analysis was performed to determine whether the encoded antigens were properly produced by cells transfected with the various DNA constructs. HEK293 cells were transfected with 10 micrograms (μg) of one of the pBI-1, pBI-10.1, pBI-11, or pBI-12 DNA constructs. 48 hours after transfection, cell lysates were collected for Western blot analysis. The results of the Western blot analysis are illustrated in FIG. 2. pBI-1, pBI-10.1, pBI-11, or pBI-12 were separated on Precast Tris-HCl protein gels (Life Technology, Rockville, MD, USA) and then transferred to nitrocellulose membranes (Bio-Rad® Laboratories, Hercules, CA). After blocking, the membrane was hybridized with anti-HPV-16-E7 monoclonal antibody (referred to as HPV16 E7 ab in FIG. 2) (8C9 clone from Invitrogen, Thermo Fisher Scientific, Waltham, MA), anti-HPV-18 / 16-E6 (referred to as HPV18 / 16 E6 ab in FIG. 2) (CIP5 clone from Abcam PLC, Cambridge, UK), or anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (referred to as GAPDH in FIG. 2) (catalog number [no.] 60004-1-Ig; Proteintech® Group, Rosemont, IL) to detect fusion proteins containing HPV-16 E7, HPV-16 / 18 Expression of fusion proteins containing E6 or GAPDH (Cat:60004-1-Ig, Proteintech® Group, Rosemont, IL) as a loading control was characterized. Peroxidase-conjugated sheep anti-mouse secondary antibody (Amersham™, Piscataway, NJ, USA) and chemiluminescence (ECL) were used. + Antibody binding was detected using a ELISA kit (Amersham™, Piscataway, NJ, USA).

[0040] As shown by the results shown in Figure 2, all DNA constructs showed expression of fusion proteins containing HPV-16 E7 antigen. Compared with pBI-10.1-transfected cells, pBI-11-transfected cells and pBI-12-transfected cells resulted in higher HPV-16 E7 expression levels, suggesting that pBI-11 and pBI-12 successfully enhanced HPV-16 E7 expression levels by the optimized HPV partial sequences.

[0041] Furthermore, HPV-16 E6 and / or HPV-18 E6 were detected in cells transfected with either pBI-10.1, pBI-11 or pBI-12. In comparison, the HPV-16 E6 and / or HPV-18 E6 expression levels in pBI-11 and pBI-12 transfected cells were higher than the HPV-16 E6 and / or HPV-18 E6 expression levels in pBI-10.1 transfected cells, indicating that the optimized HPV partial sequences of pBI-11 and pBI-12 enabled cells to express increased amounts of HPV-16 E6 and HPV-18 E6.

[0042] Taken together, the results suggest that the optimized HPV partial sequences in pBI-11 and pBI-12 achieve increased levels of expression of HPV antigens. EXAMPLES

[0043] Cells transfected with pBI-11 or pBI-12 are able to enhance presentation of HPV antigenic peptides by MHC class I molecules To compare the ability of HPV antigen presentation by cells transfected with either the pBI-10.1, pBI-11, or pBI-12 DNA constructs, an in vitro T cell activation assay was performed.

[0044] 293-D bCells or 293 K b Cells were transfected with either pBI-10.1, pBI-11, or pBI-12 DNA constructs or mock transfected using Lipofectamine 2000. After 24 hours, the cells were harvested and transformed into mouse H-2D b Restricted HPV-16 E7 (amino acids 49-57) peptide-specific CD8 + T cells (as shown in Fig. 3A) or mouse H-2K b Restricted HPV-18 E6 (amino acids 67-75) peptide-specific CD8 + T cells (shown in FIG. 3B) in the presence of GolgiPlug™ (BD Biosciences, San Diego, Calif.). Cells were then harvested and IFN-γ intracellular staining was performed to identify HPV-16 E7 antigen-specific CD8 + T cell or HPV-18 E6 antigen specific CD8 + T cell activation was determined.

[0045] As shown by the results shown in Figures 3A and 3B, cells transfected with pBI-11 or pBI-12 expressed more HPV-16 E7 peptide (amino acids 49-57)-specific CD8+ cells than cells transfected with pBI-10.1. + T cells and HPV-18 E6 peptide (amino acids 67-75)-specific CD8 + In the pseudotransfected cells, the expression of HPV antigenic peptides (e.g., E7 peptide (amino acids 49-57))-specific CD8 + T cells and E6 peptide (amino acids 67-75)-specific CD8 + Thus, the data indicate that enhanced expression of the HPV-16 / 18 E6 / E7 fusion protein in cells transfected with pBI-11 or pBI-12 promotes presentation of HPV antigens by major histocompatibility complex (MHC) class I molecules, resulting in the induction of HPV antigen-specific CD8+ / CD9+ T cells. + T cells were shown to be activated. EXAMPLES

[0046] HPV-16 E7-specific CD8 + Leads to enhanced T cell-mediated immune responses An in vivo T cell activation assay was performed to compare immune responses in mice vaccinated with either pBI-10.1, pBI-11, or pBI-12.

[0047] Figure 4A is a schematic of the experimental design. Six- to eight-week-old female C57BL / 6 mice purchased from Taconic Biosciences (Germantown, NY) were vaccinated intramuscularly three times at seven-day intervals with 25 micrograms (μg) per mouse of either pBI-1, pBI-10.1, pBI-11, or pBI-12 DNA constructs. One week after the last vaccination, CD8+ cells were detected by intracellular cytokine staining for interferon-γ followed by flow cytometry after stimulation with known MHC-I peptides within HPV-16 E6 and E7 and HPV-18 E6. + Splenocytes were harvested for analysis of T cell responses.

[0048] As shown by the results shown in FIG. 4B, both mice vaccinated with pBI-11 and mice vaccinated with pBI-12 had significantly higher E7-specific CD8+ than mice vaccinated with pBI-10.1. + We demonstrated that T cell-mediated immune responses were enhanced in vivo by pBI-11 and pBI-12, which contain optimized HPV partial sequences, and that antigen-specific CD8 + It was possible to induce a T cell-mediated immune response.

[0049] Based on the fusion protein expression levels and in vivo T cell activation assay data, it is suggested that the enhanced expression of HPV antigens in pBI-11 or pBI-12 transfected cells due to codon optimization compared to that in pBI-10.1 transfected cells contributes to stronger T cell-mediated immune responses against HPV antigens in vaccinated mice. EXAMPLES

[0050] pBI-11 DNA constructs elicit therapeutic antitumor responses in the HPV-16 E6 / E7-expressing tumor model, TC-1 The ability of the DNA constructs of the present disclosure to produce therapeutic anti-tumor effects against HPV-associated disease was examined by comparing the HPV-16 E6 / E7 + This was investigated in the TC-1 tumor model.

[0051] Figure 5A is a schematic diagram of the experimental design. Female C57BL / 6 mice aged 6 to 8 weeks were inoculated with 2 × 10 TC-1 tumor cells on day 0. 5 Tumor-bearing mice were vaccinated with pBI-10.1, pBI-11, or pBI-12 (25 μg / 50 microliters (μl) / mouse) by intramuscular (IM) injection on days 3, 6, and 10, or left untreated as controls. In some embodiments, mice can be vaccinated with alternative and / or additional vaccinations, such as TA-HPV, as a boosting vaccine as described herein.

[0052] One week after the final vaccination, PBMCs were collected for tetramer staining. For tetramer staining, mouse PBMCs were first stained with purified anti-mouse CD16 / 32, and then with FITC-conjugated anti-mouse CD8a and PE-conjugated HPV-16 E7 (amino acids 49-57) peptide-loaded H-2D. b HPV-18 E6 (amino acids 67-75) peptide-loaded H-2K tetramer or conjugated with PE bTetramers were stained for 1 hour at 4° C. After washing, cells were stained with 7-AAD. Cells were acquired on a FACSCalibur™ flow cytometer and analyzed using CellQuest Pro software (BD Biosciences, Mountain View, Calif.). The results of tetramer staining are shown in FIG. 5B and FIG. 5C.

[0053] Tumor growth was monitored twice weekly by palpation and digital caliper measurement. Tumor volume was calculated using the formula [maximum diameter x (perpendicular diameter) 2 ]×3.14 / 6. Calculated tumor volumes are shown in FIG 5D. Survival rates of tumor-bearing mice were recorded as illustrated in FIG 5E. Both natural deaths and tumor diameters greater than 2 cm leading to death were counted as deaths.

[0054] As shown in Figures 5B and 5C, tumor-bearing mice treated with pBI-10.1 showed significantly higher HPV-16 E7-specific and HPV-18 E6-specific CD8 + It is demonstrated that tumor-bearing mice treated with pBI-11 or pBI-12 exhibited significantly higher HPV-16 E7-specific and HPV-18 E6-specific CD8 T cells than mice vaccinated with pBI-10.1. + The results further showed T cell-mediated immune response. Moreover, as shown in FIG. 5C, the tumor growth in mice vaccinated with pBI-11 or pBI-12 was significantly slower than that in mice treated with pBI-10.1. Moreover, vaccination with pBI-11 or pBI-12 also resulted in better survival than mice treated with pBI-10.1 (FIG. 5D). As a result, the DNA vaccines comprising DNA constructs containing fusion genes with optimized HPV partial sequences of the present disclosure, such as pBI-11 and pBI-12, can effectively enhance anti-tumor immune responses against established TC-1 tumors and extend the survival of tumor-bearing mice.

[0055] In conclusion, DNA vaccines with the optimized HPV partial sequences encoding HPV-16 E6 (SEQ ID NO: 1), HPV-16 E7 (SEQ ID NO: 2), HPV-18 E6 (SEQ ID NO: 3), and / or HPV-18 E7 (SEQ ID NO: 4) of the present disclosure significantly induce enhanced expression of HPV antigens and improved immune responses compared to DNA vaccines without the optimized HPV partial sequences. It is surprising that the optimized HPV partial sequences of the present disclosure successfully achieve stronger antigen-specific immune responses in vivo, which further translates into stronger anti-tumor efficacy.

[0056] Those skilled in the art will readily recognize that numerous modifications and variations of the devices and methods may be made while retaining the teachings of the present disclosure. Accordingly, the above disclosure should be construed as limited only by the metes and bounds of the appended claims. [Explanation of symbols]

[0057] 101 Fragment 102 Fragment 103 Fragment

Claims

1. A DNA vaccine comprising a DNA construct containing a fusion gene, the fusion gene comprises an optimized human papillomavirus (HPV) partial sequence encoding at least one HPV antigen and a partial sequence encoding an immunostimulant; the immunostimulant is a 70 kilodalton (kDa) heat shock protein (HSP70) or a human calreticulin (CRT) protein; The optimized HPV partial sequence is HPV-16 E6 expression gene as set forth in SEQ ID NO:1, HPV-16 E7 expression gene as set forth in SEQ ID NO:2, HPV-18 E6 expression gene as set forth in SEQ ID NO:3, and HPV-18 E7 expression gene as set forth in SEQ ID NO:4 A DNA vaccine comprising:

2. the optimized HPV partial sequence comprises HPV-16 E6, HPV-16 E7, HPV-18 E6, and HPV-18 E7 expressing genes; The fusion gene comprises SEQ ID NO:

5. The DNA vaccine of claim 1.

3. the fusion gene further comprises an optimized signal sequence, The fusion gene comprises SEQ ID NO:

6. The DNA vaccine of claim 1.

4. 13. Use of a DNA vaccine according to any one of claims 1 to 3 in the manufacture of a medicament for the treatment of an HPV-associated disease in a subject in need thereof.

5. Use of a combination of (a) a DNA vaccine described in any one of claims 1 to 3 and (b) a recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 in the manufacture of a medicament for the treatment of an HPV-associated disease in a subject in need thereof, wherein the DNA vaccine is for administration to the subject as a priming vaccine, and the recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 is for administration to the subject as a boosting vaccine.

6. The use according to claim 5, wherein the recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 is TA-HPV.

7. The TA-HPV is 1×10 4 2 × 10 plaque-forming units (pfu) 8 The use according to claim 6, administered in a dose ranging from up to pfu.

8. 7. The use of claim 6, wherein the DNA vaccine is administered at a dose ranging from 100 micrograms per subject to 20 milligrams per subject.

9. 4. The DNA vaccine of any one of claims 1 to 3 for use in the treatment of an HPV-associated disease in a subject in need thereof.

10. A combination of (a) the DNA vaccine of any one of claims 1 to 3 and (b) a recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 for use in treating an HPV-associated disease in a subject in need thereof, wherein the DNA vaccine is for administration to the subject as a priming vaccine and the recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 is for administration to the subject as a boosting vaccine.

11. The combination for use according to claim 10, wherein the recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 is TA-HPV.

12. The TA-HPV is 1×10 4 2 × 10 plaque-forming units (pfu) 8 The combination for use according to claim 11, administered in a dose ranging from up to pfu.

13. The combination for use according to claim 11, wherein the DNA vaccine is administered at a dose ranging from 100 micrograms per subject to 20 milligrams per subject.

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