Vaccine for human papillomavirus and use thereof

Abstract

The present disclosure provides a vaccine for human papillomavirus (HPV) and use thereof for treating a subject with human papillomavirus-associated diseases. The vaccine may include a nucleotide having a fusion gene. The fusion gene may include an optimized DNA subsequence having an HPV-I8 E6 expressing gene set forth in SEQ ID NO: 4, or an mRNA subsequence transcribed from the optimized DNA subsequence.

Description

VACCINE FOR HUMAN PAPILLOMAVIRUS AND USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of and priority to US Provisional Patent Application Serial No. 63 / 632,333, entitled “VACCINE FOR HUMAN PAPILLOMAVIRUS AND USE THEREOF”, filed on April 10, 2024. The contents of the above-mentioned application are hereby incorporated by reference herein for all purposes.FIELD

[0002] The present disclosure generally relates to vaccines for human papillomavirus (HPV) and use thereof, and more particularly to vaccines for HPV with optimized codon and use thereof for improving efficacies on preventing and / or treating human papillomavirus-associated diseases.BACKGROUND

[0003] Human papillomavirus (HPV) serves as a common etiological agent in several human cancers, encompassing cervical, anal, penile, vulvar, vaginal, and head and neck cancers. Current vaccines against HPV, such as Gardasil® and Cervarix®, have demonstrated clinical efficacy in preventing HPV infection. However, they prove ineffective in treating individuals with existing HPV infections or HPV-associated cancers. Consequently, there is a high demand for the development of therapeutic vaccines for patients infected with HPV or those suffering from HPV-associated diseases.

[0004] Deoxyribonucleic acid (DNA) vaccination is a technique for protecting against infection or treating disease through injection with a genetically engineered plasmid that contains a DNA sequence encoding one or more antigens. DNA vaccines present theoretical advantages over conventional vaccines, including safety, speed, andpredictability of manufacture, temperature stability, flexibility in design, and the ability to induce a broader range of immune response types. Despite the aforementioned advantages, DNA vaccines generally encounter obstacles in inducing strong antigen-specific immune responses in animals. For instance, DNA vaccination with HPV E6 and E7 genes may be poorly immunogenic due to the evolved mechanisms to evade host recognition, including low levels of E6 / E7 expression. Consequently, numerous strategies to enhance DNA vaccine immunogenicity have been explored, such as vector design improvement, antigen codon optimization, use of traditional adjuvants and molecular adjuvants, electroporation (EP), co-expression of molecular adjuvants, and prime-boost strategies (L. Li and N. Petrovsky, Expen Rev Vaccines. 2016;15(3):313-29).

[0005] Codon optimization involves utilizing synonymous mutations to increase protein expression of an interested gene, such as the antigen-expressing gene in the DNA vaccine. However, codon optimization does not always positively correlate with DNA vaccine efficacy. Numerous studies have demonstrated that rare codons may not consistently be a speed-limiting step, and frequently used codons do not guarantee increased protein production. Additionally, coding regions not only specify amino acid sequences but also often contain overlapping genetic information, including RNA secondary structures that can affect protein folding. It is indicated that synonymous codons could potentially alter protein conformation and function (L. Li and N. Petrovsky, Expert Rev Vaccines. 2016;! 5(3): 313-29, V. P. Mauro and S. A. Chappell, Methods Mol Biol. 2018; 1850:275- 288). Consequently, using codon optimization to enhance the expression of encoded antigens in DNA vaccines may yield unexpected detrimental results and requires careful consideration. Furthennore, predicting whether a DNA vaccine including codon-optimized sequences can achieve a desired immunogenicity proves even more challenging.

[0006] Furthermore, before introducing a newly developed DNA vaccine to clinical settings, addressing the potential to induce autoimmunity through vaccination with novelsequences is of great concern. Adverse reactions to vaccines may result from the interaction between the susceptibility of the vaccinated subject and various vaccine components. The significant similarity7between certain pathogenic elements contained in the vaccine and specific human proteins may lead to immune cross-reactivity. In this scenario, the immune system's reaction to the pathogenic antigens may harm similar human proteins (selfantigens), thus potentially causing autoimmune disease (Cell Mol Immunol. 2018 Jun;15(6):586-594). Therefore, in addition to enhancing the immunogenicity of vaccines, it is crucial to avoid the potential induction of autoimmunity due to cross-reactivity during vaccine development.

[0007] Furthermore, the production and development of messenger ribonucleic acid (mRNA) for therapeutic and vaccine applications are now considered relatively straightforward, scalable, and exceptionally fast. The mRNA technology offers an attractive option to innovate therapeutic solutions for diseases with unmet needs. However, it is essential to note that while mRNA technology has demonstrated remarkable efficacy in various contexts, its specific effectiveness in treating HPV-related diseases remains uncertain.

[0008] In light of the reasons mentioned above, an unmet need exists for providing a DNA vaccine and mRNA vaccine with enhanced immunogenicity, better efficacy in treating HPV-associated diseases, and improved safety. However, improving the efficacy7of DNA vaccines and mRNA vaccine against HPV -associated diseases through codon optimization remains highly unpredictable and, thus, poses a problem to be addressed in the field. Additionally, there is a concern that the vaccine may potentially induce autoimmunity due to cross-reactivity, thus highlighting the need to address the risk of immune responses against junction-associated epitopes that are present in the expressed fusion protein or included in the vaccine.SUMMARY

[0009] The present disc losure is directed to vaccines for human papillomavirus (HPV) and use thereof, and more particularly to vaccines for HPV with optimized codon and use thereof for improving efficacies on preventing and / 'or treating human papillomavirus - associated diseases.

[0010] According to a first aspect of the present disclosure, a vaccine is provided. The vaccine includes a nucleotide that includes a fusion gene. The fusion gene includes: an optimized deoxyribonucleic acid (DNA) subsequence that includes an HPV- 18 E6 expressing gene (HPV- 18 E6 expressing gene) set forth in SEQ ID NO: 4; or a messenger ribonucleic acid (mRNA) subsequence which is transcribed from the optimized DNA subsequence.

[0011] In an implementation of the first aspect of the present disclosure, the optimized DNA subsequence may further include one or more of: an HPV- 16 E6 expressing gene set forth in SEQ ID NO: 1 ; an HPV-16 E7 expressing gene set forth in SEQ ID NO: 2: an HPV- 16 L2 expressing gene set forth in SEQ ID NO: 3; an HPV- 18 E7 expressing gene set forth in SEQ ID NO: 5; and an HPV-18 L2 expressing gene set forth in SEQ ID NO: 6.

[0012] In an implementation of the first aspect of the present disclosure, the optimized DNA subsequence may further include a subsequence encoding an immunostimulant, and the immunostimulant may be a 70 kilodalton (kDa) heat shock protein (HSP70) or a human calreticulin (CRT) protein.

[0013] In an implementation of the first aspect of the present disclosure, the optimized DNA subsequence may include: the HPV-16 E6 expressing gene, the HPV-16 E7 expressing gene, the HPV-16 L2 expressing gene, the HPV- 18 E6 expressing gene, and the HPV- 18 E7 expressing gene.

[0014] In an implementation of the first aspect of tire present disclosure, the optimized DNA subsequence may further include an expressing gene set forth in SEQ I D NO: 7.

[0015] In an implementation of the first aspect of the present disclosure, the optimized DNA subsequence may include the HPV-16 E6 expressing gene, the HPV-16 E7 expressing gene, the HPV-16 L2 expressing gene, the HPV-18 E6 expressing gene, the HPV-18 E7 expressing gene, and the HPV-18 L2 expressing gene.

[0016] In an implementation of the first aspect of the present disclosure, the optimized DNA subsequence may further include an expressing gene set forth in SEQ ID NO: 8.

[0017] In an implementation of the first aspect of the present disclosure, the optimized DNA subsequence may include the HPV-18 E6 expressing gene, the HPV-18 E7 expressing gene, and the HPV-18 L2 expressing gene.

[0018] In an implementation of the first aspect of the present disclosure, the optimized DNA subsequence may further include an expressing gene set forth in SEQ ID NO: 18.

[0019] According to a second aspect of the present disclosure, a method for treating an HPV-associated disease in a subject in need thereof is provided. The method includes: administering the vaccine to the subject.

[0020] In an implementation of the second aspect of the present disclosure, the method may further include: administering a recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 to the subject. The vaccine is administered as a priming vaccine and the recombinant vaccinia virus expressing the E6 and the E7 of both the HPV-16 and the HPV-18 is administered as a boosting vaccine.

[0021] In an implementation of the second aspect of the present disclosure, the recombinant vaccinia virus expressing the E6 and the E7 of both the HPV-16 and the HPV- 18 is TA-HPV.

[0022] In an implementation of the second aspect of the present disclosure, the TA-HPV is administered at a dose ranging from 1 X 104plaque-forming units (pfu) to 2x10spfu.

[0023] In an implementation of the second aspect of the present disclosure, tire vaccine is administered at a dose ranging from 100 micrograms per subject to 20 milligrams persubject.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present description will be better understood from the following detailed description when read in light of the accompanying drawings.

[0025] FIG. 1 is a schematic illustration showing DNA constructs according to some implementations of the present disclosure.

[0026] FIG. 2 is a western blot image indicating HPV- 16 E6, HPV- 16 E7, and HPV- 18 E6 expression levels in Human Embryonic Kidney (HEK) 293 cells transfected with either pBI-1, pBI-4, pBI-5, pBI-6, pBI-7, pBI-11, or pBI-19, in accordance with an implementation of the present disclosure.

[0027] FIG. 3A is a schematic illustration of an experimental design for comparing HPV- 16 E6-specific, HPV- 16 E7-specific, or HPV- 18 E6-specific CD8^ T cell responses generated by various DNA constructs, in accordance with an implementation of the present disclosure.

[0028] FIG. 3B is a bar chart comparing a number of HPV-18 E6-specific IFN-y ' CD8 ' T cells / 1.5 X IO3splenocytes after splenocytes are prepared from mice that are vaccinated with either pBI-4, pBl-5, pBI-6, pBI-7, pBI-11, or pBI-19 being stimulated by HPV-18 E6 (aa 67-75) peptide, in accordance with an implementation of the present disclosure.

[0029] FIG. 3C is a bar chart comparing a number of HPV-16 E6-specific IFN-y' CD8+T cells / ! .5x 10~ splenocytes after splenocytes are prepared from mice that are vaccinated with either pBI-4, pBI-5, pBI-6, pBI-7, pBI-11, or pBI-19 being stimulated by HPV-16 E6 (aa 50-57) peptide, in accordance with an implementation of the present disclosure.

[0030] FIG. 3D is a bar chart comparing a number of HPV-16 E7-specific IFN-y+CD8 ' T cells / 1.5XlO3splenocytes after splenocytes are prepared from mice that are vaccinated with either pBI-4, pBI-5, pBI-6, pBI-7, pBI-11, or pBI-19 being stimulated by HPV-16 E7(aa 49-57) peptide, in accordance with an implementation of the present disclosure.DETAILED DESCRIPTION

[0031] The present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary implementations of the disclosure are depicted. However, it is important to note that this disclosure may be implemented in various forms and should not be inteipreted as restricted to the exemplary implementations presented herein. Instead, these exemplary implementations are provided to ensure a comprehensive and thorough understanding of the disclosure, conveying its full scope to those skilled in the art. Like reference numerals are used to denote similar elements consistently throughout.

[0032] The terminology used herein serves the purpose of describing specific exemplary implementations and is not intended to limit the scope of the disclosure. In this context, the singular forms “a”, “an”, and “the” also encompass the plural forms, unless the context clearly indicates other-wise. It should be noted that the terms “comprises” and / or “comprising,” or “includes” and / or “including” or “has” and / or “having,” as used in this context, indicate the presence of stated features, regions, integers, steps, operations, elements, and / or components, without excluding the possibility' of additional features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0033] Unless specifically defined otherwise, all terms, including technical and scientific terms, used herein carry' the same meaning as commonly understood by those with ordinary skill in the relevant art. Additionally, it is acknowledged that terms, including those found in commonly used dictionaries, should be interpreted in a manner consistent with their meaning in the context of the relevant art and the present disclosure. Such terms will not be construed in an idealized or overly formal sense unless expressly defined as such herein.

[0034] The present disclosure provides a nucleotide vaccine (e.g., DNA vaccine or mRNAvaccine) for HPV with optimized codon and use thereof for improving efficacies on preventing and / or treating human papillomavirus-associated diseases for a subject in need. The subject, affected by an HPV-associated disease, may be a mammal (e.g., human, mice, etc.) experiencing conditions including, but not limited to, warts, papilloma, intraepithelial neoplasia, penile cancer, vaginal cancer, vulva cancer, anal cancer, oropharyngeal cancer, non-melanoma skin cancer, conjunctival cancer, or cervical cancer.

[0035] The nucleotide vaccine of the present disclosure may include a fusion gene that contains a codon-optimized DNA subsequence from human papillomavirus, or an mRNA subsequence transcribed from the codon-optimized DNA subsequence. In the context described herein, the nucleotide vaccine may also be referred to as an optimized DNA vaccine or an optimized mRNA vaccine.

[0036] A synthetic fusion gene, encompassing a codon-optimized DNA subsequence that encodes at least one HPV antigen (e.g., an optimized DNA subsequence), may be provided. More specifically, the optimized DNA subsequence may include one or more of the following: an HPV- 16 E6 expressing gene encoding the HPV- 16 E6 antigen as set forth in SEQ ID NO:1; an HPV-16 E7 expressing gene encoding the HPV-I6 E7 antigen as set forth in SEQ ID NO: 2; an HPV-16 L2 expressing gene encoding the HPV-16 L2 antigen as set forth in SEQ ID NO: 3; an HPV-18 E6 expressing gene encoding the HPV-18 E6 antigen as set forth in SEQ ID NO: 4; an HPV-18 E7 expressing gene encoding the HPV- 18 E7 antigen as set forth in SEQ ID NO: 5: and an HPV-18 L2 expressing gene encoding the HPV-18 L2 antigen as set forth in SEQ ID NO: 6.

[0037] In some implementations, tire fusion gene containing the optimized DNA subsequence may be chemically synthesized or cloned into a plasmid, such as pNGVL4a and pcDNA3 plasmids, serving as a vector, to generate a DNA construct. Thus, the fusion gene constitutes a subsegment of the DNA construct. In other implementations, other plasmids, either alternative or additional, may be utilized to provide a vector as describedherein. In some implementations, the fusion gene containing the mRNA subsequence may be synthesized by in vitro transcription (IVT). For example, the DNA construct containing the optimized DNA subsequence may be used as a template for IVT.

[0038] In some implementations, the nucleotide vaccine may farther include an ingredient, such as an adjuvant, to elicit a more robust immune response in the subject receiving the vaccine.

[0039] In some implementations, the nucleotide vaccine may be administered as a priming vaccine, followed by the subsequent administration of a recombinant vaccinia vims expressing E6 and E7 of both HPV-16 and HPV-18, as a boosting vaccine. This combination therapy is employed in a heterologous prime -boost regimen to enhance the immune responses of a subject against HPV-associated diseases. For instance, the nucleotide vaccine may' be administered at a dosage ranging from 100 to 20 milligrams per subject, and the recombinant vaccinia vims expressing E6 and E7 of both HPV-16 and HPV- 18 may be administered at a dosage ranging from 1 x 104pfu to 2 x 10spfu.

[0040] In some implementations, the recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV-18 may be TA -HPV. TA-HPV is a recombinant vaccinia viral vaccine expressing the oncogenes E6 and E7 of HPV types 16 and 18. The HPV-16 and HPV-18 oncogenes E6 and E7 are inserted in a head-to-head orientation under the control of the p7.5 and H6 promoters at a neutral site in the vaccinia vims Wyeth strain genome (L. K, Borysiewicz el al., Lancet., 1996 Jun l ;347(9014): 1523-7). For both the HPV-16 and HPV- 18 genes, the E6 termination codon may be altered to create an E6ZE7 fused open reading frame, and a defined mutation is introduced to inactivate the Rb -binding site in E7. TA-HPV may be administered at a dosage ranging from 1 X IO4pfa to 2 x 108pfu, preferably at a dosage ranging from 2x 104pfu to 5x 107pfu.

[0041] Examples of the DNA constructs of the nucleotide vaccines against HPV are described as follows. FIG. 1 illustrates seven different DNA constructs, including pBI-1,pBI-4, pBI-5, pBI-6, pBI-7, pBI-11 , and pBI-19, where the DNA constructs pBI-1 and pBI- 11 include a fusion gene that contains a subsequence encoding a signal peptide (denoted as "S” in FIG. 1), a subsequence encoding an immunostimulant (e.g., HSP70 in FIG. 1), and a subsequence encoding one or more HPV antigens (e.g., HPV-16 E7 (denoted as E7(16)in FIG. I), HPV-18 E7 (denoted as E7(18)in FIG. 1), HPV-16 E6 (denoted as E6(!6)in FIG. 1), and / or HPV-18 E6 (denoted as E6(18) in FIG. 1)). The DNA constructs pBI-4, pBI-5, pBI-6, pBI-7, and pBI-19 include a fusion gene that contains a subsequence encoding an immunostimulant (e.g., CRT in FIG. 1) and a subsequence encoding HPV antigens (e.g., HPV- 16 E6 (denoted as E6(16) in FIG. 1 ), HPV-16 E7 (denoted as E7(16) in FIG. 1 ), HPV- 16 L2 (denoted as L2(16) in FIG. 1), HPV-18 E6 (denoted as E6(16) in FIG. 1), HPV-18 E7 (denoted as E7(18) in FIG. 1), and / or HPV-18 L2 (denoted as L2(18) in FIG. 1)). In some implementations, the immunostimulant may include, but is not limited to, a 70-kilodalton heat shock protein (HSP70) or a human calreticulin (CRT) protein.

[0042] In some implementations, pBI-1, with a nucleotide sequence set forth in SEQ ID NO: 10, is a DNA construct that incorporates a fusion gene containing: a subsequence encoding a signal peptide, a DNA subsequence with a "detox" form of HPV'- 16 E7 expressing gene, and a subsequence encoding HSP70. The term "detox" signifies that the HPV oncogenes (e.g., E6 or E7) DNA is modified by mutation to express proteins incapable of oncogenic transformation.

[0043] To enhance the expression of HPV antigens and further develop nucleotide vaccines against HPV-associated diseases with safety and improved immunogenicity, optimized DNA subsequences encoding HPV-16 E6, HPV-16 E7, HPV-16 L2, HPV-18 E6, HPV-18 E7, and HPV-18 L2, or mRNA subsequences transcribed from the optimized DNA subsequences may be designed by mutation to express proteins incapable of oncogenic transformation and by codon optimization.

[0044] The optimized HPV-16 E6 expressing gene may have a nucleotide sequence setforth in SEQ ID NO: I for the DNA constructs pBI-4, pBI-6, pBI-7, and pBI-19; and the optimized HPV-16 E6 expressing gene may have a nucleotide sequence set forth in SEQ ID NO: 11 for the DNA construct pBI- 11. The optimized HPV-16 E7 expressing gene may have a nucleotide sequence set forth in SEQ ID NO: 2 for the DNA constructs pBI-4, pBI- 6, pBI-7, and pBI-19: and the optimized HPV-16 E7 expressing gene may have a nucleotide sequence set forth in SEQ ID NO: 12 for the DNA construct pBI-11. The optimized HPV-16 L2 expressing gene may have a nucleotide sequence set forth in SEQ ID NO: 3 for the DNA constructs pBI-4, pBI-6, pBI-7, and pBI-19. The optimized HPV- 18 E6 expressing gene may have a nucleotide sequence set forth in SEQ ID NO: 4 for the DNA constructs pBI-5, pBI-6, and pBI-7: and the optimized HPV-18 E6 expressing gene may have a nucleotide sequence set forth in SEQ ID NO: 13 for the DNA construct pBI- 11. The optimized HPV-18 E7 expressing gene may have a nucleotide sequence set forth in SEQ ID NO: 5 for the DNA constructs pBI-5, pBI-6, pBI-7, pBI-11, and pBI-19. The optimized HPV-18 L2 expressing gene may have a nucleotide sequence set forth in SEQ ID NO: 6 for the DNA constructs pBI-5 and pBI-7.

[0045] DNA constructs may be designed to incorporate a fusion gene, which may include an optimized DNA subsequence encoding at least one HPV antigen selected from a group of: an HPV-16 E6 expressing gene set forth in SEQ ID NO: 1, an HPV-16 E7 expressing gene set forth in SEQ ID NO: 2, an HPV-16 L2 expressing gene set forth in SEQ ID NO: 3, an HPV-18 E6 expressing gene set forth in SEQ ID NO: 4, an HPV-18 E7 expressing gene set forth in SEQ ID NO: 5, and an HPV-18 L2 expressing gene set forth in SEQ ID NO: 6.

[0046] In some implementations, pBI-4, with a nucleotide sequence outlined in SEQ ID NO: 14, is a DNA construct that incorporates a fusion gene containing: a subsequence encoding CRT, an optimized DNA subsequence that contains an HPV-16 E6 expressing gene set forth in SEQ ID NO: 1, an HPV-16 E7 expressing gene set forth in SEQ ID NO:2, and an HPV-16 L2 expressing gene outlined in SEQ ID NO: 3.

[0047] In some implementations, pBI-5, with a nucleotide sequence outlined in SEQ ID NO: 18, is a DNA construct that incorporates a fusion gene containing: a subsequence encoding CRT, an optimized DNA subsequence that contains an HPV-I8 E6 expressing gene outlined in SEQ ID NO: 4, an HPV-18 E7 expressing gene outlined in SEQ ID NO: 5, and an HPV-18 L2 expressing gene outlined in SEQ ID NO: 6.

[0048] In some implementations, pBI-6, with a nucleotide sequence outlined in SEQ ID NO: 7, is a DNA construct that incorporates a fusion gene containing: a subsequence encoding CRT, an optimized DNA subsequence that contains an HPV-16 E6 expressing gene set forth in SEQ ID NO: 1, an HPV-16 E7 expressing gene set forth in SEQ ID NO: 2, an HPV-16 L2 expressing gene outlined in SEQ ID NO: 3, an HPV-18 E6 expressing gene outlined in SEQ ID NO: 4, and an HPV-18 E7 expressing gene outlined in SEQ ID NO: 5.

[0049] In some implementations, pBI-7, with a nucleotide sequence outlined in SEQ ID NO: 8, is a DNA construct that incorporates a fusion gene containing: a subsequence encoding CRT, an optimized DNA subsequence that contains an HPV-16 E6 expressing gene set forth in SEQ ID NO: 1, an HPV-16 E7 expressing gene set forth in SEQ ID NO: 2, an HPV-16 L2 expressing gene outlined in SEQ ID NO: 3, an HPV-18 E6 expressing gene outlined in SEQ ID NO: 4, an HPV-18 E7 expressing gene outlined in SEQ ID NO: 5, and an HPV-18 L2 expressing gene outlined in SEQ ID NO: 6.

[0050] In some implementations, pBI-19, with a nucleotide sequence outlined in SEQ ID NO: 15, is a DNA construct that incorporates a fusion gene containing: a subsequence encoding CRT, an optimized DNA subsequence that contains an HPV-16 E6 expressing gene set forth in SEQ ID NO: 1, an HPV-16 E7 expressing gene set forth in SEQ ID NO: 2, an HPV-16 L2 expressing gene outlined in SEQ ID NO: 3, a ’’detox" form of HPV-18 E6 expressing gene set forth in SEQ ID NO: 17, and an HPV-18 E7 expressing geneoutlined in SEQ ID NO: 5.

[0051] In some implementations, pBI-11, with a nucleotide sequence outlined in SEQ ID NO: 16, is a DNA construct incorporates a fusion gene containing: a subsequence encoding a signal peptide; an optimized DNA subsequence that contains an HPV-16 E7 expressing gene outlined in SEQ ID NO: 12, an HPV-18 E7 expressing gene outlined in SEQ ID NO: 5, an HPV-16 E6 expressing gene outlined in SEQ ID NO: 11, and an HPV-18 E6 expressing gene outlined in SEQ ID NO: 13; and a subsequence encoding HSP70.Example 1: Design and synthesis of HPV DNA vaccine constructs

[0052] The DNA construct pBI-1 has been previously described as pNGVL4a- SigE7(detox)HSP70 (C. Trimpie et al. Vaccine 2003, 21:4036-4042). All of DNA constructs pBI-4, pBI-5, pBI-6, pBI-7, and pBI-19 included a synthesized DNA fragment encoding a fusion protein that contained CRT and subsequent HPV antigens. In respect of HPV antigens, the DNA construct pBI-4 included HPV-16 E6, HPV-16 E7, and HPV-16 L2. In contrast, the DNA construct pBI-5 included HPV-18 E6, HPV-18 E7, and HPV-18 L2. Altogether, the DNA construct pBI-6 included HPV-16 E6, HPV-16 E7, HPV-16 L2, FIPV-18 E6, and HPV-18 E7. Moreover, the DNA construct pBI-7 included HPV-16 E6, HPV-16 E7, HPV-16 L2, HPV-18 E6, HPV-18 E7, and HPV-18 L2. The DNA construct pBI-19 included an identical DNA sequence to the DNA construct pBI-6, except for HPV 18 E6 expressing gene, which was designed as a wild type for the DNA construct pBI- 19, instead of being codon-optimized for the DNA construct pBI-6. Furthermore, the DNA construct pBl-11 included a synthesized DNA fragment encoding a fusion protein that contained the signal peptide, HPV-16 E7, HPV-18 E7, HPV-16 E6, and HPV-18 E6, along with HSP70. The synthesized DNA fragment of pBI-11 was cloned into the pBI-1 to replace the fragment between EcoRI and Tth 11 II, in frame with HSP70. In relation to CRT- related plasmids, CRT in frame with the synthesized DNA fragment of pBI-4 was cloned into pNGVL4a between Sall and BsaBL For CRT in frame with the synthesized DNAfragment of pBI-5, pBI-6, pBI-7, and pBI-19, Sall and Xbal were used for cloning into pNGVL4a.

[0053] The genes in the synthesized DNA fragment of each DNA construct have been either optimized for gene expression. As shown in FIG. i, the fragments, marked as 105, indicate the native sequences, while fragments, marked as 101, 102, or 103 , represent DNA sequences that were codon-optimized by different algorithm at different durations and fragments marked as 104 represent DNA sequences that were mutated for detox.Example 2: HPV antigen expression level under codon optimization

[0054] To assess the production of the encoded antigen in cells that are transfected with the various DNA constructs, western blot analysis was performed. HEK 293 cells were transfected with 10 micrograms (pg) of the DNA constructs pBI-1, pBI-4, pBI-5, pBI-6, pBI-7, pBI-11, and pBI-19. After 48 hours of transfection, cell lysates were collected for western blot analysis, and the results are depicted in FIG. 2. The DNA constructs pBI-1, pBI-4, pBI-5, pBI-6, pBI-7, pBI-11, and pBI-19 were separated on a Precast Tris-HCl protein gel (Life Technology, Rockville, MD, USA) and then transferred onto a nitrocellulose membrane (Bio-Rad8Laboratories, Hercules, CA). After blocking, the membrane was hybridized with anti -HPV- 16 E6 monoclonal antibody (denoted as Anti- F1PV- 16 E6) (E6-6F4 clone from EUROMEDEX, Souffelweyersheim, France; 1 :500 (4°C, overnight)), anti-HPV-16 E7 monoclonal antibody (denoted as Anti-HPV-16 E7) (8C9 clone Invitrogen, Thermo Fisher Scientific®, Waltham, MA; 1 .100 (4°C, overnight)), anti- HPV-18 E6 (denoted as Anti-HPV-18 E6) (G-7 clone from Santa Cruz Biotechnology Inc, Dallas, TX; 1:100 (4°C, overnight)), or anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (denoted as GAPDFI) (catalog number [no.] 60004- 1-Ig; Proteintech1® Group, Rosemont, IL; 1.5000 (4°C, overnight)) to assess the expression levels of the fusi on protein containing HPV-16 E6, the fusion protein containing HPV-16 E7, the fusion protein containing HPV-18 E6, or GAPDH as a loading control. Antibody binding wasdetected using a peroxidase-conjugated goat anti-mouse secondary antibody ( Amersham1M, Piscataway, NJ, USA; 1 :10000 (RT, Ihr)) and chemiluminescence (ECL+detection kit; Amersham™, Piscataway, NJ, USA).

[0055] The expression of the encoded antigen HPV-16 E6, HPV-16 E7, and HPV-18 E6 in cells that are transfected with the DNA constructs pBI-1, pBI-4, pBI-5, pBI-6, pBI-7, pBI-11, or pBI-19 are shown in FIG. 2. Furthermore, the signal intensity of the western blot, as shown in FIG. 2, was quantified by Visionworks acquisition and analysis, and the results of HPV-16 E6, HPV-16 E7, and HPV-18 E6 protein expression are presented in Table 1 , Table 2, and Table 3, respectively. The intensity of the top band of the construct pBI-19 was defined as 100 to establish the values for the other groups relative to the construct pBI-19.

[0056] As indicated in the results shown in Table 1, the DNA constructs pBI-4, pBI-6, pBI-7, pBI-11, and pBI-19 demonstrated the expression of a fusion protein containing the HPV-16 E6 antigen, where the HPV-16 E6 expression level shows: pBI-4 = pBI-6 > pBI- 19 = pBI-11 > pBI-7.Table 1

[0057] As indicated in the results shown in Table 2, the DNA constructs pBI-1 , pBI-4, pBI-6, pBI-7, pBI-11, and pBI-19 demonstrated the expression of a fusion protein containing the HPV-16 E7 antigen, where the HPV-16 E7 expression level shows: pBI-4 > pBI-11 > pBI-6 > pBI-1 = pBI-19 > pBI-7.Table 2

[0058] As indicated in the results shown in Table 3, the DNA constructs pBI-5, pBI-6, pBI-7, pBI-11, and pBI-19 demonstrated the expression of a fusion protein containing the HPV-18 E6 antigen, where the HPV-18 E6 expression level shows: pBI-6 > pBI-5 > pBI- 11 > pBI-19 > pBI-7.Table 3

[0059] In comparison, the expression levels of HPV-16 E6, HPV-16 E7 and HPV-18 E6 in cells transfected with pBI-6 were higher than those in cells transfected with pBI-19, thus indicating that the optimized DNA subsequence of pBI-6 enabled cells to express HPV-16 E6, HPV-16 E7 and HPV-18 E6 in increased amounts. Furthermore, the expression levels of HPV-18 E6 in cells transfected with pBI-5 were higher than those in cells transfected with pBI-19, thus indicating that the optimized DNA subsequence of pBI-5 enabled cells to express HPV-18 E6 in increased amounts. Taken together, the results suggest that the optimized DNA subsequences in pBI-5 and pBI-6 achieved an elevated expression level of HPV antigen.Example 3: Specific CD8+T cell immune responses under codon optimization

[0060] T cell activation assays w'ere conducted to compare the immune responses in mice vaccinated with either the DNA constructs pBI-4, pBI-5, pBI-6, pBI-7, pBI-11, or pBI-19.

[0061] FIG. 3A presents a schematic illustration of the experiment design. Six-to eight- week-old female C57BL / 6 mice obtained from Taconic Biosciences (Germantown, NY) were vaccinated intramuscularly three times at 7 -day intervals with 25 micrograms (pg) / mouse of the DNA constructs pBI-4, pBI-5, pBI-6, pBI-7, pBI-I l, or pBI-19. One week after the last vaccination, splenocytes were collected for the analysis of CD8+T cell response, which is determined by intracellular cytokine staining for interferon-y followed by flow cytometry after stimulation with either HPV-16 E6 (aa 50-57) peptide, HPV-16 E7 (aa 49-57) peptide, or HPV-18 E6 (aa 67-75) peptide. The results were shown in FIG. 3B, FIG. 3C, and FIG. 3D.

[0062] As indicated in FIG. 3B, mice vaccinated with any of the DNA constructs pBI-5, pBI-6, and pBI-7 exhibited potent and strong HPV-16 E6-specific CD8+T cell-mediatedimmune responses. In contrast, mice vaccinated with the DNA construct pBI-19, which differs from the DNA construct pBI-6 only in the HPV-18 E6 subsequence, demonstrated weak HPV-18 E6-specific CDS+T cell-mediated immune responses.

[0063] Among the mice vaccinated with the DNA constructs, which contains the HPV-18 E6 expressing gene set forth in SEQ ID NO: 4, the DNA construct pBI-6 elicited the strongest HPV-18 E6-specific-CD84T cell-mediated immune response compared to the DNA constructs pBI-5 or pBI-7. Furthermore, the DNA construct pBI-11, which includes another type of codon-optimized HPV-18 E6 subsequence, as a positive control, also exhibited elevated HPV-18 E6-specific CD84T cell-mediated immune responses, and the DNA construct pBI-6 unexpectedly elicited an even stronger HPV-18 E6-specific-CD84T cell-mediated immune response than pBI-11.

[0064] Upon amalgamating the results from FIG. 2 and Table 3, it is unexpected that while the HPV-18 E6 expression levels of the DNA constructs pBI-6 and pBI-5 were merely around 1.5 times higher than that of the DNA construct pBI-19, and the HPV-18 E6 expression level of the DNA construct pBI-7 was even lower than that of the DNA construct pBI-19, the HPV-18 E6-specific CD8" T cell-mediated immune responses led by the DNA constructs pBI-5, pBI-6, and pBI-7, which contain the optimized DNA subsequence of HPV-18 E6 expressing gene set forth in SEQ ID NO: 4, significantly surpass that led by the DNA construct pBI-19.

[0065] The results demonstrate that the fusion gene containing the optimized DNA subsequence with the HPV-18 E6 expressing gene set forth in SEQ ID NO: 4 surprisingly induced significantly heightened HPV-18 E6-specific CD8+T cell-mediated immune responses.

[0066] As indicated in the results shown in FIG. 3C, mice vaccinated with any of the DNA constructs pBI-4, pBI-6, and pBI-7 exhibited potent and comparable HPV-16 E6-specific CD8 T cell-mediated immune responses compared with those with the DNA constructpBI-19 and those with the DNA construct pBI-11.

[0067] As indicated in FIG. 3D, mice vaccinated with any of the DNA constructs pBI-4, pBI-6, and pBI-7 exhibited potent HPV-16 E7-specific CD8+T cell-mediated immune responses compared with those with the DNA construct pBI- 19. The DNA constructs pBI- 4 and pBI-6 even elicited a stronger HPV-16 E7-specific-CD8+T cell-mediated immune response compared to the DNA construct pBI-11, which includes a different codon- optimized HPV-16 E7 subsequence, as a positive control.

[0068] The results demonstrate that the DNA constructs pBI-6 and pBI-7, which include the optimized DNA subsequence containing HPV-18 E6 expressing gene set forth in SEQ ID NO: 4 and HPV-16 antigens expressing genes, could induce significantly enhanced antigen-specific CD8+T cell-mediated immune responses not only for HPV-18 but also for HPV-16.

[0069] Considering the expression levels of the fusion protein and the data obtained from the in vivo T cell activation assay, it is suggested that the HPV DNA vaccine featuring a codon-optimized sequence encoding HPV-18 E6 as in pBI-5, pBI-6, and pBI-7 unexpectedly led to significantly heightened HPV-18 E6-specific CD8+T cell-mediated immune responses. Notably, this enhanced response was achieved while maintaining the capacity to generate potent CD8^ T cell-mediated immune responses specific to HPV-16 E6 and HPV-16 E7.Example 4: Analysis of potential expression of novel peptides derived from HPV-18 E6 / E7 proteins identical to endogenous self-peptides

[0070] To identify potential vaccine epitopes capable of inducing cross-reactivity towards self-antigens, the sequences of vaccine peptides with those of human proteins were compared. Considering that peptide antigens are presented as fragments of either 8 to 11 amino acids on MHC class I to CD8 T cells or 12 to 20 amino acids on MHC class II to CD4 T cells, a comprehensive search for all linear sequences comprising 8 amino acids (8-mers) was conducted. These were extracted from HPV-18 E6 / E7 peptides and the junctional regions in the pBI-6-encoded fusion protein and compared with human protein sequences found in the UniProt protein database (https: / / uww.uniprot.org / ).

[0071] First, 258 8-mers from amino acid 845 to 1,109 of pBI-6 were generated, encompassing the final 7 amino acids of the HPV-I6 L2, HPV-18 E6, and HPV-18 E7. These sequences were submitted to the UniProt protein database to search for exact matches for human proteins. The search results did not reveal any exact match between vaccine and endogenous human peptide sequences that are at least 8 amino acids in length. A secondary search for all 259 7-mers yielded 92 entries, corresponding to 9 unique sequences mapped to 10 unique endogenous human proteins. The results were shown as Table 4. However, these 7-mers are below the minimum size for a T cell epitope and thus are unable for MHC presentation.

[0072] As an alternative approach, the Immune Epitope Database (https: / / www.iedb.org / ) was searched using the same region of the pBI-6 protein sequence (265 amino acids). The "substring" function for linear epitope was opted to obtain any epitope sequences that are mapped to the sequence. Search results revealed no match between the pBI-6 protein sequence and the epitope sequences in the database. Collectively, these analyses suggest that the incorporation of HPV-18 E6 and E7 into pBI-4 (e.g., the pBI-6) is unlikely to produce peptides that could elicit cross-reactive T cell immunity against self-antigens.Table 4

[0073] In summary, the optimized HPV-18-E6 (103 fragment) segment contained in theDNA construct pBI-6 not only enhances protein expression but also contributes to asignificantly augmented immune response. Furthermore, the DNA construct pBI-6 as a whole generate peptides is unlikely to elicit cross-reactive T-cell immunity against selfantigens, thus leading to a superior immune response with improved safety.

[0074] Those skilled in the art will readily observe that numerous modifications and alterations to the device and method may be made while retaining the teachings of the disclosure. Accordingly, the above disclosure should be construed as limited only by the metes and bounds of the appended claims.

[0075] The implementations shown and described above and below are only examples. Many details are often found in the art. Therefore, many such details are neither shown nor described herein for the sake of brevity. Even though numerous characteristics and advantages of the present disclosure have been set forth in the foregoing description, together with details of the structure and function of the present disclosure, the present disclosure is illustrative only, and changes may be made in the details. It will therefore be appreciated that the implementations described above and below may be modified within the scope of the claims.

Claims

WHAT IS CLAIMED IS:

1. A vaccine, comprising a nucleotide including a fusion gene, wherein the fusion gene comprises: an optimized deoxyribonucleic acid (DNA) subsequence comprising a human papillomavirus (HPV)-18 E6 expressing gene (HPV-18 E6 expressing gene) set forth in SEQ ID NO: 4; or a messenger ribonucleic acid (mRNA) subsequence transcribed from the optimized DNA subsequence.

2. The vaccine according to claim 1, wherein the optimized DNA subsequence further comprises one or more of: an HPV-16 E6 expressing gene set forth in SEQ ID NO: 1 ; an HPV-16 E7 expressing gene set forth in SEQ ID NO: 2; an HPV-16 L2 expressing gene set forth in SEQ ID NO: 3; an HPV-18 E7 expressing gene set forth in SEQ ID NO: 5; and an HPV-18 L2 expressing gene set forth in SEQ ID NO: 6.

3. The vaccine according to claim 2, wherein the optimized DNA subsequence further comprises a subsequence encoding an immunostimulant.

4. The vaccine according to claim 3, wherein the immunostimulant is a 70 kilodalton (kDa) heat shock protein (HSP70) or a human calreticulin (CRT) protein.

5. The vaccine according to claim 3, wherein the optimized DNA subsequence comprises: the HPV-16 E6 expressing gene, the HPV-16 E7 expressing gene, the HPV-16 L2 expressing gene, the HPV-18 E6 expressing gene, and the HPV-18 E7 expressing gene.

6. The vaccine according to claim 5, wherein the optimized DNA subsequence further comprises an expressing gene set forth in SEQ ID NO: 7.

7. The vaccine according to claim 3, wherein the optimized DNA subsequence comprises the HPV-16 E6 expressing gene, the HPV-16 E7 expressing gene, the HPV-16 L2 expressing gene, the HPV- 18 E6 expressing gene, the HPV- 18 E7 expressing gene, and the HPV- 18 L2 expressing gene.

8. The vaccine according to claim 7, wherein the optimized DNA subsequence further comprises an expressing gene set forth in SEQ ID NO: 8.

9. The vaccine according to claim 3, wherein the optimized DNA subsequence comprises the HPV- 18 E6 expressing gene, the HPV- 18 E7 expressing gene, and the HPV- 18 L2 expressing gene.

10. The vaccine according to claim 9, wherein the optimized DNA subsequence further comprises an expressing gene set forth in SEQ ID NO: 18.

11. A method for treating an HPV-associated disease in a subject thereof, comprising: administering the vaccine according to claim 2 to the subject.

12. The method according to claim 11, further comprising: administering a recombinant vaccinia virus expressing E6 and E7 of both HPV-16 and HPV- 18 to the subject, wherein the vaccine is administered as a priming vaccine and the recombinant vaccinia virus expressing the E6 and the E7 of both the HPV-16 and the HPV-18 is administered as a boosting vaccine.

13. The method according to claim 12, wherein the recombinant vaccinia virus expressing the E6 and the E7 of both the HPV-16 and the HPV-18 is a therapeutic antigen HPV (TA-HPV).

14. The method according to claim 13, wherein the TA-HPV is administered at a dose ranging from 1 x 104plaque-forming units (pfu) to 2x 108pfu.

15. The method according to claim 13, wherein the vaccine is administered at a dose ranging from 100 micrograms per subject to 20 milligrams per subject.

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