AU2022200326A1 - Vaccine combination and method for using the same - Google Patents

Abstract

A vaccine combination may include first and second vaccines. The first vaccine may include a first fusion protein or a first polynucleotide encoding the first fusion protein. The first fusion protein may include an E7 protein of HPV-16; an E7 protein of HPV-18; an E6 protein of HPV-16; an E6 protein of HPV-18; and a heat shock protein. The second vaccine may include second and third fusion proteins, or a second polynucleotide encoding the second and third fusion proteins. The second fusion protein may include an E6 protein of HPV-16 and an E7 protein of HPV-16. The third fusion protein may include an E6 protein of HPV-18 and an E7 protein of HPV-18. A functional variant may be employed for one or more of the proteins. Amino acid sequences of junction regions in the first fusion protein may be different from those in the second and third fusion proteins. 24/24

Description

VACCINE COMBINATION AND METHOD FOR USING THE SAME REFERENCE TO SEQUENCE LISTING

[0001] Accompanying this application is a sequence listing in an American Standard Code for

Information Interchange (ASCII) text file named "211223-US86679-Sequence Listing-vlF.txt",

created December 07, 2021, and having a size of 18,253 bytes. The sequence listing is hereby fully

incorporated by reference herein.

STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR UNDER 37 C.F.R.

1.77(b)(6)

[0002] A prior disclosure, "Development of DNA Vaccine Targeting E6 and E7 Proteins of

Human Papillomavirus 16 (HPV16) and HPV18 for Immunotherapy in Combination with

Recombinant Vaccinia Boost and PD-i Antibody," mBio, January/February 2021, Volume 12,

Issue 1, pages 1-19, published January 19, 2021, discusses work related to the claimed subject

matter of this application. The inventor of the present application, Yung-Nien Chang,

unequivocally states that he is the sole inventor of the subject matter discussed in the prior

disclosure and that he provided key materials to conduct experiments related to the prior disclosure.

The inventor is also a co-author of the prior disclosure. The remaining co-authors of the prior

disclosure conducted and recorded various aspects of the experiments under the direction of the

inventor, and thus contributed to the prior disclosure in that capacity. A copy of the prior disclosure

is provided in association with an Information Disclosure Statement submitted in conjunction with

this application.

1/24

BACKGROUND

1. Field

[0003] The present disclosure generally relates to vaccine combinations, particularly for a

human papillomavirus (HPV)-associated disease, and more specifically to improving the vaccine

combination by the particular design of a priming vaccine and a boosting vaccine.

2. Related Art

[0004] HPV is a common etiological agent in several human cancers, including cervical, anal,

penile, vulvar, vaginal, and head and neck cancers. Current vaccines against HPV, such as

Gardasil© and Cervarix©, have shown clinical efficacy in preventing HPV infection, but they are

ineffective in treating patients with existing HPV infection or HPV-associated cancers. As such,

development of therapeutic vaccines for patients infected with HPV or even suffering from HPV

associated diseases are highly demanded.

[0005] Deoxyribonucleic acid (DNA) vaccination is a technique for protecting against

infection or treating disease by injection with a genetically engineered plasmid containing a

DNA sequence encoding one or more antigens. DNA vaccines have theoretical advantages over

conventional vaccines, including safety, speed, and predictability of manufacture; temperature

stability; flexibility in design; and the ability to induce a wider range of immune response types.

[0006] An effective vaccine usually requires more than one-time immunization in the form of

prime-boost. Traditionally the same vaccines are given multiple times as homologous boosts.

Some findings suggest that prime-boost can be done with different types of vaccines containing

the same antigens. In many cases such heterologous prime-boost can be more immunogenic than

homologous prime-boost (Curr Opin Immunol. 2009 Jun; 21(3): 346-351). Heterologous prime

boost may include administration of two different vectors or delivery systems expressing the same

or overlapping antigenic inserts. It has been known that using certain vector combinations could

2/24 increase both antibody and T cell immunity. However, while a priming vaccine and a boosting vaccine, which both can be either a fusion protein of antigens or a vector encoding the same, may have the same antigen arrangement in the fusion protein, the enhanced immunity generated by the prime-boost regimen may also include boosting an immune response against the junction associated epitopes in the fusion protein of antigens and may lead to an impaired immune response against the target pathogen or other undesired effects.

[0007] Furthermore, before providing a newly developed DNA vaccine to the clinics, the

potential to induce autoimmunity by vaccinating with novel sequences is a great concern that

should be addressed. Such adverse reaction to vaccines may be viewed as a result of the interaction

between susceptibility of the vaccinated subject and various vaccine components. The significant

similarity between certain pathogenic elements contained in the vaccine and specific human

proteins may lead to immune cross-reactivity, wherein the reaction of the immune system towards

the pathogenic antigens may harm the similar human proteins (self-antigens), essentially causing

autoimmune disease (Cell Mol Immunol. 2018 Jun;15(6):586-594). Therefore, in addition to

elevating the immunogenicity of vaccines, it is essential as well to avoid potential induction of

autoimmunity due to cross-reactivity when developing vaccines.

[0008] In view of the reasons mentioned above, there exists an unmet need for providing a

vaccine for treating HPV-associated diseases with enhanced immunogenicity and improved safety,

as reflected in a reduced risk to generate an immune response against the junction-associated

epitopes potentially in the fusion protein expressed by or contained in the vaccine and to induce

cross-reactivity against self-antigens.

BRIEF SUMMARY OF THE DISCLOSURE

[0009] In a first aspect of the present disclosure, a vaccine combination may be provided. The

vaccine combination may include a first vaccine and a second vaccine. The first vaccine may

include a first fusion protein or a first polynucleotide encoding the first fusion protein. The first

3/24 fusion protein, in an order from N to C terminus, may include: (i) an E7 protein of human papillomavirus type 16 (HPV-16) or a functional variant thereof; (ii) an E7 protein of human papillomavirus type 18 (HPV-18) or a functional variant thereof; (iii) an E6 protein of HPV-16 or a functional variant thereof; (iv) an E6 protein of HPV-18 or a functional variant thereof; and (v) a heat shock protein or a functional variant thereof. The E7 protein of HPV-16 or a functional variant thereof may comprise SEQ ID NO: 1 at a beginning of the protein and SEQ ID NO: 2 at an end of the protein. The E7 protein of HPV-18 or a functional variant thereof may comprise SEQ

ID NO: 3 at a beginning of the protein and SEQ ID NO: 4 at an end of the protein. The E6 protein

of HPV-16 or a functional variant thereof may comprise SEQ ID NO: 5 at a beginning of the

protein and SEQ ID NO: 6 at an end of the protein. The E6 protein of HPV-18 or a functional

variant thereof may comprise SEQ ID NO: 7 at a beginning of the protein and SEQ ID NO: 8 at

an end of the protein. The second vaccine may include a second fusion protein and a third fusion

protein, or a second polynucleotide encoding the second fusion protein and the third fusion protein.

The second fusion protein may include an E6 protein of HPV-16 or a functional variant thereof;

and an E7 protein of HPV-16 or a functional variant thereof. The third fusion protein may include

an E6 protein of HPV-18 or a functional variant thereof; and an E7 protein of HPV-18 or a

functional variant thereof. Amino acid sequences ofjunction regions in the first fusion protein may

be different from those in the second fusion protein and the third fusion protein.

[0010] In some embodiments, the E7 protein of HPV-16 may comprise SEQ ID NO: 9; the

E7 protein of HPV-18 may comprise SEQ ID NO: 10; the E6 protein of HPV-16 may comprise

SEQ ID NO: 11; and the E6 protein of HPV-18 may comprise SEQ ID NO: 12.

[0011] In some embodiments, the E7 protein of HPV-16 may be encoded by SEQ ID NO: 13;

the E7 protein of HPV-18 may be encoded by SEQ ID NO: 14; the E6 protein of HPV-16 may be

encoded by SEQ ID NO: 15; and the E6 protein of HPV-18 may be encoded by SEQ ID NO: 16.

[0012] In some embodiments, the first polynucleotide may include SEQ ID NO: 17.

[0013] In some embodiments, the first polynucleotide may include SEQ ID NO: 18.

4/24

[0014] In some embodiments, the second polynucleotide may be contained within a

recombinant virus.

[0015] In some embodiments, the recombinant virus may be TA-HPV.

[0016] In a second aspect of the present disclosure, a method for treating an HPV-associated

disease in a subject in need thereof may be provided. The method may include administering the

aforementioned vaccine combination to the subject. The first vaccine may be administered as a

priming vaccine, and the second vaccine may be administered as a boosting vaccine.

[0017] In some embodiments, the method may further include administering the first vaccine

to the subject as a boosting vaccine after the administration of the first vaccine as a priming vaccine

and prior to the administration of the second vaccine as a boosting vaccine.

[0018] In some embodiments, the first vaccine may comprise the first polynucleotide. In some

embodiments, the first vaccine may be administered at a dose ranging from 10 micrograms per

subject to 20 milligrams per subject.

[0019] In some embodiments, the second vaccine may comprise TA-HPV. In some

embodiments, the second vaccine may be administered at a dose ranging from 1 x 104 plaque

forming units (pfu) to 2 x 10 pfu.

[0020] In some embodiments, the method may further include administering a chemotherapy,

radiotherapy, chemo-radiotherapy, cryotherapy, thermotherapy, targeted therapy, cellular therapy,

gene therapy, or immunotherapy in combination with the administration of the vaccine

combination.

[0021] In some embodiments, the chemotherapy, radiotherapy, chemo-radiotherapy,

cryotherapy, thermotherapy, targeted therapy, cellular therapy, gene therapy, or immunotherapy

may be administered prior to and/or simultaneously with the administration of the first vaccine.

[0022] In some embodiments, the immunotherapy may comprise administering an immune

checkpoint inhibitor to the subject.

[0023] In some embodiments, the immune checkpoint inhibitor may be an immune modulator

5/24 targeting programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic

T-lymphocyte-associated antigen 4 (CTLA-4), inducible costimulator (ICOS), T-cell

immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3) or T cell

immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT).

[0024] In some embodiments, the immune checkpoint inhibitor may be an anti-PD-i antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] So that the manner in which the above-recited features of the present disclosure can be

understood in detail, a more particular description of the disclosure, briefly summarized above,

may be had by reference to embodiments, some of which are illustrated in the appended drawings.

It is to be noted, however, that the appended drawings illustrate only typical embodiments of this

disclosure and are therefore not to be considered limiting of its scope, for the disclosure may admit

to other equally effective embodiments.

[0026] FIG. IA is a schematic illustration showing a DNA construct encoding the first fusion

protein of the first vaccine of the vaccine combination according to an embodiment of the present

disclosure.

[0027] FIG. iB is a schematic illustration showing a cassette for expression of an HPV-16

E6-E7 fusion protein and an HPV-18 E6-E7 fusion protein inserted into the genome of a vaccinia

virus of the vaccine combination according to an embodiment of the present disclosure.

[0028] FIG. 2A is a schematic illustration of the experimental design for characterizing HPV

antigen-specific CD8' T cell-mediated immune responses in mice vaccinated with the vaccine

combination of the present disclosure in a prime and boost strategy (two DNA doses and one

recombinant virus dose, DDV regimen) or a DNA vaccine alone in a prime and boost strategy

(three DNA doses, DDD regimen), in accordance with an embodiment of the present disclosure.

[0029] FIG. 2B is a bar chart showing the percentage of HPV-16 E7-specific CD8' T cells/

total CD8+ cells in peripheral blood mononuclear cells (PBMCs) collected from naive mice or

6/24 mice treated with the vaccine combination of the present disclosure (DDV regimen) or the DNA vaccine alone (DDD regimen) using HPV-16 E7 (aa 49-57) peptide-loaded tetramer staining, using a two-tailed Student's t-test, in accordance with an embodiment of the present disclosure.

[0030] FIG. 2C is a bar chart of the number of HPV-16 E7-specific IFN-' CD8' T cells/3 x

105 splenocytes from mice vaccinated with the vaccine combination of the present disclosure

(DDV regimen) or the DNA vaccine alone (DDD regimen) 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, in

accordance with an embodiment of the present disclosure.

[0031] FIG. 3A is a schematic illustration of the experimental design for characterizing the

HPV antigen-specific immune response and antitumor effects in TC-1 tumor-bearing mice treated

with the vaccine combination of the present disclosure in a prime and boost strategy with or

without anti-PD-i antibody, in accordance with an embodiment of the present disclosure.

[0032] FIG. 3B is a bar chart showing the percentage of HPV-16 E7-specific CD8' T cells/

total CD 8' T cells in peripheral blood mononuclear cells (PBMCs) prepared from untreated mice,

mice treated with anti-PD-i antibody alone, mice treated with the vaccine combination of the

present disclosure (DDV), or mice treated with the vaccine combination of the present disclosure

in combination with anti-PD-i antibody (Anti-PD-i+DDV), using HPV-I6 E7 (aa 49-57) peptide

loaded tetramer staining, using a two-tailed Student's t-test, in accordance with an embodiment of

the present disclosure.

[0033] FIG. 3C is a curve chart showing TC- Itumor volumes in the untreated mice, the mice

treated with anti-PD-i antibody alone, the mice treated with the vaccine combination of the present

disclosure (DDV), or the mice treated with the vaccine combination of the present disclosure in

combination with anti-PD-i antibody (Anti-PD-I+DDV), in accordance with an embodiment of

the present disclosure.

[0034] FIG. 3D is a Kaplan-Meier survival curve showing the probability of survival in the

untreated mice, the mice treated with anti-PD-i antibody alone, the mice treated with the vaccine

7/24 combination of the present disclosure (DDV), or the mice treated with the vaccine combination of the present disclosure in combination with anti-PD- antibody (Anti-PD-l+DDV), in accordance with an embodiment of the present disclosure.

[0035] In accordance with common practice, the various described features are not drawn to

scale and are drawn to emphasize features relevant to the present disclosure. Like reference

characters denote like elements throughout the figures and text.

DETAILED DESCRIPTION

[0036] The present disclosure will now be described more fully hereinafter with reference to

the accompanying drawings, in which exemplary embodiments of the disclosure are shown. This

disclosure may, however, be embodied in many different forms and should not be construed as

limited to the exemplary embodiments set forth herein. Rather, these exemplary embodiments are

provided so that this disclosure will be thorough and complete, and will fully convey the scope of

the disclosure to those skilled in the art. Like reference numerals refer to like elements throughout.

[0037] The terminology used herein is for the purpose of describing particular exemplary

embodiments only and is not intended to be limiting of the disclosure. As used herein, the singular

forms "a", "an", and "the" are intended to include the plural forms as well, unless the context

clearly indicates otherwise. It will be further understood that the terms "comprises" and/or

"comprising," or "includes" and/or "including" or "has" and/or "having", when used herein,

specify the presence of stated features, regions, integers, steps, operations, elements, and/or

components, but do not preclude the presence or addition of one or more other features, regions,

integers, steps, operations, elements, components, and/or groups thereof.

[0038] The term "functional variant", as used herein, refers to a protein (i) having at least

about 80%, 85%, 90%, 95,6%9, 97%, 98%, 99% or more identical in amino acid sequence to

the wild-type protein; and (ii) retaining the immunogenicity of the wild-type protein, for example,

the ability to induce protein-specific CD8'T cells.

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[0039] The term "in combination with", as used herein, includes the administration of two or

more vaccines or therapeutic agents simultaneously, or sequentially in any order within no specific

time limits, unless otherwise indicated, in the course of treating the same disease in the same

patient.

[0040] Unless otherwise defined, all terms (including technical and scientific terms) used

herein have the same meaning as commonly understood by one of ordinary skill in the art to which

this disclosure belongs. It will be further understood that terms, such as those defined in commonly

used dictionaries, should be interpreted as having a meaning that is consistent with their meaning

in the context of the relevant art and the present disclosure, and will not be interpreted in an

idealized or overly formal sense unless expressly so defined herein.

[0041] A vaccine combination of the present disclosure is provided for a subject having a

human papillomavirus (HPV)-associated disease. The subject having an HPV-associated disease

may be a mammal (e.g., human, mice, etc.) suffering from, 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.

[0042] The vaccine combination may include a first vaccine and a second vaccine. The first

vaccine includes a first fusion protein or a first polynucleotide that is designed to encode the first

fusion protein. The first fusion protein includes a plurality of HPV antigens and a heat shock

protein having a specific arrangement and thereby encompasses specific junction regions between

the HPV antigens and between the HPV antigen and the heat shock protein. The second vaccine

includes a second fusion protein and a third fusion protein, or a second polynucleotide that is

designed to encode the second fusion protein and the third fusion protein. The second fusion

protein includes two HPV antigens having a specific arrangement and thereby encompasses a

specific junction region between the HPV antigens. The third fusion protein also includes two

HPV antigens having a specific arrangement and thereby encompasses a specific junction region

9/24 between the HPV antigens. The amino acid sequences of the junction regions in the first fusion protein are different from those in the second fusion protein and the third fusion protein.

[0043] Specifically, the specific arrangement of HPV antigens of the first fusion protein, the

second fusion protein and the third fusion protein are described as follows:

[0044] The first fusion protein, in an order from N to C terminus, may include (i) an E7 protein

of human papillomavirus type 16 (HPV-16) or a functional variant thereof, wherein the E7 protein

of HPV-16 or a functional variant thereof comprises SEQ ID NO: 1 at the beginning of the protein

and SEQ ID NO: 2 at the end of the protein; (ii) an E7 protein of human papillomavirus type 18

(HPV-18) or a functional variant thereof, wherein the E7 protein of HPV-18 or a functional variant

thereof comprises SEQ ID NO: 3 at the beginning of the protein and SEQ ID NO: 4 at the end of

the protein; (iii) an E6 protein of HPV-16 or a functional variant thereof, wherein the E6 protein

of HPV-16 or a functional variant thereof comprises SEQ ID NO: 5 at the beginning of the protein

and SEQ ID NO: 6 at the end of the protein; (iv) an E6 protein of HPV-18 or a functional variant

thereof, wherein the E6 protein of HPV-18 or a functional variant thereof comprises SEQ ID NO:

7 at the beginning of the protein and SEQ ID NO: 8 at the end of the protein; and (v) a heat shock

protein or a functional variant thereof. Thereby, junctions are formed between the adjacent HPV

antigens (e.g., E7 protein of HPV-16 and E7 protein HPV-18; E7 protein HPV-18 and E6 protein

of HPV-16; and E6 protein of HPV-16 and E6 protein HPV-18) and between the E6 protein of

HPV-18 and the heat shock protein in the first fusion protein.

[0045] The second fusion protein, in an order from N to C terminus, may include an E6 protein

of HPV-16 or a functional variant thereof; and an E7 protein of HPV-16 or a functional variant

thereof. The third fusion protein, in an order from N to C terminus, may include an E6 protein of

HPV-18 or a functional variant thereof; and an E7 protein of HPV-18 or a functional variant

thereof. Thereby, a junction is formed between the adjacent HPV antigens (i.e., E6 protein of HPV

16 and E7 protein of HPV-16; or E6 protein of HPV-18 and E7 protein of HPV-18) in the second

fusion protein or the third fusion protein.

10/24

[0046] Due to the aforementioned arrangement of the first fusion protein, the second fusion

protein and the third fusion protein, the amino acid sequences of the junction regions in the first

fusion protein are different from those in the second fusion protein and the third fusion protein.

[0047] Examples of the first polynucleotide of the first vaccine and the second polynucleotide

of the second vaccine according to the vaccine combination are illustrated in FIG. 1A and FIG. 1B,

respectively.

[0048] FIG. 1A illustrates an exemplary first polynucleotide (denoted as PBI-11) that encodes

the first fusion protein. The first polynucleotide includes a fusion gene encoding the first fusion

protein, which in an order from N to C terminus comprises: a signal peptide (denoted as "S"), an

E7 protein of HPV-16 (denoted as E7(16)), an E7 protein of HPV-18 (denoted as E7(18)), an E6

protein of HPV-16 (denoted as E6(16)), an E6 protein of HPV-18 (denoted as E6(18), and a 70

kilodalton heat shock protein (HSP70).

[0049] The first fusion protein thereby has junction regions formed between the adjacent HPV

antigens (e.g., E7 protein HPV-16 and E7 protein HPV-18, E7 protein HPV-18 and E6 protein of

HPV-16, E6 protein of HPV-16 and E6 protein of HPV-18).

[0050] In some embodiments, the E7 protein of HPV-16 may comprise SEQ ID NO: 9, the

E7 protein of HPV-18 may comprise SEQ ID NO: 10, the E6 protein of HPV-16 may comprise

SEQ ID NO: 11, and the E6 protein of HPV-18 may comprise SEQ ID NO: 12.

[0051] In some embodiments, the E7 protein of HPV-16 may be encoded by SEQ ID NO: 13,

the E7 protein of HPV-18 may be encoded by SEQ ID NO: 14, the E6 protein of HPV-16 may be

encoded by SEQ ID NO: 15, and the E6 protein of HPV-18 may be encoded by SEQ ID NO: 16.

[0052] In some embodiments, pBI-11 may be used as the first vaccine of the instant vaccine

combination. pBI-11, which has a nucleotide sequence as set forth in SEQ ID NO: 17, is a DNA

construct including a fusion gene which includes: a subsequence encoding a signal peptide; an

optimized HPV subsequence (FIG. 1A, fragments 101) encoding an E7 protein of HPV-16 as set

forth in SEQ ID NO: 9, an E7 protein of HPV-18 as set forth in SEQ ID NO: 10, an E6 protein of

11/24

HPV-16 as set forth in SEQ ID NO: 11, and an E6 protein of HPV-18 as set forth in SEQ ID NO:

12; and a subsequence encoding an HSP70.

[0053] In some embodiments, alternatively and/or additively, pBI-12 may be used as the first

vaccine of the instant vaccine combination. pBI-12, which has a nucleotide sequence as set forth

in SEQ ID NO: 18, is a DNA construct comprising a fusion gene which includes: an optimized

signal sequence encoding a signal peptide; an optimized HPV subsequence encoding an E7 protein

of HPV-16 as set forth in SEQ ID NO: 9, an E7 protein of HPV-18 as set forth in SEQ ID NO: 10,

an E6 protein of HPV-16 as set forth in SEQ ID NO: 11, and an E6 protein of HPV-18 as set forth

in SEQ ID NO: 12; and a subsequence encoding an HSP70.

[0054] In some embodiments, the first vaccine may comprise the first polynucleotide, and the

first vaccine may be administered at a dose ranging from 10 micrograms per subject to 20

milligrams per subject.

[0055] FIG. 1B illustrates an exemplary second polynucleotide that encodes the second fusion

protein and the third fusion protein. As shown in FIG. 1B, a cassette for expression of the second

fusion protein and the third fusion protein is contained within the vaccinia virus genome (denoted

as TA-HPV). The second polynucleotide includes a first open reading frame for encoding an E6

and an E7 protein of HPV-16 (denoted as HPV16 E67 in FIG. IB); and a second open reading

frame for encoding an E6 and an E7 protein of HPV-18 (denoted as HPV18 E67 in FIG. IB).

[0056] In some embodiments, the second polynucleotide may be contained in a recombinant

virus.

[0057] In some embodiments, the recombinant virus which contains the second

polynucleotide may be, but not limited to TA-HPV. The TA-HPV may be administered at a dose

ranging from 1 x 104 pfu to 2 x 10' pfu. The TA-HPV may be administered preferably at a dose

ranging from 2 x 104 pfu to 5 x 107 pfu.

[0058] In some embodiments, the vaccine combination of the present disclosure may be

administered to a subject for treating an HPV-associated disease.

12/24

[0059] In some embodiments, the first vaccine of the vaccine combination may be

administered as a priming vaccine and the second vaccine of the vaccine combination may be

administered subsequently as a boosting vaccine, so as to provide a combination therapy being

used in a heterologous prime-boost regimen to enhance a subject's immune responses against HPV.

For example, the first vaccine may comprise the first polynucleotide and be administered at a dose

ranging from 10 micrograms per subject to 20 milligrams per subject, and the second vaccine may

comprise TA-HPV and be administered at a dose ranging from 1 x 104 pfu to 2 x 10 pfu.

[0060] In some embodiments, the first vaccine of the vaccine combination may be

administered as a priming vaccine and be administered subsequently as a boosting vaccine, and

after the administration of the first vaccine as the boosting vaccine, the second vaccine of the

vaccine combination may be administered subsequently also as a boosting vaccine, so as to provide

a combination therapy being used in a heterologous prime-boost regimen to enhance a subject's

immune responses against HPV.

[0061] In some embodiments, the vaccine combination may further include an immune

checkpoint inhibitor. The immune checkpoint inhibitor may be administered simultaneously

and/or sequentially in any order with the administration of the first vaccine and/or the second

vaccine. For example, the immune checkpoint inhibitor may be administered prior to and/or

simultaneously with the administration of the first vaccine.

[0062] In some embodiments, the immune checkpoint inhibitor may be an immune modulator

that targets programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-Li),

cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), inducible costimulator (ICOS), T-cell

immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3) or T cell

immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT).

For instance, the immune checkpoint may be an antibody targeting PD-i (also called "anti-PD-1

antibody").

[0063] In some embodiments, the first vaccine and the second vaccine of the vaccine

13/24 combination may further include an ingredient, such as an adjuvant, to create a stronger immune response in the subject receiving the vaccine.

Example 1: Design and synthesis of the vaccine combination

[0064] A pBI-1 DNA construct has been described previously as pNGVL4a

SigE7(detox)HSP70 (C. Trimple et al. Vaccine 2003, 21:4036-4042). In one example, the pBI-ll

DNA construct was derived by Gibson assembly of a synthesized DNA fragment encoding a fusion

protein of a signal peptide, an E7 protein of HPV-16, an E7 protein of HPV-18, an E6 protein of

HPV-16, an E6 protein of HPV-18, and a 5' portion of HSP70 (up to the Tth111I site), flanked 5'

by anEcoRI and Kozak site and 3'with a Tth111 site. The synthesized DNA fragment was cloned

into the pBI-1 to replace the fragment between EcoRI and Tth111I in frame with HSP70. The

expressing genes for the E7 protein of HPV-16, the E7 protein of HPV-18, the E6 protein of HPV

16, and the E6 protein of HPV-18 in pBI- IIwere codon-optimized and encoded by SEQ ID NO:

1-4 respectively. In another example, the pBI-12 DNA construct also included a synthesized DNA

fragment encoding a fusion protein of the signal peptide, an E7 protein of HPV-16, an E7 protein

of HPV-18, an E6 protein of HPV-16, an E6 protein of HPV-18, and a HSP70. The expressing

genes for the signal peptide, HPV-16 E7, HPV-18 E7, HPV-16 E6, and HPV-18 E6 in pBI-12 were

codon-optimized. The synthesized DNA fragment of pBI-12 was cloned into the pBI-1 to replace

the fragment between EcoRI and Tth111 in frame with HSP70.

[0065] The genes in the synthesized DNA fragment of each DNA construct that have been

either optimized for gene expression (FIG. 1A, fragments 101) or based on native papillomaviral

sequences (FIG. 1A, fragments 103) are shown in FIG. 1A.

[0066] TA-HPV is a recombinant vaccinia viral vaccine expressing oncogenes E6 and E7 of

HPV types 16 and 18. The HPV-16 and HPV-18 oncogenes E6 and E7 may be inserted in a head

to-head orientation under the control of the p7.5 and H6 promoters (denoted as P7.5 and H6,

respectively, in FIG. 1B) at a neutral site in the vaccinia virus Wyeth strain genome (L. K.

14/24

Borysiewicz et al., Lancet., 1996 Jun 1;347(9014):1523-7). For both the HPV-16 and HPV-18

genes, the E6 termination codon may be altered to create an E6/E7 fused open reading frame and

defined mutation introduced to inactivate the Rb-binding site in E7.

Example 2: The arrangement of the HPV antigens in the fusion protein encoded in the

vaccine combination does not include peptides with sequences common to host proteins that

can induce cross-reactive immunity against self-antigens.

[0067] To identify vaccine epitopes that might induce cross-reactivity against self-antigens, the

sequence of the first fusion protein encoded by the first vaccine to those of human proteins were

compared. Considering that peptide antigens are presented as fragments of either 8-11 amino acids

on MHC class I to CD8 T cells, or 12-20 amino acids on MHC class II to CD4 T cells, a search

for all 8-mers generated from the HPV16/18 E6/E7 peptides plus thejunctional regions in pBI-1

encoded fusion protein against human protein sequences in UniProt TM , which contains the Swiss

ProtTM and TrEMBLTM databases, was carried out.

[0068] Specifically, in order to search for potential novel peptides that may be identical to

endogenous peptides, all linear sequences of 5-8 amino acids in length (5-mers to 8-mers) from

amino acid 24-550 of pBI- I1were generated. The linear sequences span the last 7 amino acids of

the signal peptide, HPV-16 E7, HPV-18 E7, HPV-18 E6, HPV-16 E6, and the first 11 amino acids

of HSP70. In total, 520 8-mers, 521 7-mers, 522 6-mers, and 523 5-mers were generated.

[0069] Furthermore, the sequences were submitted to the UniProtTM protein database

(https://www.uniprot.org/) in groups of 80-100 sequences using the Peptide Search Tool provided

at the website and searched for exact matches against the Swiss-ProtT and TrEMBLTM databases

filtered for human proteins. The results revealed no exact match between the pBI- encoded

fusion protein and endogenous human peptide sequences that are at least 8 amino acids in length.

[0070] An alternative searching approach was also conducted. The Immune Epitope Database

(https://www.iedb.org/) was searched by using the same region of the pBI- protein sequence.

15/24

"Substring" for Linear Epitope to obtain any epitope sequences that are mapped to the pBI-10.1

sequence (disclosed in U.S. Patent Application No. 17/534,256, filed on November 23, 2021) were

selected. The search identified a B cell epitope derived from HPV-16 E7 in pBI-11 (see Table 1

below). This epitope is also present in pBI-1, which has been tested for its safety in human subjects.

Taken together, these results indicate that the arrangement of the fusion protein encoded by the

first vaccine of the instant application (e.g., pBI-11 or pBI-12) is unlikely to generate peptides that

could induce cross-reactive T cell immunity against self-antigens.

[0071] Table 1. Peptide epitope from pBI-1 protein identified in the Immune Epitope Database HPV Protein Epitope Human Protein Uniprot ID HPV16 E7 RTLED Glutamate decarboxylase 2 (GAD2) A0A3B3IU09

[0072] In addition, pBI-1 encodes a fusion protein of the signal peptide, HPV-16 E7, HPV-18

E7, HPV-16 E6, and HPV-18 E6 (E7(16/E7( 8)/E6(16)/E6( 8), see FIG. 1A), arranged in an order

different from that in TA-HPV (see FIG. 1B). Such arrangement can further avoid boosting

immune response against junction-associated epitopes potentially contained within the fusion

protein encoded by pBI-11.

[0073] As a result, the vaccine combination of the instant application reduces the risk to induce

cross-reactive immune response against self-antigens and the possibility to generate immunity

against junction-associated epitopes in the fusion protein comprised within or encoded by the

vaccine, and thus has an improved safety.

Example 3: HPV antigen-specific CD8* T cell-mediated immune responses generated by the

pBI-11 vaccine can be further enhanced by boost with TA-HPV vaccinia virus vaccine.

[0074] In vivo T cell activation assays were performed to compare immune response in mice

vaccinated with either pBI-11 alone, or pBI- 1in combination with TA-HPV.

[0075] FIG. 2A is a schematic illustration of the experiment design. 6- to 8-week-old female

C57BL/6 mice purchased from Taconic Biosciences (Germantown, NY) were vaccinated pBI-11

16/24

(25 mg/50 ml/mouse) through intramuscular (I.M.) injection. The mice were divided into a control

group (naive group), a DDD group and a DDV group. Each mouse in each group was boosted with

the same regimen 7 days later. One week after the second vaccination, the DDD group of the mice

(denoted as DDD in FIG.2A) was vaccinated with pBI-11 (25 mg/50 ml/mouse) through I.M.

injection. The DDV group of mice (denoted as DDV in FIG. 2A) was vaccinated with TA-HPV (1

X 106 pfu/50 ml/mouse) through I.M. injection. Six days after the last vaccination, peripheral blood

was collected from the vaccinated or naive mice for HPV-16 E7 tetramer staining. Fourteen days

after the last vaccination, splenocytes were prepared from the vaccinated mice and stimulated with

either HPV-16 E6 (aa 50 to 57), HPV-16 E7 (aa 49 to 57), or HPV-18 E6 (aa 67 to 75) peptide in

the presence of GolgiPlug T M (BD Pharmingen, San Diego, CA). It is known that the presentation

of epitope HPV-16 E6 (aa 50 to 57) is suppressed by the immunodominant epitope of HPV-16 E7

(aa 49 to 57). Intracellular IFN-y cytokine staining assay was performed to detect antigen-specific

CD8' T cells. The cells were acquired with a FACSCaliburTM flow cytometer, and data were

analyzed with CellQuest Pro software (BD Biosciences, Mountain View, CA).

[0076] As shown in FIG. 2B, mice vaccinated with the DDV regimen had significantly higher

percentages of E7-specific CD8+ T cells than mice vaccinated with pBI- IIalone (DDD) or naive

mice.

[0077] Furthermore, FIG. 2C shows that mice in a prime-vaccinia boost (DDV) had

significantly higher numbers of HPV-16 E7-specific T cells (P = 0.0428) and higher HPV-18 E6

specific T cells (P = 0.2116) than those that only received pBI-11. In fact, HPV16 E7-specific

CD8+ T cell-mediated immune responses in mice vaccinated with TA-HPV alone was previously

characterized (Virology 2018 Dec; 525:205-215). It is found that mice vaccinated with TA-HPV

alone did not generate appreciable HPV16 E7-specific CD8+ T cell-mediated immune responses

(Virology 2018 Dec; 525:205-215). However, our result indicates that TA-HPV booster

vaccination is capable of simultaneously enhancing HPV-16 and HPV-18 antigen-specific CD8'

T cell immune responses generated after priming with pBI- 1DNA vaccine.

17/24

[0078] Based on the above results, the heterologous prime-boost vaccination using the first

vaccine (e.g., pBI-ll) in combination with the second vaccine (e.g., TA-HPV) according to the

vaccine combination of the present disclosure induces an elevated HPV antigen-specific CD8+ T

cell-mediated immune responses compared to administration with the first vaccine (e.g., pBI-1)

alone.

Example 4: The vaccine combination of the present disclosure can be combined with anti

PD-1 immune checkpoint blockade to improve therapeutic antitumor response.

[0079] The ability of the vaccine combination of the present disclosure in combination with

an anti-PD-i immune checkpoint inhibitor to generate therapeutic antitumor effects against HPV

associated diseases were examined in the HPV-16 E6/E7+ TC-i tumor model.

[0080] FIG. 3A is a schematic illustration of the experiment design. 6- to 8-week-old female

C57BL/6 mice were injected with 2 x 105 of TC-i tumor cells subcutaneously on day 0. On day 3,

the mice were divided into 4 groups (untreated group, anti-PD-i group, DDV group and anti-PD

i+DDV group). The mice in the anti-PD-1 group were injected with purified anti-mouse PD-i

monoclonal antibody (MAb; clone 29F.iA12, 200mg/mouse) via intraperitoneal injection. The

treatment was repeated every other day. The mice in the DDV group were vaccinated with pBI-II

(25mg/ 50ml/mouse) through I.M. injection and boosted once 3 days later, and were further

boosted with TA-HPV vaccinia virus 3 days later through skin scarification. The mice in the anti

PD-i+DDV group were treated with both anti-mouse PD-i MAb and pBI-i IDNA vaccine prime

followed by TA-HPV vaccinia virus boost. On day 27, PBMCs were collected for the

characterization of HPV-16 E7-specific CD8' T cell-mediated immune responses using HPV-16

E7 peptide (aa 49 to 57)-loaded tetramer staining.

[008i] In one example, schematic illustration of the experiment in FIG. 3A shows that the

immune checkpoint inhibitor (e.g., anti-PD-i antibody) was administered prior to heterologous

prime-boost vaccination (e.g., DDV). However, in other examples, additively and/or alternatively,

18/24 the immune checkpoint inhibitor may be administered during the course of heterologous prime boost vaccination.

[0082] For tetramer staining, mouse PBMCs were stained with purified anti-mouse CD16/32

first and then stained with FITC-conjugated anti-mouse CD8a and PE-conjugated HPV-16 E7 (aa

49-57) peptide-loaded H-2Db tetramer at4C for 1 hour. After washing, the cells were stained with

7-AAD. The cells were acquired with the FACSCaliburTM flow cytometer and analyzed with

CellQuest Pro software (BD Biosciences, Mountain View, CA). The results of tetramer staining

are shown in FIG. 3B.

[0083] The growth of the tumor was monitored twice a week by palpation and digital caliper

measurement. Tumor volume was calculated using the formula [largest diameter x (perpendicular

diameter) 2 ]x 3.14/6 and is shown in FIG. 3C. The survival rate of the tumor-bearing mice was

recorded, as illustrated in FIG. 3D, where both natural death and a tumor diameter greater than

2 cm leading to death were counted as death.

[0084] As shown in Fig. 3B, mice receiving DDV regimen, either alone or with anti-PD-i

antibody, displayed HPV-16 E7-specific CD8+T cell-mediated immune responses, whereas in the

absence of vaccination with anti-PD-i antibody, treatment of anti-PD-i antibody did not elicit a

detectable HPV-16 E7-specific CD8+ T cell response.

[0085] Furthermore, FIG. 3C demonstrated that addition of anti-PD-i antibody treatment to the

DDV regimen significantly enhanced the therapeutic antitumor effects. This suggests synergy of

vaccination and anti-PD-i antibody treatment and that the latter is not effective without a prior

immune response. Additionally, FIG. 3D shows that the combinational treatment (anti-PD-I+DDV)

translated into significantly (P = 0.0073 when compared to DDV, and P = 0.0002 when compared

to anti-PD-1) better survival of the tumor-bearing mice.

[0086] In conclusion, the vaccine combination of the present disclosure including a first vaccine

which comprises a first fusion protein with an indicated arrangement of HPV antigens, or a first

polynucleotide encoding the same; and a second vaccine which comprises a second fusion protein

19/24 and a third fusion protein both with an arrangement of HPV antigens different from that in the first fusion protein, or a second polynucleotide encoding the second fusion protein and the third fusion protein, not only reduces the risk to induce cross-reactive immunity against self-antigens and to boost an immune response against junction-associated epitopes in the fusion protein contained in or encoded by the vaccine, but also exhibits a significant HPV antigen-specific immune response.

Moreover, the vaccine combination of the present disclosure in combination with an immune

checkpoint inhibitor (e.g., anti-PD-i antibody) elicits a stronger HPV antigen-specific antitumor

response in vivo and further translates into more potent antitumor efficacy.

[0087] Those skilled in the art will readily observe that numerous modifications and alterations

of 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.

[0088] In the claims which follow and in the preceding description of the invention, except

where the context requires otherwise due to express language or necessary implication, the word

"comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to

specify the presence of the stated feature but not to preclude the presence or addition of further

features in various embodiments of the invention.

[0089] It is to be understood that, if any prior art publication is referred to herein, such

reference does not constitute an admission that the publication forms a part of the common general

knowledge in the art, in Australia or any other country.

20/24

SEQUENCE LISTING

<110> Papivax Biotech Inc.

<120> VACCINE COMBINATION AND METHOD FOR USING THE SAME

<130> US86679 2022200326

<160> 18

<170> PatentIn version 3.5

<210> 1 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> HPV16 E7

<400> 1

Met His Gly Asp Thr Pro Thr Leu 1 5

<210> 2 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> HPV16 E7

<400> 2

Gly Pro Ile Cys Ser Gln Lys Pro 1 5

<210> 3 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> HPV18 E7

<400> 3

Met His Gly Pro Lys Ala Thr Leu 1 5

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

<210> 4 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> HPV18 E7 2022200326

<400> 4

Gly Pro Trp Cys Ala Ser Gln Gln 1 5

<210> 5 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> HPV16 E6

<400> 5

Met His Gln Lys Arg Thr Ala Met 1 5

<210> 6 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> HPV16 E6

<400> 6

Cys Cys Arg Ser Ser Arg Thr Arg 1 5

<210> 7 <211> 8 <212> PRT <213> Artificial Sequence

<220> <223> HPV18 E6

<400> 7

Met Ala Arg Phe Glu Asp Pro Thr

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

1 5

<210> 8 <211> 8 <212> PRT <213> Artificial Sequence 2022200326

<220> <223> HPV18 E6

<400> 8

Arg Gln Glu Arg Leu Gln Arg Arg 1 5

<210> 9 <211> 98 <212> PRT <213> Artificial Sequence

<220> <223> HPV16 E7

<400> 9

Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln 1 5 10 15

Pro Glu Thr Thr Asp Leu Tyr Gly Tyr Gly Gln Leu Asn Asp Ser Ser 20 25 30

Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp 35 40 45

Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr 50 55 60

Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu 65 70 75 80

Asp Leu Leu Met Gly Thr Leu Gly Ile Val Gly Pro Ile Cys Ser Gln 85 90 95

Lys Pro

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

<210> 10 <211> 105 <212> PRT <213> Artificial Sequence

<220> <223> HPV18 E7 2022200326

<400> 10

Met His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His Leu Glu 1 5 10 15

Pro Gln Asn Glu Ile Pro Val Asp Leu Leu Gly His Gly Gln Leu Ser 20 25 30

Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp Gly Val Asn His Gln His 35 40 45

Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met 50 55 60

Cys Cys Lys Cys Glu Ala Arg Ile Glu Leu Val Val Glu Ser Ser Ala 65 70 75 80

Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe 85 90 95

Val Gly Pro Trp Cys Ala Ser Gln Gln 100 105

<210> 11 <211> 153 <212> PRT <213> Artificial Sequence

<220> <223> HPV16 E6

<400> 11

Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro 1 5 10 15

Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

20 25 30

Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu 35 40 45

Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly 2022200326

50 55 60

Asn Pro Tyr Ala Val Gly Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile 65 70 75 80

Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu 85 90 95

Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn 100 105 110

Gly Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys 115 120 125

Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met 130 135 140

Ser Cys Cys Arg Ser Ser Arg Thr Arg 145 150

<210> 12 <211> 153 <212> PRT <213> Artificial Sequence

<220> <223> HPV18 E6

<400> 12

Met Ala Arg Phe Glu Asp Pro Thr Arg Arg Pro Tyr Lys Leu Pro Asp 1 5 10 15

Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys 20 25 30

Val Tyr Cys Lys Thr Val Leu Glu Leu Thr Glu Val Phe Glu Phe Ala

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

35 40 45

Phe Lys Asp Leu Phe Val Val Tyr Arg Asp Ser Ile Pro His Ala Ala 50 55 60

Gly His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg His 2022200326

65 70 75 80

Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys Leu Thr Asn Thr 85 90 95

Gly Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Gly Gln Lys Pro Leu 100 105 110

Asn Pro Ala Glu Lys Leu Arg His Leu Asn Glu Lys Arg Arg Phe His 115 120 125

Asn Ile Ala Gly His Tyr Arg Gly Gln Cys His Ser Cys Cys Asn Arg 130 135 140

Ala Arg Gln Glu Arg Leu Gln Arg Arg 145 150

<210> 13 <211> 294 <212> DNA <213> Artificial Sequence

<220> <223> Optimized HPV16 E7(detox) DNA sequence

<400> 13 atgcacgggg atacacccac actgcacgag tacatgctgg atctgcagcc cgagaccacc 60

gacctgtacg gctacggcca gctgaacgat tccagcgagg aggaggatga gattgacggg 120

cccgccggcc aggccgagcc cgatagggcc cactacaaca tcgtgacatt ctgctgcaag 180

tgcgatagca ccctgaggct gtgcgtccag agcacccacg tggacatcag gacactggag 240

gacctgctga tgggcaccct ggggatcgtg gggcccatct gcagccagaa gccc 294

<210> 14 <211> 315 <212> DNA

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

<213> Artificial Sequence

<220> <223> Optimized HPV18 E7(detox) DNA sequence

<400> 14 atgcacggcc ctaaggccac cctgcaggac atcgtgctgc acctggagcc tcagaacgag 60 2022200326

atccccgtgg acctgctggg gcacggccag ctgtccgatt ccgaggagga gaacgatgag 120

attgacggag tgaaccacca gcacctgcct gctaggaggg ccgaacccca gcggcacaca 180

atgctgtgca tgtgttgcaa gtgtgaggcc cggatcgagc tggtggtgga gagctcagcc 240

gatgacctgc gggccttcca gcagctgttc ctgaacacac tgagctttgt ggggccctgg 300

tgcgccagcc agcag 315

<210> 15 <211> 459 <212> DNA <213> Artificial Sequence

<220> <223> Optimized HPV16 E6(detox) DNA sequence

<400> 15 atgcaccaga agaggacagc catgttccag gacccccagg agcggccgag gaagctgccc 60

caactgtgca ccgagctgca gacaaccatc cacgacatca tcctggagtg cgtgtactgc 120

aagcagcagc tgctgaggag agaggtctac gattttgcct ttagagacct gtgcattgtg 180

taccgggatg gcaacccata cgccgtgggg gataaatgtt tgaagtttta cagcaagatt 240

tctgagtaca gacattactg ttattccctg tacggaacta cactggagca gcagtacaac 300

aagcccctgt gcgatctgct gattagatgc attaacggcc agaagccact gtgccctgag 360

gagaagcaga gacatctgga taagaagcag cggttccata acattagagg aagatggaca 420

ggcaggtgca tgtcatgctg cagaagctcc aggaccagg 459

<210> 16 <211> 459 <212> DNA <213> Artificial Sequence

<220> <223> Optimized HPV18 E6(detox) DNA sequence

<400> 16

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

atggcccggt ttgaggaccc cacaaggagg ccctacaagc tgcctgacct gtgcacagag 60

ctaaacacaa gcctccagga tattgagatc acctgcgtgt actgcaagac agtcctggag 120

ctgaccgagg tgttcgagtt cgcctttaag gatctgttcg tggtgtaccg ggatagcatc 180

ccccacgccg ccggccacaa gtgcatcgac ttctacagca ggatccggga gctgaggcac 240 2022200326

tacagcgata gcgtgtacgg ggacacactg gagaagctga ccaacacagg gctgtacaac 300

ctgctgatcc ggtgcctgag ggggcagaag cccctgaacc ccgccgagaa gctgaggcac 360

ctgaacgaga agaggcggtt ccacaacatc gccgggcact acaggggcca gtgccacagc 420

tgctgcaaca gggccaggca ggagaggctg cagcggcgc 459

<210> 17 <211> 3422 <212> DNA <213> Artificial Sequence

<220> <223> pBI-11 DNA Sequence

<400> 17 atggcggccc ccggcgcccg gcggccgctg ctcctgctgc tgctggcagg ccttgcacat 60

ggcgcctcag cactctttga ggatctaatc atgcacgggg atacacccac actgcacgag 120

tacatgctgg atctgcagcc cgagaccacc gacctgtacg gctacggcca gctgaacgat 180

tccagcgagg aggaggatga gattgacggg cccgccggcc aggccgagcc cgatagggcc 240

cactacaaca tcgtgacatt ctgctgcaag tgcgatagca ccctgaggct gtgcgtccag 300

agcacccacg tggacatcag gacactggag gacctgctga tgggcaccct ggggatcgtg 360

gggcccatct gcagccagaa gcccatgcac ggccctaagg ccaccctgca ggacatcgtg 420

ctgcacctgg agcctcagaa cgagatcccc gtggacctgc tggggcacgg ccagctgtcc 480

gattccgagg aggagaacga tgagattgac ggagtgaacc accagcacct gcctgctagg 540

agggccgaac cccagcggca cacaatgctg tgcatgtgtt gcaagtgtga ggcccggatc 600

gagctggtgg tggagagctc agccgatgac ctgcgggcct tccagcagct gttcctgaac 660

acactgagct ttgtggggcc ctggtgcgcc agccagcaga tgcaccagaa gaggacagcc 720

atgttccagg acccccagga gcggccgagg aagctgcccc aactgtgcac cgagctgcag 780

acaaccatcc acgacatcat cctggagtgc gtgtactgca agcagcagct gctgaggaga 840

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

gaggtctacg attttgcctt tagagacctg tgcattgtgt accgggatgg caacccatac 900

gccgtggggg ataaatgttt gaagttttac agcaagattt ctgagtacag acattactgt 960

tattccctgt acggaactac actggagcag cagtacaaca agcccctgtg cgatctgctg 1020

attagatgca ttaacggcca gaagccactg tgccctgagg agaagcagag acatctggat 1080 2022200326

aagaagcagc ggttccataa cattagagga agatggacag gcaggtgcat gtcatgctgc 1140

agaagctcca ggaccaggat ggcccggttt gaggacccca caaggaggcc ctacaagctg 1200

cctgacctgt gcacagagct aaacacaagc ctccaggata ttgagatcac ctgcgtgtac 1260

tgcaagacag tcctggagct gaccgaggtg ttcgagttcg cctttaagga tctgttcgtg 1320

gtgtaccggg atagcatccc ccacgccgcc ggccacaagt gcatcgactt ctacagcagg 1380

atccgggagc tgaggcacta cagcgatagc gtgtacgggg acacactgga gaagctgacc 1440

aacacagggc tgtacaacct gctgatccgg tgcctgaggg ggcagaagcc cctgaacccc 1500

gccgagaagc tgaggcacct gaacgagaag aggcggttcc acaacatcgc cgggcactac 1560

aggggccagt gccacagctg ctgcaacagg gccaggcagg agaggctgca gcggcgcatg 1620

gctcgtgcgg tcgggatcga cctcgggacc accaactccg tcgtctcggt tctggaaggt 1680

ggcgacccgg tcgtcgtcgc caactccgag ggctccagga ccaccccgtc aattgtcgcg 1740

ttcgcccgca acggtgaggt gctggtcggc cagcccgcca agaaccaggc ggtgaccaac 1800

gtcgatcgca ccgtgcgctc ggtcaagcga cacatgggca gcgactggtc catagagatt 1860

gacggcaaga aatacaccgc gccggagatc agcgcccgca ttctgatgaa gctgaagcgc 1920

gacgccgagg cctacctcgg tgaggacatt accgacgcgg ttatcacgac gcccgcctac 1980

ttcaatgacg cccagcgtca ggccaccaag gacgccggcc agatcgccgg cctcaacgtg 2040

ctgcggatcg tcaacgagcc gaccgcggcc gcgctggcct acggcctcga caagggcgag 2100

aaggagcagc gaatcctggt cttcgacttg ggtggtggca ctttcgacgt ttccctgctg 2160

gagatcggcg agggtgtggt tgaggtccgt gccacttcgg gtgacaacca cctcggcggc 2220

gacgactggg accagcgggt cgtcgattgg ctggtggaca agttcaaggg caccagcggc 2280

atcgatctga ccaaggacaa gatggcgatg cagcggctgc gggaagccgc cgagaaggca 2340

aagatcgagc tgagttcgag tcagtccacc tcgatcaacc tgccctacat caccgtcgac 2400

gccgacaaga acccgttgtt cttagacgag cagctgaccc gcgcggagtt ccaacggatc 2460

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

actcaggacc tgctggaccg cactcgcaag ccgttccagt cggtgatcgc tgacaccggc 2520

atttcggtgt cggagatcga tcacgttgtg ctcgtgggtg gttcgacccg gatgcccgcg 2580

gtgaccgatc tggtcaagga actcaccggc ggcaaggaac ccaacaaggg cgtcaacccc 2640

gatgaggttg tcgcggtggg agccgctctg caggccggcg tcctcaaggg cgaggtgaaa 2700 2022200326

gacgttctgc tgcttgatgt taccccgctg agcctgggta tcgagaccaa gggcggggtg 2760

atgaccaggc tcatcgagcg caacaccacg atccccacca agcggtcgga gactttcacc 2820

accgccgacg acaaccaacc gtcggtgcag atccaggtct atcaggggga gcgtgagatc 2880

gccgcgcaca acaagttgct cgggtccttc gagctgaccg gcatcccgcc ggcgccgcgg 2940

gggattccgc agatcgaggt cactttcgac atcgacgcca acggcattgt gcacgtcacc 3000

gccaaggaca agggcaccgg caaggagaac acgatccgaa tccaggaagg ctcgggcctg 3060

tccaaggaag acattgaccg catgatcaag gacgccgaag cgcacgccga ggaggatcgc 3120

aagcgtcgcg aggaggccga tgttcgtaat caagccgaga cattggtcta ccagacggag 3180

aagttcgtca aagaacagcg tgaggccgag ggtggttcga aggtacctga agacacgctg 3240

aacaaggttg atgccgcggt ggcggaagcg aaggcggcac ttggcggatc ggatatttcg 3300

gccatcaagt cggcgatgga gaagctgggc caggagtcgc aggctctggg gcaagcgatc 3360

tacgaagcag ctcaggctgc gtcacaggcc actggcgctg cccaccccgg ctcggctgat 3420

ga 3422

<210> 18 <211> 3422 <212> DNA <213> Artificial Sequence

<220> <223> pBI-12 DNA Sequence

<400> 18 atggccgccc caggagctag aaggccactt ttgttgctgt tgctggccgg attagcccat 60

ggagctagcg ctctgtttga ggatctgatc atgcacgggg atacacccac actgcacgag 120

tacatgctgg atctgcagcc cgagaccacc gacctgtacg gctacggcca gctgaacgat 180

tccagcgagg aggaggatga gattgacggg cccgccggcc aggccgagcc cgatagggcc 240

cactacaaca tcgtgacatt ctgctgcaag tgcgatagca ccctgaggct gtgcgtccag 300

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

agcacccacg tggacatcag gacactggag gacctgctga tgggcaccct ggggatcgtg 360

gggcccatct gcagccagaa gcccatgcac ggccctaagg ccaccctgca ggacatcgtg 420

ctgcacctgg agcctcagaa cgagatcccc gtggacctgc tggggcacgg ccagctgtcc 480

gattccgagg aggagaacga tgagattgac ggagtgaacc accagcacct gcctgctagg 540 2022200326

agggccgaac cccagcggca cacaatgctg tgcatgtgtt gcaagtgtga ggcccggatc 600

gagctggtgg tggagagctc agccgatgac ctgcgggcct tccagcagct gttcctgaac 660

acactgagct ttgtggggcc ctggtgcgcc agccagcaga tgcaccagaa gaggacagcc 720

atgttccagg acccccagga gcggccgagg aagctgcccc aactgtgcac cgagctgcag 780

acaaccatcc acgacatcat cctggagtgc gtgtactgca agcagcagct gctgaggaga 840

gaggtctacg attttgcctt tagagacctg tgcattgtgt accgggatgg caacccatac 900

gccgtggggg ataaatgttt gaagttttac agcaagattt ctgagtacag acattactgt 960

tattccctgt acggaactac actggagcag cagtacaaca agcccctgtg cgatctgctg 1020

attagatgca ttaacggcca gaagccactg tgccctgagg agaagcagag acatctggat 1080

aagaagcagc ggttccataa cattagagga agatggacag gcaggtgcat gtcatgctgc 1140

agaagctcca ggaccaggat ggcccggttt gaggacccca caaggaggcc ctacaagctg 1200

cctgacctgt gcacagagct aaacacaagc ctccaggata ttgagatcac ctgcgtgtac 1260

tgcaagacag tcctggagct gaccgaggtg ttcgagttcg cctttaagga tctgttcgtg 1320

gtgtaccggg atagcatccc ccacgccgcc ggccacaagt gcatcgactt ctacagcagg 1380

atccgggagc tgaggcacta cagcgatagc gtgtacgggg acacactgga gaagctgacc 1440

aacacagggc tgtacaacct gctgatccgg tgcctgaggg ggcagaagcc cctgaacccc 1500

gccgagaagc tgaggcacct gaacgagaag aggcggttcc acaacatcgc cgggcactac 1560

aggggccagt gccacagctg ctgcaacagg gccaggcagg agaggctgca gcggcgcatg 1620

gctcgtgcgg tcgggatcga cctcgggacc accaactccg tcgtctcggt tctggaaggt 1680

ggcgacccgg tcgtcgtcgc caactccgag ggctccagga ccaccccgtc aattgtcgcg 1740

ttcgcccgca acggtgaggt gctggtcggc cagcccgcca agaaccaggc ggtgaccaac 1800

gtcgatcgca ccgtgcgctc ggtcaagcga cacatgggca gcgactggtc catagagatt 1860

gacggcaaga aatacaccgc gccggagatc agcgcccgca ttctgatgaa gctgaagcgc 1920

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

gacgccgagg cctacctcgg tgaggacatt accgacgcgg ttatcacgac gcccgcctac 1980

ttcaatgacg cccagcgtca ggccaccaag gacgccggcc agatcgccgg cctcaacgtg 2040

ctgcggatcg tcaacgagcc gaccgcggcc gcgctggcct acggcctcga caagggcgag 2100

aaggagcagc gaatcctggt cttcgacttg ggtggtggca ctttcgacgt ttccctgctg 2160 2022200326

gagatcggcg agggtgtggt tgaggtccgt gccacttcgg gtgacaacca cctcggcggc 2220

gacgactggg accagcgggt cgtcgattgg ctggtggaca agttcaaggg caccagcggc 2280

atcgatctga ccaaggacaa gatggcgatg cagcggctgc gggaagccgc cgagaaggca 2340

aagatcgagc tgagttcgag tcagtccacc tcgatcaacc tgccctacat caccgtcgac 2400

gccgacaaga acccgttgtt cttagacgag cagctgaccc gcgcggagtt ccaacggatc 2460

actcaggacc tgctggaccg cactcgcaag ccgttccagt cggtgatcgc tgacaccggc 2520

atttcggtgt cggagatcga tcacgttgtg ctcgtgggtg gttcgacccg gatgcccgcg 2580

gtgaccgatc tggtcaagga actcaccggc ggcaaggaac ccaacaaggg cgtcaacccc 2640

gatgaggttg tcgcggtggg agccgctctg caggccggcg tcctcaaggg cgaggtgaaa 2700

gacgttctgc tgcttgatgt taccccgctg agcctgggta tcgagaccaa gggcggggtg 2760

atgaccaggc tcatcgagcg caacaccacg atccccacca agcggtcgga gactttcacc 2820

accgccgacg acaaccaacc gtcggtgcag atccaggtct atcaggggga gcgtgagatc 2880

gccgcgcaca acaagttgct cgggtccttc gagctgaccg gcatcccgcc ggcgccgcgg 2940

gggattccgc agatcgaggt cactttcgac atcgacgcca acggcattgt gcacgtcacc 3000

gccaaggaca agggcaccgg caaggagaac acgatccgaa tccaggaagg ctcgggcctg 3060

tccaaggaag acattgaccg catgatcaag gacgccgaag cgcacgccga ggaggatcgc 3120

aagcgtcgcg aggaggccga tgttcgtaat caagccgaga cattggtcta ccagacggag 3180

aagttcgtca aagaacagcg tgaggccgag ggtggttcga aggtacctga agacacgctg 3240

aacaaggttg atgccgcggt ggcggaagcg aaggcggcac ttggcggatc ggatatttcg 3300

gccatcaagt cggcgatgga gaagctgggc caggagtcgc aggctctggg gcaagcgatc 3360

tacgaagcag ctcaggctgc gtcacaggcc actggcgctg cccaccccgg ctcggctgat 3420

ga 3422

220118-148100-US86679-WP-Sequence Listing-v1F.txt[19/01/2022 11:16:55 am]

Claims (16)

What is claimed is:

1. A vaccine combination comprising a first vaccine comprising: a first fusion protein or afirst polynucleotide encoding the first fusion protein, wherein the first fusion protein, in an order from N to C terminus, comprises: (i) an E7 protein of human papillomavirus type 16 (HPV-16) or a functional variant thereof, the E7 protein of HPV-16 or a functional variant thereof comprising SEQ ID NO: 1 at a beginning of the protein and SEQ ID NO: 2 at an end of the protein; (ii) an E7 protein of human papillomavirus type 18 (HPV-18) or a functional variant thereof, the E7 protein of HPV-18 or a functional variant thereof comprising SEQ ID NO: 3 at a beginning of the protein and SEQ ID NO: 4 at an end of the protein; (iii) an E6 protein of HPV-16 or a functional variant thereof, the E6 protein of HPV-16 or a functional variant thereof comprising SEQ ID NO: 5 at a beginning of the protein and SEQ ID NO: 6 at an end of the protein; (iv) an E6 protein of HPV-18 or a functional variant thereof, the E6 protein of HPV-18 or a functional variant thereof comprising SEQ ID NO: 7 at a beginning of the protein and SEQ ID NO: 8 at an end of the protein; and (v) a heat shock protein or a functional variant thereof; and a second vaccine comprising: a second fusion protein and a third fusion protein, or a second polynucleotide encoding the second fusion protein and the third fusion protein, wherein the second fusion protein comprises: an E6 protein of HPV-16 or a functional variant thereof; and an E7 protein of HPV-16 or a functional variant thereof; and wherein the third fusion protein comprises: an E6 protein of HPV-18 or a functional variant thereof; and an E7 protein of HPV-18 or a functional variant thereof, wherein amino acid sequences of junction regions in the first fusion protein are different from those in the second fusion protein and the third fusion protein.

2. The vaccine combination according to claim 1, wherein the E7 protein of HPV-16 comprises SEQ ID NO: 9;

21/24 wherein the E7 protein of HPV-18 comprises SEQ ID NO: 10; wherein the E6 protein of HPV-16 comprises SEQ ID NO: 11; and wherein the E6 protein of HPV-18 comprises SEQ ID NO: 12.

3. The vaccine combination according to claim 1, wherein the E7 protein of HPV-16 is encoded by SEQ ID NO: 13; wherein the E7 protein of HPV-18 is encoded by SEQ ID NO: 14; wherein the E6 protein of HPV-16 is encoded by SEQ ID NO: 15; and wherein the E6 protein of HPV-18 is encoded by SEQ ID NO: 16.

4. The vaccine combination according to claim 1, wherein the first polynucleotide comprises SEQ ID NO: 17.

5. The vaccine combination according to claim 1, wherein the first polynucleotide comprises SEQ ID NO: 18.

6. The vaccine combination according to claim 1, wherein the second polynucleotide is contained within a recombinant virus.

7. The vaccine combination according to claim 6, wherein the recombinant virus is TA-HPV.

8. A method for treating an HPV-associated disease in a subject in need thereof, comprising: administering the vaccine combination according to claim 1 to the subject, wherein the first vaccine is administered as a priming vaccine; and the second vaccine is administered as a boosting vaccine.

9. The method according to claim 8, further comprising: administering the first vaccine to the subject as a boosting vaccine after the administration of the first vaccine as a priming vaccine and prior to the administration of the second vaccine as a boosting vaccine.

10. The method according to claim 8, wherein the first vaccine comprises thefirst polynucleotide, and wherein the first vaccine is administered at a dose ranging from 10 micrograms per subject to 20 milligrams per subject.

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11. The method according to claim 8, wherein the second vaccine comprises TA-HPV, and wherein the second vaccine is administered at a dose ranging from 1 x 104 plaque-forming units (pfu) to 2 x 10 pfu.

12. The method according to claim 8, further comprising: administering a chemotherapy, radiotherapy, chemo-radiotherapy, cryotherapy, thermotherapy, targeted therapy, cellular therapy, gene therapy, or immunotherapy in combination with the administration of the vaccine combination.

13. The method according to claim 12, wherein the chemotherapy, radiotherapy, chemo radiotherapy, cryotherapy, thermotherapy, targeted therapy, cellular therapy, gene therapy, or immunotherapy is administered prior to and/or simultaneously with the administration of the first vaccine.

14. The method according to claim 12, wherein the immunotherapy comprises administering an immune checkpoint inhibitor to the subject.

15. The method according to claim 14, wherein the immune checkpoint inhibitor is an immune modulator targeting programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD LI), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), inducible costimulator (ICOS), T-cell immunoglobulin and mucin domain 3 (TIM-3), lymphocyte activation gene 3 (LAG-3) or T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT).

16. The method according to claim 14, wherein the immune checkpoint inhibitor is an anti-PD-I antibody.

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