The use of pokeweed antiviral protein isoform one (PAP-i) for the treatment of human papilloma virus (HPV) infection and HPV-associated diseases

Abstract

The invention refers to the use of PAP-I, a plant ribosome inactivating protein (RIP), or a functional variant thereof for treatment of human papillomavirus (HPV) infection and preventing or treating diseases or conditions associated with HPV infection.

Description

[0001] THE USE OF POKEWEED ANTIVIRAL PROTEIN ISOFORM ONE (PAP-I) FOR THE TREATMENT OF HUMAN PAPILLOMA VIRUS (HPV) INFECTION AND HPV-ASSOCIATED DISEASES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the treatment of virus infection. In particular, the invention refers to the treatment of human papilloma virus (HPV) infection and the prophylaxis or treatment of HPV-associated diseases.

[0004] BACKGROUND OF THE INVENTION

[0005] HPV is a virus that infects epithelial cells and has about 200 subtypes. It is classified into low-risk and high-risk types based on its potential to cause tumors. High-risk HPV (hrHPV) types, such as HPV 16, HPV18 and others, are major factors in the development of cervical, vaginal, penile, and oral cancers, as well as high-grade intraepithelial lesions. Low-risk HPV (IrHPV) types, such as HPV6 and HPVl l, lead to conditions like low-grade squamous intraepithelial lesions (LSIL), flat warts, and genital warts. Over 80% of women has HPV infection during their lifetime, but most infections are transient and cleared up on their own in a short time. However, 10-20% of infections with hrHPV are persistent, potentially involving integration of the viral DNA into the human genome, thereby causing cancers such as cervical cancer. Research shows that 99.7% of cervical cancers are due to high-risk HPV infections. Cervical cancer represents about 5% of all human cancers, with approximately 600,000 new cases and 340,000 deaths worldwide annually, according to WHO statistics from 2020.

[0006] The most concerning aspect of persistent hrHPV infections is the random integration of viral DNA into the human genome. This integration can inactivate the HPV E2 gene, leading to the uncontrolled overexpression of the HPV E6 and E7 oncogenes. These oncoproteins disrupt the functions of tumor suppressor proteins such as p53 and retinoblastoma protein (pRb), leading to the development of cancers like cervical cancer. Recent studies have found that the HPV virus is integrated into the human genome in more than 80% of cervical cancer cases. Therefore, the timely elimination of persistent hrHPV infection is crucial for preventing the occurrence of associated cancers such as cervical cancer. Preventing the HPV virus from entering host cells is the primary goal of developing HPV preventive vaccines. Currently, 2-valent, 4-valent, and 9-valent HPV vaccines have been developed and are available on the market. These preventive vaccines use the virus's major capsid protein, LI, as the immune antigen. Through genetic engineering, the recombinant L1 protein is expressed and forms virus-like particles. These recombinant virus-like particles are used as antigens to immunize the human body, inducing the production of anti -HPV LI protein antibodies. These antibodies bind to the HPV LI capsid protein, neutralizing the virus and preventing it from entering cells, thereby preventing HPV infection. However, for patients already infected with HPV, where the virus has entered cells and may have established a persistent infection, preventive vaccines are ineffective.

[0007] Currently, conventional treatments for persistent hrHPV infections include cryotherapy, carbon dioxide therapy, laser therapy, loop electrosurgical excision procedure, and cold knife conization. There is no antiviral drug currently available that specifically targets HPV infection. International experts agree that there is a significant gap in therapeutic drugs for persistent HPV infection.

[0008] Thus, there is an urgent need for drugs that can eliminate persistent HPV infections and treat diseases resulting from HPV infection.

[0009] SUMMARY OF THE INVENTION

[0010] In a first aspect, there is provided a use of pokeweed antiviral protein isoform one (PAP-I), a plant ribosome inactivating protein (RIP), or a functional variant thereof in the manufacture of a pharmaceutical composition for treating human papilloma virus (HPV) infection.

[0011] In some embodiments, the pharmaceutical composition is for clearing HPV persistent infection or for the prophylaxis or treatment of HPV-associated diseases or conditions.

[0012] In some embodiments, PAP-I or a functional variant thereof comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0013] In some embodiments, the pharmaceutical composition is for prophylaxis or treatment of HPV-associated diseases or conditions. Such HPV-associated diseases or conditions may be selected from a group consisting of genital tract inflammation, cervical polyp, warts, intraepithelial neoplasia, and HPV associated cancer Preferably, the genital tract inflammation is selected from a group consisting of viral vaginitis, vaginal cysts, and viral cervicitis; the cervical polyp is selected from polyps formed by excessive proliferation of cervical mucosa due to chronic inflammation and cervical infections, protruding towards the external cervical ; the warts are selected from the group consisting of condyloma acuminatum, anal warts, common warts, plantar warts, flat warts, verruca filiformis, subungual and periungual warts; the intraepithelial neoplasia is selected from the group consisting of cervical intraepithelial neoplasia (CIN, including CIN I, CIN II, CIN III, which is also known as squamous intraepithelial lesion (SIL) that can be classified as low-grade SIL (LSIL) or high-grade SIL(HSIL)), vaginal intraepithelial neoplasia (ValN, including ValN I, ValN II, ValN III), vulvar intraepithelial neoplasia (VIN, including VIN I, VIN II, VIN III), penile intraepithelial neoplasia (PIN, including PIN I, PIN II, PIN III), and anorectal intraepithelial neoplasia (AIN, including AIN I, AIN II, AIN III); and HPV associated cancers such as cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, laryngeal cancer, and oral cancer.

[0014] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient. For example, the excipient may be selected from adhesives, wetting agents, suspending agents, buffers, preservatives, pH control agents and glidants. Preferably, the pharmaceutical composition further comprises the following excipients, including but not limited to, the cellulose derivatives such as carboxymethyl cellulose, sodium alginate, carrageenan, sodium starch glycolate, carbomer, trehalose, dextran, cyclodextrin, and the like.

[0015] In some embodiments, the pharmaceutical composition is in the form of a powder, solution, liniment, gel, dressing, tablet, pill, capsule, emulsion, or ointment.

[0016] In certain embodiments, the pharmaceutical composition is administered topically to either lower genital tract or skin. The pharmaceutical composition may also be administered via inhalation or parenteral injection (such as intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, or intracavernous injection). In a second aspect, there is provided a pharmaceutical composition comprising a PAP-I or a functional variant thereof and at least one pharmaceutically acceptable excipient for use in treating HPV infection. In particular, the pharmaceutical composition is used for the prophylaxis or treatment of HPV-associated diseases or conditions.

[0017] In a third aspect, there is provided a method for treating HPV infection in a subject, comprising administering a therapeutic effective amount of a composition comprising PAP-I or a functional variant to said subject. In particular, the method is for the prophylaxis or treatment of HPV-associated diseases or conditions

[0018] In a fourth aspect, there is provided an in vitro method for inhibiting the expression of HPV E6 / E7 oncogenes in a sample (such as cultured cells, isolated sample and the like), comprising contacting the sample with PAP-1 or a functional variant thereof.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Certain embodiments of the present disclosure are described more fully hereinafter with reference to the accompanying drawings.

[0021] Figure 1 shows the effects of PAP-I on the growth of cervical cancer cells Ca Ski (A) compared to a non-cervical cell line 293 T (B) in the presence or absence of anti -PAP-I antibody for up to 6 days. Data shows mean ± SD of three independent experiments.

[0022] Figure 2 shows the effects of PAP-I and PAP-II on the growth of cervical cancer cells Ca Ski. Histograms show mean ± SD of three independent experiments. ** p<0.01 versus control analyzed by one-way analysis of variance followed by Bonferroni test.

[0023] Figure 3 shows the effects of PAP-I on the expression of HPV16 E7 oncoprotein in Ca Ski cells (A) and HPV18 E6 oncoprotein in HeLa cells (B) in the presence or absence of anti- PAP-I antibody for up to 6 days. The expressions of -actin (Actin), HPVI6 E7 in Ca Ski (A) and HPV18 E6 in HeLa (B) were detected by western blot. For each set, a representative image from three repeated experiments is shown.

[0024] Figure 4 shows PAP-I -mediated inhibition of tumor growth in SCID mice. Hella cells were treated by different concentrations of PAP-I for 3 days, and then the cells were harvested and used as tumor inducer. Histograms show mean ± SD of three independent experiments. **p < 0.01; ***p < 0.001 vs. control (0 pg / ml PAP-I) analyzed by one-way analysis of variance followed by Bonferroni test.

[0025] Figure 5 shows the effects of a pharmaceutical composition including PAP-I on inhibition of HPV E6 / E7 mRNA. After the treatment of one course (3 months), the expression level of E6 / E7 mRNAs in cervical specimens was evaluated using Aptima HPV assay (Gen-Probe Inc., San Diego, CA) according to the manufacturing introduction. Specimens with S / CO values of 2=0.5 were considered as positive. ***p < 0.001 vs. control (T test).

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] Further details of the invention will now be described with reference to the following nonlimiting examples. Unless otherwise defined herein, technical and scientific terms used in the present description have meanings that are commonly understood by those of ordinary skill in the art.

[0028] A. Definitions

[0029] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0030] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of’. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases that require the presence of the named features / steps and permit the presence of other features / steps. However, such description should be construed as also describing compositions, mixtures, or processes as “consisting of’ and “consisting essentially of’ the enumerated features / steps, which allows the presence of only the named features / steps, along with any impurities that might result therefrom, and excludes other features / steps.

[0031] As used herein, the term “therapeutically effective amount” or “efficacious amount” of a drug refers to an amount of the drug that is an amount sufficient to obtain a pharmacological response such as activating a biological target (e g., inhibiting HPV infection); or alternatively, is an amount of the drug that, when administered to a subject with a specified disorder or disease, is sufficient to have the intended effect, e g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the specified disorder or disease in the subject A therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The therapeutically effective amount will depend in part on the nature of the drug, the manner and route of administration, the stage and severity of the disease being treated, the weight and general state of health of the subject, and the judgment of the prescribing physician.

[0032] As used herein, the term “subject” refers to animals, typically mammals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates, domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human.

[0033] As used herein, “preventing” or “prophylaxis” is an approach for preventing the occurrence of a specific disease or condition (such as HPV associated cancer). For example, some patients may be diagnosed as having HPV infection while not exhibiting observable symptoms. Timely clearance of HPV in such patients thus would be necessary to prevent the occurrence of severe diseases or conditions (such as HPV associated cancers) at a later stage.

[0034] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and / or prolonging survival. The treatment may be achieved by eliminating or clearing the virus such as HPV which causes the disease or condition. As used herein, terms “eliminating”, “elimination”, “clearing” and “clearance” are used interchangeably in the present invention, and mean that the virus (such as HPV) is no longer detectable (i.e., becoming HPV negative) by known methods such as PCR after treatment with the PAP-T protein or a functional variant thereof in an environment such as in a subject. Also encompassed by “treatment” is a reduction of pathological consequence of the disease. The methods of the present disclosure contemplate any one or more of these aspects of treatment.

[0035] As used herein, the term "pharmaceutically acceptable", as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a human).

[0036] As used herein, the term “excipient” broadly refers to any component other than the active therapeutic ingredient (i.e., RIP such as PAP-I). The excipient may be an inert substance, an inactive substance, and / or a not medicinally active substance The excipient may serve various purposes, e.g. as a excipient, vehicle, filler, binder, lubricant, glidant, disintegrant, flow control agents, crystallization retarders, solubilizers, stabilizer, enzyme inhibitors, basifies, acidifiers and / or to improve administration, and / or absorption of the active substance. A person skilled in the art may select one or more of the aforementioned excipients with respect to the particular desired properties of the dosage form by routine experimentation and without any undue burden. The amount of each excipient used may vary within ranges conventional in the art.

[0037] As used herein, the term “functional variant” refers to a sequence similarity to a reference protein and retains substantial biological activity of reference protein of which it is a variant. Such functional variants can have one or more amino acid additions, substitutions, deletions and / or chemical modifications when compared to the reference protein sequence. Deletions and insertions may be internal and / or at one or more termini. Substitutions may be a conservative substitution that includes the replacement of one or more amino acids with a similar or homologous amino acid(s) that have little or no effect on the overall net charge, polarity, or hydrophobicity of the protein. Substitutions may also be with either the L- or the D-form of an amino acid. The functional variant may comprise an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference polypeptide, while maintaining at least similar or sometimes having even superior biological activity as compared to the reference protein.

[0038] B. Use of plant ribosome inactivating protein (RIP)

[0039] Plant RlPs are EC3.2.2.22 N-glycosidases, found among most plant species encoded as small gene families, distributed in several tissues being endowed with defensive functions against fungal or viral infections. Based on their physical properties, the number of polypeptide chains, and posttranslational modifications, RIPs are classified into three types. Type I RIPs have a single RNA N-glycosidase domain. They are strongly basic proteins that are clearly distinct in their global sequence homology and posttranslational alterations, yet share several active site residues and secondary structure elements. The majority of characterized RIPs fall into this category. Examples of type I RIPs include pokeweed antiviral protein (PAP), saporin, and barley (Hordeum vulgare) translational inhibitor. Type II RIPs have two structurally and functionally distinct domains: the catalytic RNA N-glycosidase domain and the carbohydrate (lectin properties) binding domain Examples of type II RIPs include ricin, abrin and modeccin. Type III RIPs are synthesized as inert precursors (pro-RIPs), and undergo proteolytic modifications to acquire their enzymatically activity These RIPs are much less common than either type I or type II RIPs. Type III RIPs have been identified from maize (Zea mays) and barley (from H. vulgare).

[0040] PAP-I is a 29 kDa type I RIP found in pokeweed plants. It removes specific purine residues from the sarcin / ricin (S / R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP-I is thought to play an important role in the plant’s defence mechanism against foreign pathogens. Several different forms of PAP have been isolated from Phytolacca americana L. , namely PAP-I from the spring leaves, PAP -II from the early summer leaves, PAP-III from late summer leaves, PAP-S and its two isoforms PAP-SI and PAP-S2 from the seeds, PAP-R from roots, PAP-H from hairy roots, a-PAP from all organs. It was reported that PAP-I shares 76% sequence identity with PAP-S 1 and PAP-S, 39% identity with PAP-II, 37% identity with PAP-III, and 74% identity with a-PAP. The variance of sequence identity among different PAPs suggests that they may have some different functions and activities. The present invention provides the use of PAP-I, which is a plant ribosome inactivating protein (RIP) or a functional variant thereof in the manufacture of a pharmaceutical composition for treating human papilloma virus (HPV) infection.

[0041] The present invention also provides a pharmaceutical composition for treating human papilloma virus (HPV) infection comprising PAP-I or a functional variant thereof and at least one pharmaceutically acceptable excipient.

[0042] The present invention further provides a method for treating HPV infection in a subject, comprising administering a therapeutic effective amount of a pharmaceutical composition comprising PAP-I or a functional variant thereof to said subject.

[0043] The amino acid sequence of PAP-I is as shown in SEQ ID NO: 1 . Thus, PAP-I or a functional variant thereof preferably comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence set forth in SEQ ID NO: 1. More preferably, PAP-I or a functional variant thereof consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0044] The PAP-I or a functional variant thereof may be derived by direct isolation from plant tissues. For example, the PAP-I may be isolated from plants of the Phytolacca family, preferably but not limited to Phytolacca americana L. , Phytolacca acinosa Roxb. , Phytolacca dioica or Phytolacca dodecandra. Alternatively, PAP-1 or a functional variant may also be obtained by recombinant expression.

[0045] The PAP-I, a plant RIP or a functional variant thereof may be used for treating or inhibiting HPV infection in vivo or in vitro, in particular persistent high-risk HPV infection. For example, it may be used as an inhibitor of HPV infection in some in vitro experiments. More importantly, it may also be used to eliminate HPV infection in a subject (i.e., in vivo), thus to treat HPV-associated diseases or conditions Examples of HPV-associated diseases or conditions that can be prevented or treated by the present invention include but not limited to genital tract inflammation, cervical polyp, warts, intraepithelial neoplasia and HPV associated cancer. For example, the genital tract inflammation may be selected from a group consisting of viral vaginitis, vaginal cysts, and viral cervicitis; cervical polyp may be selected from polyps formed by excessive proliferation of cervical mucosa due to chronic inflammation and cervical infections, protruding towards the external cervical; the warts may be selected from the group consisting of condyloma acuminatum, anal warts, common warts, plantar warts, flat warts, verruca filiformis , subungual and periungual warts; and intraepithelial neoplasia may be selected from the group consisting of cervical intraepithelial neoplasia (CIN, including CIN I, CIN II, CIN III, which is also known as squamous intraepithelial lesion (SIL) that can be classified as low-grade SIL (LSIL) or high-grade SZL(HSIL)), vaginal intraepithelial neoplasia (ValN, including ValN I, ValN II, ValN III), vulvar intraepithelial neoplasia (VIN, including VIN I, VIN II, VIN III), penile intraepithelial neoplasia (PIN, including PIN I, PIN II, PIN III) ,and anorectal intraepithelial neoplasia(AIN, including AIN I, AIN 11, AIN III); and HPV associated cancers such as cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, laryngeal cancer, and oral cancer.

[0046] With the PAP-I or a functional variant thereof being the active ingredient for treating HPV infection, the pharmaceutical composition may further comprise at least one pharmaceutically acceptable excipient.

[0047] Pharmaceutically acceptable excipients suitable for use in the present invention may be selected based on the chosen route of administration and standard pharmaceutical practice.

[0048] Examples of excipients may include, but not limited to the following:

[0049] (a) a binder or a filler, which is selected from acacia, agar, alginic acid, carmellose sodium, dextrin, veegum or gel white, gellan gum, sodium alginate, hydroxypropyl starch, maltodextrin, modified starch, pectin, potassium alginate, polyvinyl pyrrolidone, carboxymethyl cellulose or an alkali metal salt thereof, microcrystalline cellulose, bentonite, col loidal silicon dioxide, microcrystalline cellulose / sodium carboxy methylcellulose, gum tragacanth, corn starch, or gelatin;

[0050] (b) a wetting agent, which is selected from alcohol, glycerin, propylene glycol, polyethylene glycol, mineral oil, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, docusate sodium, nonoxynol 9, octoxynol, poloxamer, poloxamer 124, poloxamer 188, 237, 338, 407, polyoxyl 35 castor oil, polyoxyl 40;

[0051] (c) a suspending agent (sometimes also known as thickening agents), which is selected from gelatin, crosslinked polyacrylic acid, polymethacrylic acid, polyhydroxyethyl methacrylic acid, hydroxypropyl methyl cellulose, polyethylene glycol, sodium carboxymethyl cellulose, hyaluronic acid, chitosan, polycarbophil, pectin, copolymers of dextran, polyacrylamide, acacia, copolymer of caprolactone and ethylene oxide, carbopol 934, tragacanth, eudragit, polyvinyl pyrrolidone, polyacrylate and polyacrylate copolymer resins, celluloses and cellulose derivatives for example methyl-, ethyl- and propyl celluloses; hydroxyalkylcelluloses, hydroxyl propyl celluloses, hydroxyl propyl alkyl celluloses and the like including xanthan gum, polyvinyl resins, polyethylene glycol, polyethylene oxide, sorbitol, sucrose, xylitol, dextrose, carbomer, trehalose, fructose, maltitol, sugar, sodium alginate, carrageenan, sodium starch glycolate;

[0052] (d) a buffering agent, which is selected from a group comprising acetate, amino acids, ammonium sulfate, benzoate, bicarbonate, borate, citrate, citric acid monohydrate, disodium hydrogen phosphate, glutamate, lactate, meglumine, potassium citrate, sodium acetate, sodium citrate, sodium phosphate, sulfate, tartrate, triethanolamine, TRIS, and trisodium citrate dehydrate,

[0053] (e) a preservative, which is selected from a group comprising vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben, chlorhexidine gluconate, chlorhexidine acetate, chlorhexidine hydrochloride, triclosan, benzalkonium bromide, and polyhexamethylene biguanide hydrochloride;

[0054] (f) a pH control agent, which is selected from a group comprising citric acid, tartaric acid, fumaric acid, sodium citrate, dibasic calcium phosphate, and dibasic sodium phosphate;

[0055] (g) a glidant, which is selected from a group comprising silica gel, fumed silica, talc, and magnesium carbonate; and

[0056] (h) a freeze-drying protectant, which is selected from a group comprising sucrose, mannitol, polyvinylpyrrolidone (PVP), amino acids, serum albumin, and glycoproteins.

[0057] In one embodiment, the pharmaceutical composition comprises one or more excipients selected from a group comprising cellulose derivatives such as carboxymethyl cellulose, sodium alginate, carrageenan, sodium starch glycolate, carbomer, trehalose, dextran, cyclodextrin, and a freeze-drying protectant (such as sucrose, mannitol, polyvinylpyrrolidone (PVP), amino acids, serum albumin, and glycoproteins).

[0058] The pharmaceutical composition may be formulated into various dosage forms that are suitable for administration. In some embodiments, the pharmaceutical composition may be in the form of a powder (such as freeze-dried powder), solution, liniment, gel, dressing, tablet, pill, capsule, emulsion, or ointment. The pharmaceutical composition may also be administered via various administration routes depending on the specific dosage form used. In some embodiments, the pharmaceutical composition may be administered by topical administration to the lower genital tract (such as vaginal or cervical topical use) or skin. In other embodiments, the pharmaceutical composition may also be administered via inhalation or parenteral injection (such as intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, or intracavemous injection).

[0059] It should be understood that any and all embodiments of the present disclosure can be combined with technical features in any other embodiment or multiple other embodiments to obtain additional embodiments under the premise of no conflict. The invention includes such combinations resulting in further embodiments.

[0060] EXAMPLES

[0061] The following examples are intended to exemplify the present disclosures and are not limitations of the claimed invention. All molecules, compositions, methods, assays, and results disclosed in the examples and other sections of the specification, figures, and claims form part of the disclosure of the invention.

[0062] Example 1. Isolation of PAP-I

[0063] PAP-I protein was purified from pokeweed using a method known in the art. Briefly, the leaves of Phytolacca americana were homogenized with deionized water, and then subjected to acid precipitation and ammonium sulfate precipitation. The obtained crude extract was purified by DEAE-Sepharose Fast Flow anion exchange chromatography and CM-Sepharose Fast Flow cation exchange chromatography. The purified extract was then analysed by high- performance liquid chromatography (HPLC) using Agilent 1260 plus the ion exchange chromatography column (Tskgel, SP-5PW 7.5 mm l.D><7.5 cm, 10pm). Seven peaks were detected, and proteins corresponding to each peak were further separated, purified and sequenced. Three peaks were found to have the same amino acid sequence of PAP-I (SEQ ID NO: 1). The different HPLC chromatograms may due to post-translation modification, such as glycosylation variants. The purified PAP-1 was used for further tests.

[0064] Example 2. PAP-I inhibited cell growth of cervical cancer cells To prepare a rabbit anti-PAP-I antibody, the purified PAP-I was used as an antigen to generate a polyclonal antibody (Wuhan Bioyeargene Biotechnology)

[0065] Ca Ski cells or 293T cells were plated in a 24-well plate (at a concentration of 5x104cells per well) and treated with 0.29 pg / ml (i.e., 10 nM) PAP-I in the presence or absence of an anti- PAP-I antibody for up to 6 days, respectively, and the total number of alive cells in each well was counted every 24 hours. Cells without PAP-I treatment were used as control. During each count, the total number of alive cells in each control well was set as 100%, and the total number of alive cells in treated wells was normalized to its control.

[0066] As shown in Figure 1 , PAP-I treatment significantly inhibited the growth of Ca Ski cells while this effect was not detected in a non-cervical cell line 293T. Five days after the treatment, there were only a few alive cells in the PAP-1 -treated group compared to the confluent cells in the control group without PAP-I treatment. It was also observed that this inhibition effect of PAP-I was blocked in the presence of anti-PAP-I antibody, indicating that the inhibition of Ca Ski cell growth was indeed caused by the PAP-I treatment (Figure 1 A).

[0067] Example 3. PAP-I inhibits the growth of cervical cancer cells more effectively than PAP-II.

[0068] PAP-II was isolated from pokeweed using the same method as described in Example 1, and was further confirmed by sequencing. The effects of both PAP-I and PAP-II on the growth of Ca Ski cells were tested using the method disclosed in Example 2. Cells without any treatment were used as control. After one day of treatment, PAP-I significantly inhibited the growth of Ca Ski cells compared with PAP-II at the same dose (Figure 2).

[0069] Example 4. PAP-I inhibited the expression of HPV E6 / E7 oncoproteins

[0070] Ca Ski cells or HeLa cells in 6-well plate (at a concentration of 2.5 x 105cells per well) were treated with 0.29 pg / ml PAP-I in the absence or presence of the anti-PAP-I antibody from Example 2 (1: 100 dilution of anti-serum) for up to 6 days. The expression of -actin (Actin), HPV16 E7 in Ca Ski cells and / or HPV18 E6 in HeLa cells were detected by western-blot (anti-HPV16 E7 and anti-p-Actin antibodies from Santa Cruz Biotechnology; anti -HP VI 8 E6 antibody from Merck Millipore). With PAP-I treatment, the expressions of HPV16 E7 and HPV18 E6 oncoproteins were gradually decreased as the culture time increased. Five days after the treatment, oncoprotein E7 and E6 were no longer detectable (Figure 3A and 3B, left panel). Further, the PAP-I - mediated inhibition of HPV E7 and E6 expression was counteracted when co-cultured with the anti-PAP-I antibody (Figure 3A and 3B, right panels).

[0071] Example 5. HPV-induced tumour growth was inhibited after PAP-I treatment

[0072] It has been reported that subcutaneous injection of cells expressing HPV E6 / E7 into severe combined immunodeficiency (SCID) mice induces tumor development. The effects of PAP-I on the tumor growth were examined using the SCID animal model.

[0073] HeLa cells were treated with various concentrations (0.12, 0.29, 0.73, 1.81 pg / ml) of purified PAP-I protein for 3 days. Subsequently, 100 pl of these treated cells (at a concentration of 5 x 105cells / ml) were inoculated into 6-week-old male SCID mice (3 mice per group) via subcutaneous injection at the right armpit of the mice on days I, 14 and 28. On day 29, at the end of tumor induction period, the SCID mice were euthanized by cervical dislocation. Tumor blocks were excised, weighted and analysed. It was observed that PAP-I treatment significantly inhibited the tumour growth in a dose-dependent manner (Figure 4), suggesting that PAP-I was able to inhibit the malignant transformation induced by HeLa cells.

[0074] Example 6. The clinical effects of PAP-I inhibition on HPV E6 / E7 mRNA expression

[0075] A composition comprising PAP-I with carbomer as the preferred carrier excipient was prepared. The composition is in the dosage form of a 0.5 g freeze-dried powder, comprising 0.06% purified PAP-I, 3% arginine, 96.28% polysaccharide, and 0.66% carbomer (carboxypolymethylene). Prior to administration, the composition was reconstituted by adding 3 ml of physiological saline, and thoroughly mixed to achieve a gel. Then the gel was administered vaginally using a disposable applicator. Eight participants with persistent high- risk HPV infection (i.e., diagnosed as having hrHPV infection for at least one year), but without a high level of cervical lesions (i.e., diagnosed as having no more than CIN I according to cervical cytology and histopathology examination) were enrolled in this clinical test, age ranging from 25 to 50 years, with an average age of 38 years. One dose of the composition was administered intravaginally every other day for three months, except during the menstrual period. Just before and after the treatment, the HPV E6 / E7 mRNA level in cervical specimens of all patients was assayed with Aptima HPV assay (Gen-Probe Inc., San Diego, CA) according to the manufacturing introduction. Assay results were interpreted on the basis of the signal-to-cutoff ratio (S / CO), and specimens with S / CO values of 3=0.5 were considered positive. As shown in Figure 5, after the treatment, the E6 / E7 mRNA level in high-risk HPV-infected patients was significantly reduced compared to levels just before treatment, with most patients showing negative results.

[0076] Example 7. The clinical effects of PAP-I on both hrHPV clearance and CIN I regression. The effects of PAP-I were further investigated to determine whether it can eliminate high-risk HPV infections and lead to the regression of cell lesions, 180 patients with hrHPV and CIN 1 were enrolled. 149 of the patients were treated, while 31 served as the control group. The treatment group received intravaginal treatment of the PAP-1 composition prepared in Example 6 for one course (i.e., one dose every other day for three months, except during the menstrual period). After 1-3 months of drug withdrawal, both groups were tested for hrHPV using the Tellgenplex HPV27 genotyping Assay (Tellgen Life Science, Shanghai, China), and subjected to cervical cytology and histopathology examination. As show in Table 1, the HPV DNA clearance rate was 59.06% (88 / 149) while their CIN I regression rate was 79. 19% (119 / 149) in the treatment group. In the control group, 25.81% (7 / 31) of patients showed HPV DNA self-clearance while 22.58% (8 / 31) showed CIN I regression. This example demonstrated that the treatment significantly increased both hrHPV clearance and CIN I regression rates compared to the control group.

[0077] Table 1. Effects of PAP-I on both hrHPV clearance and CIN I regression.

[0078] Example 8. The safety of PAP-T treatment

[0079] From cell to mouse experiments, no harmful effects were observed or detected. Moreover, no significant side effect during and after the intervention in the clinical trials was reported so far. Moreover, routine physical examinations of all participants before and after intervention indicated that the participant’s blood routine, liver function (Alanine amino transferase (ALT) and Aspartate transaminase (AST) test), kidney function (Blood urea nitrogen (BUN) and creatinine (Cr) test) and other tests were in the normal range. However, mild vaginal itch was occasionally observed during treatment.

[0080] While the subject matter of this disclosure has been described and shown in considerable detail with reference to certain illustrative aspects, including various combinations and subcombinations of features, those skilled in the art will readily appreciate other aspects and variations and modifications thereof as encompassed within the scope of the present disclosure. Moreover, the descriptions of such aspects, combinations, and sub-combinations is not intended to convey that the claimed subject matter requires features or combinations of features other than those expressly recited in the claims. Accordingly, the scope of this disclosure is intended to include all modifications and variations encompassed within the spirit and scope of the following appended claims.

[0081] Table 2. Sequences used in the present invention

Claims

Claims1. Use of pokeweed antiviral protein isoform one (PAP-I), which is a plant ribosome inactivating protein (RIP), or a functional variant thereof in the manufacture of a pharmaceutical composition for treatment of human papilloma virus (HPV) infection.

2. The use of claim 1, wherein the pharmaceutical composition is used for eliminating HPV infection, in particular for clearance of high-risk HPV persistent infection.

3. The use of claim 1 or 2, wherein the pharmaceutical composition is for the prophylaxis or treatment of HPV-associated diseases or conditions.

4. The use of any one of claims 1-3, wherein the PAP-I or a functional variant thereof is isolated from the Phytolacca family, preferably from Phytolacca americana , Phytolacca acinosa Roxb. , Phytolacca dioica or Phytolacca dodecandra, or is obtained by recombinant expression.

5. The use of any one of claims 1-4, wherein PAP-I or a functional variant thereof comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence set forth in SEQ ID NO: 1.

6. The use of claim 5, wherein PAP-I consists of the amino acid sequence set forth in SEQ ID NO: 1.

7. The use of any one of claims 3-6, wherein the HPV-associated diseases or conditions is selected from genital tract inflammation, cervical polyp, warts, intraepithelial neoplasia, and HPV associated cancer.

8. The use of claim 7, wherein the genital tract inflammation is selected from a group consisting of viral vaginitis, vaginal cysts, and viral cervicitis; the cervical polyps is selected from polyps formed by excessive proliferation of cervical mucosa due to chronic inflammation and cervical infections, protruding towards the external cervical; the warts are selected from the group consisting of condyloma acuminatum, anal warts, common warts, plantar warts, flat warts, verruca filiformis, subungual and periungual warts; the intraepithelial neoplasia is selected from the group consisting of cervical intraepithelial neoplasia (CIN, including CIN I, CIN II, CIN III, which is also known as squamous intraepithelial lesion (SIL) that can be classified as low-grade SIL (LSIL) or high-grade SIL(HSIL)), vaginal intraepithelial neoplasia (ValN, including ValN I, ValN II, ValN III), vulvar intraepithelial neoplasia (VIN, including VIN I, VIN II, VIN III), penile intraepithelial neoplasia (PIN, including PIN I, PIN II, PIN III)? anorectal intraepithelial neoplasia(AIN, including AIN I, AIN II, AIN III), and the HPV associated cancer is selected from a group consisting of cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, laryngeal cancer, and oral cancer.

9. The use of any of claims 1 -8, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

10. The use of claim 9, wherein the at least one pharmaceutically acceptable excipient is selected from a group comprising a binder, a wetting agent, a suspending agent, a buffering agent, a preservative, a pH control agent, a glidant and a freeze-drying protectant.

11. The use according to claim 10, wherein the pharmaceutical composition comprises one or more excipients selected from a group consisting of cellulose derivatives, sodium alginate, carrageenan, trehalose, carbomer, sodium starch glycolate, dextran, cyclodextrin, and freeze- drying protectants, wherein the freeze-drying protectants are selected from a group consisting of sucrose, mannitol, polyvinylp yrrolidone (PVP), amino acids, serum albumin, and glycoproteins.

12. The use of any one of claims 1-11, wherein the pharmaceutical composition is in the form of a powder, solution, liniment, gel, dressing, tablet, pill, capsule, emulsion, or ointment.

13. The use of any one of claims l-12wherein the pharmaceutical composition is administered topically through the lower genital tract or skin, or administered via inhalation or parenteral injection.

14. A pharmaceutical composition comprising a PAP-I or a functional variant thereof and at least one pharmaceutically acceptable excipient for treating HPV infection.

15. The pharmaceutical composition of claim 14, wherein PAP-I consists of the amino acid sequence set forth in SEQ ID NO: 1.

16. A method for treating HPV infection in a subject, comprising administering a therapeutic effective amount of a composition comprising PAP-I or a functional variant thereof to said subject.

17. The method of claim 16, wherein PAP-I consists of the amino acid sequence set forth in SEQ ID NO: 1.

18. An in vitro method of inhibiting the expression of HPV E6 / E7 oncogenes in a sample, comprising contacting the sample with PAP-I or a functional variant thereof.

19. The in vitro method of claim 18, wherein PAP-I consists of the amino acid sequence set forth in SEQ ID NO: 1.

Images