Use of cell wall skeleton of isolated <i>Rhodococcus ruber </i>for preparing human papilloma virus infection treatment drug

Abstract

Disclosed is a use of a cell wall skeleton of isolated Rhodococcus ruber or a composition containing the same for preparing a human papillomavirus infection treatment drug. The cell wall skeleton of Rhodococcus ruber isolated from Rhodococcus ruber was stored and preserved at China General Microbiological Culture Collection Center, No. 1, West Beichen Road, Chaoyang District, Beijing on Mar. 22, 2019 with accession number CGMCC 17431.

Claims

1. A method for the treatment of human papilloma virus infection, said method comprising administration of a therapeutically effective amount of an isolated Rhodococcus ruber cell wall skeleton or a pharmaceutical composition comprising said isolated Rhodococcus ruber cell wall skeleton to a subject in need, wherein: said isolated Rhodococcus ruber cell wall skeleton is derived from Rhodococcus ruber deposited at China General Microbiological Culture Collection Center, No. 1-3 West Beichen Road, Chaoyang District, Beijing, on Mar. 22, 2019, under deposit number CGMCC 17431.

2. The method according to claim 1, wherein said therapeutically effective amount is at a unit dose of 1 ?g to 1000 ?g.

3. The method according to claim 2, wherein said therapeutically effective amount is at a unit dose of 15 ?g to 500 ?g.

4. The method according to claim 3, wherein said therapeutically effective amount is at a unit dose of 30 g to 60 g.

5. The method according to claim 1, wherein said isolated Rhodococcus ruber cell wall skeleton is obtained by the following steps: a) providing Rhodococcus ruber CGMCC 17431; b) culturing said Rhodococcus ruber; c) collecting the cultured Rhodococcus ruber; d) disrupting the collected Rhodococcus ruber to obtain a disrupted product; and e) removing lipids, nucleic acids and proteins from the obtained disrupted product.

6. The method according to claim 1, wherein said treatment is performed at a frequency selected from the group consisting of: administering 1 to 3 times a day, 1 to 6 times per two days, 1 to 9 times per three days, 1 to 14 times a week, and 1 to 60 times a month.

7. The method according to claim 1, wherein said treatment lasts between 2 days and 2 months.

8. The method of claim 1, wherein said administration is topical administration.

9. The method of claim 8, wherein said topical administration is skin or mucosa coating, or subcutaneous or submucosal injection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: The colony morphology of Rhodococcus ruber.

(2) FIG. 2: The identification results of 16S rRNA.

DETAILED DESCRIPTION OF THE INVENTION

(3) Isolation refers to the separation of the Rhodococcus ruber of the present disclosure from its original growth environment.

(4) The skilled person knows that the cell wall structures of gram-positive bacteria and gram-negative bacteria are different. Specifically, the cell wall of gram-positive bacteria is thicker (usually 20 nm to 80 nm), comprising about 90% peptidoglycan and about 10% teichoic acid (a polymer formed by alcohol and phosphoric acid molecules, usually existing in the form of sugar ester or amino acid ester). The peptidoglycan layer is dense, even as many as 20 layers. However, the cell wall of gram-negative bacteria is much thinner than that of gram-positive bacteria, and the structure is more complex, divided into outer membrane and peptidoglycan layer (usually 2 nm to 3 nm).

(5) The peptidoglycan layer is a unique component of the bacterial cell wall and is a derivative of heteropolysaccharide. Each peptidoglycan monomer comprises 3 parts: the sugar unit (for example, at least two sugar molecules are connected by glycosidic bonds to form the framework of peptidoglycan), the peptide tail (a short peptide chain formed by linking several amino acids, which is connected to a N-acetylmuramic acid molecule), and the peptide bridge (which crosslinks the adjacent peptide tails to form a high-strength network structure). Different bacteria have different peptide bridges, peptide tails and cross-linking manners.

(6) Isolated Rhodococcus ruber Cell Wall Skeleton

(7) In the present disclosure, isolated Rhodococcus ruber cell wall can be understood as either a complete cell wall or an incomplete cell wall (for example, disrupted or partially degraded). Under the instruction of the present disclosure, the skilled person will understand that the ingredients exhibiting the desired activity are derived from Rhodococcus ruber cell wall (for example, the cell wall itself or composition thereof). Therefore, various forms are allowed to be used in clinical applications, including complete cell walls, disrupted cell walls, incompletely degraded products of cell walls, cell wall components, cell wall extracts, etc., which are all included in the scope of the present disclosure.

(8) Cell Wall Skeleton

(9) A component that constitute the main structure of the cell wall; however, it cannot be understood as merely representing the cross-linked network-like entity in the cell wall, and the skilled person understands that other cell wall compositions adsorbed by, bound to, or carried by the cross-linked network-like entity cannot be excluded.

(10) Rhodococcus ruber

(11) The Rhodococcus ruber used in the embodiments of the present disclosure refers to the Rhodococcus ruber species of the Rhodococcus genus, and is not limited to a specific cell strain.

(12) Non-limiting examples include the TOY7 strain (Agricultural Environment Microbiological Culture Collection, Nanjing Agricultural University), CGMCC No. 4795, DSM43338, CCTCC No. 2012035, CGMCC No. 16640 and CGMCC 17431.

(13) Identification of Rhodococcus ruber

(14) According to the known or future microbial identification techniques, the skilled person can perform taxonomic identification on a strain of bacteria. For example, the available identification techniques include morphology, physiological and biochemical characteristics, 16S rRNA, and the like. The skilled person understands that with the development of science and technology, identification techniques involve different methods. In the earlier period, morphological and biochemical identification methods were mainly used, but the reliability of these methods is not high. After the advent of sequencing technology, the skilled person can identify bacteria strains in a more reliable way. For example, when the DNA sequences of 16S rRNA are identified as having more than 97% (inclusive) of identity, it is determined that the two bacteria belong to the same species (Hua Gougen et al., The taxonomy and application of Rhodococcus, Microbiology China, 2003: 30 (4)). In terms of Rhodococcus ruber, the known strains deposited in international (or national) culture collection units are used as model strains, and are compared with the strain to be identified.

(15) Dosage Form

(16) The medicament or pharmaceutical composition or active ingredient or product of the present disclosure can be embodied in, but not limited to, the following forms: ointment, cream, plaster, gel, lotion, tincture, liniment, oil, paste, lyophilized powder, aerosol, suppository, patch, suspension, oral liquid, buccal tablet and skin care product (cleanser, toning lotion, essence, lotion, cream and mask).

(17) Excipient

(18) An excipient suitable for the present disclosure is for example, but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethyl cellulose, erythritol, gelatin, magnesium stearate, mannitol, propellant, humectant, solvent, solubilizer, emulsifier, antioxidant, pH regulator and preservative. Specifically, non-limiting examples also include: white vaseline, carbomer, hydroxypropyl methylcellulose, methyl cellulose, sodium hydroxymethyl cellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerin, propylene glycol, deionized water, water for injection, distilled water, ethanol, hexadecanol, octadecanol, p-aminobenzoic acid, acetamide, isopropanol, Tween, polyoxyethyl hydrogenated castor oil, stearic acid, glyceryl monostearate, triglycerol monostearate, sucrose fatty acid ester, sucrose ester, sucrose acetate isobutyrate, sorbitan tristearate, isopropyl myristate, cholesterol, squalene, squalane, n-butanol, ethylene glycol, ethanol, propylene glycol, polyglycerol ester, sulfite, cysteine, di-tert-butyl hydroxytoluene, potassium sorbate, phosphate buffer solution, triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, nipagin, thimerosal, chlorocresol, trichlorobutanol, benzoic acid and sodium salt thereof.

(19) Preparation Unit

(20) The medicament or pharmaceutical composition or active ingredient or product of the present disclosure can be prepared in the form of a unit preparation (unit form).

(21) In some embodiments, the medicament (or preparation, or therapeutic agent, or medical apparatus) comprises a unit dose of: 0.001 mg to 500 mg of the product derived from Rhodococcus ruber; or 0.001 mg to 500 mg of the Rhodococcus ruber cell wall; or 0.001 mg to 500 mg of the Rhodococcus ruber cell wall skeleton.

(22) Specific example of the unit dose is 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 mg?10%, or the range between any two of the above values.

(23) Administering, giving, providing to and treating, when applied to animals, humans, cells, tissues, organs or biological samples, refer to the contact of the medicament (therapeutic agent, active ingredient or composition) with the animals, humans, cells, tissues, organs or biological samples.

(24) Treatment means administering an internal or external medicament (therapeutic agent, active ingredient or composition) (such as the Rhodococcus ruber cell wall or pharmaceutical composition thereof according to the present disclosure) to a subject, for alleviating (relieving, delaying, improving, curing) one or more disease symptoms to a clinically measurable degree in the subject (or population) treated, wherein the subject has, is suspected of having, or is susceptible to one or more diseases or symptoms thereof.

(25) The amount of the medicament (therapeutic agent, active ingredient or composition) that effectively alleviates any disease symptom is called a therapeutically effective amount. It can vary depending on a variety of factors, such as the disease state, age and body weight of the subject. It should be understood that the medicament (therapeutic agent, active ingredient or composition) may be ineffective in alleviating the target disease or symptoms thereof in a single subject, but it can be determined according to any statistical test method known in the art (such as Student's t test, chi-square test and U test according to Mann and Whitney) that, the medicament (therapeutic agent, active ingredient or composition) is statistically effective for the target disease or symptoms thereof.

(26) Optionally means that what is described later can happen, but does not have to happen; it depends on the situation. For example, optionally, performing sub-packaging means that the product is allowed to be sub-packaged, but it is not required to be sub-packaged; whether the product is sub-packaged or not does not affect the achievement of the technical effects.

(27) A, an, single and the, if not explicitly stated, also include plural forms.

(28) The present disclosure is further described below with reference to the examples, preparation examples and test examples. However, these examples, preparation examples and test examples do not limit the scope of the present disclosure. When the specific conditions are not specified, operation should be done in accordance with the normal conditions and the conditions recommended by the raw material supplier. The reagents for which the sources are not specifically indicated are conventional reagents purchased on the market.

(29) The skilled person especially understands that although a specific cell line is used in the following specific examples, the achievement of the technical effects is not limited to the specific cell line, and any species belonging to the Rhodococcus genus, Rhodococcus ruber species is applicable.

EXAMPLES

Example 1. Deposit of the Strain

(30) The inventors deposited the laboratory-preserved master strain at China General Microbiological Culture Collection Center (CGMCC), No. 1-3 West Beichen Road, Chaoyang District, Beijing, on Mar. 22, 2019, under deposit number CGMCC 17431. Tests showed that the deposited strain is viable.

Example 2. Identification of the Strain

(31) 1. Morphological Characteristics of the Colony Observed by the Naked Eyes

(32) The strain was cultured on a glycerol agar medium at 30 to 37? C. (specifically at 32 to 35? C.) for 12 to 72 (specifically 36 to 60, such as 40 to 50) hours, and the colonies were observed (FIG. 1): swelled up; in orange-red color (slightly different depending on influences of light, the color of the culture medium, etc.); with a dry and wrinkled surface, slightly shiny (slightly different depending on differences in culture conditions); fragile to the touch; with a colony size of about 1 mm to 2 mm (slightly different depending on differences in culture conditions).
2. Observation Under Microscope The bacteria were branched and had diaphragms, to form mycelium (slightly different depending on differences in culture conditions); The hyphae were divided to form regular short and thick cells (slightly different depending on differences in culture conditions); After culturing for 4 to 5 days, the bacteria became short rod-shaped or spherical (slightly different depending on differences in culture conditions).
3. Staining Property

(33) The strain was gram stain positive.

(34) 4. Biochemical Reactions

(35) The strain was cultured on a glycerol agar slant medium at 30 to 37? C. (specifically at 32 to 35? C.) for 12 to 72 (specifically 36 to 60, such as 40 to 50) hours. Then, the following tests were performed on the culture.

(36) 4.1 Acid Production from Carbohydrates:

(37) Positive: glycerin, mannitol, sorbitol, D-arabitol, D-fructose and D-glucose; Negative: inositol, inulin, lactose, sucrose, starch, maltose, glycogen, xylitol, gluconate, trehalose, erythritol, melezitose, melibiose, raffinose, cellobiose, amygdalin, gentiobiose, adonol, arbutin, D-arabinose, L-arabinose, ?-methyl-D-glucoside, ?-methyl-D-mannoside, D-ribose, D-xylose, L-xylose, N-acetyl-glucosamine, D-turanose, D-lyxose, ?-methyl-D-xyloside, D-galactose, D-tagatose, D-fucose, L-fucose, D-mannose, L-sorbose, L-arabinitol, L-rhamnose and 2-keto-gluconate.
4.2 Enzyme Activity Assay (API ZYM): Positive: alkaline phosphatase, lipoid esterase (C8), lipase (C14), leucine arylaminase, valine arylaminase, cystine arylaminase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-B1-phosphohydrolase and ?-glucosidase; Negative: N-acetyl-glucosaminidase, esterase (C4), ?-galactosidase, ?-uronidase, ?-glucosidase, ?-galactosidase, ?-mannosidase and ?-fucosidase.
4.3 Nitrate reduction reaction: positive; catalase: positive; tyrosinase: positive; amylase: negative; oxidase: negative; gelatin liquefaction: negative.
4.4 Sole Carbon Source:
Biolog Gen II Growth Experiment: Positive for glucuronamide, ?-hydroxy-DL butyric acid, D-fructose-6-phosphate, ?-D-glucose, D-fructose, D-mannitol, D-arabitol, D-sorbitol, quinic acid, ?-aminobutyric acid, citric acid, L-malic acid, bromosuccinic acid, Tween 40, propionic acid and acetic acid;
Biolog Gen III Chemical Sensitivity Experiment Sensitive to p-dimethylamine tetracycline, sodium tetradecyl sulfate, rifamycin SV, pH 5.0, 8% sodium chloride, lincomycin, fusidic acid, D-serine, vancomycin, tetrazolium violet and tetrazolium blue; Insensitive to sodium bromate, 1% sodium lactate, pH 6.0, 1%-4% sodium chloride, nalidixic acid, lithium chloride, potassium tellurite, aztreonam and sodium butyrate.
4.5. 16S rRNA Identification

(38) The 15 strains isolated from the working seed tube and the 10 different strains isolated from the original seed tube were subjected to genome extraction, 16S rRNA amplification and sequencing. The 16S rRNA genes of the total of 25 strains have 100% identity. This means that the 25 strains are of the same species (FIG. 2).

(39) At the same time, the neighbor-joining strain phylogenetic tree constructed based on the Kimura2-parameter algorithm showed that the strain was classified as Rhodococcus ruber.

Preparation Examples

Preparation Example 1. Culture Methods

(40) 1. The Rhodococcus ruber with the deposit number CGMCC 17431 can be cultured by conventional microbial production methods. 2. The culture method can be solid culture or liquid culture. 3. The nutrient sources in the culture medium are no specifically indicated. The culture medium can contain carbon sources, nitrogen sources and other nutrient sources that are commonly used for microbial culture. The carbon source can be any carbon source that can be used by Rhodococcus ruber, for example, fructose, glucose, and the like. The nitrogen source can be meat extract, peptone, ammonium salt, nitrate and other organic or inorganic nitrogen-containing compounds. For other nutrient sources, some inorganic salts can be added appropriately, for example, NaCl and phosphates. 4. The culture conditions (temperature, time, etc.) are not strictly limited. The skilled person can choose the conditions that maximize the yield based on the preliminary small-scale pilot test data. 5. As an example, the following culture condition was used to ferment Rhodococcus ruber:
(1) The Medium Composition Included:

(41) Peptone, beef extract, sodium chloride, phosphate, glycerin (and, optionally agar, when in solid culture).

(42) (2) Parameters of the Culture Method:

(43) After the working strain was recovered, it was transferred to a solid culture medium for 3-5 days, and then transferred to liquid culture (30-37? C., maintained for 3-5 days). The fed-batch semi-continuous mode or the batch mode can be used. The pH, bacterial density, dissolved oxygen and carbon source consumption were monitored during culture.

Preparation Example 2. Bacteria Disruption

(44) The bacteria obtained in Preparation Example 1 were collected and the cells were disrupted (for example, but not limited to sonication). Any appropriate well-known method in the art was also allowed to be used for bacteria disruption, such as CN101250490A or CN101323865A.

(45) The disruption state was checked under a microscope. There should be no more than 5 intact bacteria visible in each visual field. The disruption was considered as qualified when several (10 to 30) visual fields checked met this standard.

Preparation Example 3. Removal of Nucleic Acids, Lipids, Proteins and Cell Membranes

(46) 1. Removal of Nucleic Acids:

(47) The supernatant after disruption was centrifuged. DNase and RNase were added to the obtained precipitate, and nucleic acids were removed according to the operation recommended by the supplier of the enzymes.

(48) 2. Removal of Proteins:

(49) Common protease (such as trypsin) was added to the precipitate, and proteins were removed according to the operation recommended by the supplier of the enzyme.

(50) 3. Removal of Lipids:

(51) Organic reagents (for example, but not limited to, one of acetone, ether and ethanol or the combination thereof) were added to the precipitate, and lipids were removed according to conventional operations in the art.

(52) 4. Removal of Cell Membranes:

(53) Triton X-100 was added to the precipitate, and the precipitate was collected by centrifugation according to conventional operations in the art, and rinsed with PBS.

(54) It should be understood that the skilled person can adjust the orders of the above steps of removing impurities to make them compatible. After removing the non-cell wall components, the precipitate was reconstituted in water for injection for later use. Optionally, it could be sterilized at 115? C. for 20-30 minutes as a stock solution of the cell wall skeleton (mainly comprising the cell wall skeleton and components thereof).

(55) 5. Yield

(56) A total of 653 mL of bacterial liquid (after disruption) was collected from 159 Kolle flasks. The wet weight yield was 138 g; the cell wall skeleton yield was about 0.87 g/Kolle flask.

Preparation Example 4. Preparation Methods of the Pharmaceutical Compositions

(57) 1. Excipients (such as dextran 40, mannitol or trehalose) were added to the product obtained in Preparation Example 3, filled into a vial to result in the pharmaceutical composition.

(58) TABLE-US-00001 TABLE l The pharmaceutical composition can be formulated in various forms Composition Capacity of each vial Component and amount Composition 1 2 mL Active ingredient Dextran 40 60 ?g 15 mg Composition 2 2 mL Active ingredient Dextran 40 240 ?g 48 mg Composition 3 2 mL Active ingredient Dextran 40 120 ?g 36 mg Composition 4 2 mL Active ingredient Trehalose 60 ?g 12 mg Composition 5 2 mL Active ingredient Trehalose 120 ?g 36 mg Composition 6 2 mL Active ingredient Mannitol 120 ?g 36 mg Composition 7 2 mL Active ingredient Mannitol 60 ?g 12 mg 2. The product obtained in Preparation Example 3 (active ingredient 30 ?g to 120 ?g) was coated on the dressing to prepare medical apparatus for external use. 3. The pharmaceutical compositions in Table 1 were lyophilized to prepare lyophilized powders (numbered as compositions 1 to 7, respectively). 4. Quality inspection (composition 1 was taken as an example)

(59) TABLE-US-00002 TABLE 2 Quality inspection items Appearance White unconsolidated solid or powder Water content ?6% Solubility Qualified if it was dissolved within 1 minute after 2.0 mL of NaCl injection was added; Identification of sugars The solution was blue-green in color Content of muramic acid 2.0 ?g/vial (criteria: ?1.0 ?g/vial) Amount of residual proteins 0.4 ?g/vial (criteria: ?9.0 ?g/vial) Amount of residual RNAs 0.8% (criteria: no more than 5%) Amount of residual DNAs 0.9% (criteria: no more than 5%) Amount of residual Triton X-100 Undetected (criteria: no more than 5%) Amount of residual lipids 3.8% (criteria: no more than 5%) Phagocytosis rate 75% (criteria: ?40%) Phagocytic index 1.05 (criteria: ?0.50) Abnormal toxicity in mice The composition was considered as qualified if all the mice were alive and had no abnormal reactions during the observation period, and the body weight of each mouse was increased at the end of the inspection. Abnormal toxicity in guinea pigs . The composition was considered as qualified if all the guinea pigs were alive and had no abnonnal reactions during the observation period, and the body weight of each guinea pig was increased at the end of the inspection.

Test Examples

Test Example 1. Pharmacological Tests

(60) 1. After intravenous injection of each composition at the doses 20, 40 and 80 times higher than the human clinical dose, the blood pressure, respiration, heart rate and electrocardiogram of the anesthetized cats were monitored, and the compositions had no significant effect; 2. After intravenous injection of each composition at the dose 1000 times higher than the human clinical dose, the compositions had no significant effect on the coordinated movement and learning and memory function of mice.

(61) It can be seen that the pharmaceutical compositions of the present invention (composition 1 to composition 7) have no significant effect on the mental state, nervous system, cardiovascular system and respiratory system of animals.

Test Example 2. Safety Tests

(62) 1. Sterility Test:

(63) The results were negative, proving the sterility of the compositions.

(64) 2. Acute Toxicity Test in Mice:

(65) The compositions were administered to the experimental groups by subcutaneous injection and intraperitoneal injection, and the dose was 5 times higher than the dose used for human. The control group was treated with sterile normal saline 0.5 mL/animal. The animals were continuously observed for 7 to 8 days. The mice were in good condition and had no abnormality in their body weight or their organs.

(66) 3. Long-Term Toxicity Test:

(67) The compositions were vaginally administered at the dose 30 times higher than that for clinical use once a day continuously for three months. No toxic effects were found in dogs; the electrocardiograms and blood biochemical indicators were within the normal range. After stopping the administration for two weeks, no delayed toxicity was observed (composition 1 to composition 7).

Test Example 3. Stability Tests

(68) The pharmaceutical compositions were placed at room temperature (18-25? C.) for 0, 1, 2, 3, 8, 14 and 21 months, and the alanine content, muramic acid content, phagocytosis rate and phagocytic index had no statistically significant difference compared to those at the start of the test (three batches were tested).

(69) In summary, the lyophilized powder formulation of the pharmaceutical composition can be stored stably for 24 months (composition 1 to composition 7).

Test Example 4. Phagocytosis Tests

(70) Macrophages are the main cells of the mononuclear phagocyte system. The activation of phagocytes after antigen stimulation can significantly enhance the phagocytic function. After inducing the production of peritoneal macrophages in the mice, the mice were intraperitoneally injected with chicken red blood cells. After 30 minutes, the mice were sacrificed and the peritoneal fluid was taken out and stained. The percentage of the phagocytized red blood cells was counted under a microscope to determine the killing ability of the phagocytes, indirectly measuring the non-specific immunity level of the body.

(71) Composition 1 of the present application was used for testing. For composition 1, the phagocytosis rate was 75% and the phagocytic index was 1.05. While, the phagocytosis rate and phagocytic index for the negative control (excipient) and blank control (normal saline) were all relatively low. These results show that the cell wall skeleton of the present application has a strong ability to promote immunophagocytosis.

Effect Example: Use of the Rhodococcus ruber Cell Wall Product of the Present Invention in the Treatment of HPV Infected Subjects

(72) In view of the immunophagocytosis-promoting ability of the Rhodococcus ruber cell wall skeleton of the present invention discovered by the inventors, the inventors used the following methods to identify the effect of the Rhodococcus ruber cell wall product obtained by the method of the present invention on the treatment of human papilloma virus (HPV) infection.

(73) It is known that the HPV detection methods in the art usually include the following:

(74) 1. Cytological Smear Testing:

(75) It was usually named as the Papanicolaou staining detection method in the past, also known as the smear detection method. In the method, the collected cervical secretions are smeared on glass slides and observed under a microscope, mainly to check whether there are vacuolar cells or keratinocytes. The detection rate is 70-76%.

(76) 2. Thin-Prep Liquid Based Cytology Test (TCT):

(77) Samples are collected at the cervix using special brushes. Then the impurities in the collected samples are separated using the cell preservation solution to form an ultra-thin clear cell smear, which can detect the presence of HPV virus and determine the type of the virus. The method is also known as TCT. The detection rate is 70-95%.

(78) 3. Immunohistochemical Detection Methods:

(79) After immunizing the animal with HPV, the polyclonal antibody produced is used to check the tissue HPV antigen, and the PAP method is used to display the virus protein to prove the presence of the virus antigen. When HPV protein is positive, light red color appeared in epithelial cells is considered as weak positive reaction, and brown granular sedimentation observed in the vacuole cell nucleus is considered as positive. But the detection rate is low (40-60%), the sensitivity is not high, and the type of the virus cannot be determined.

(80) 4. HPV DNA Detection Methods:

(81) (1) Polymerase Chain Reaction (PCR):

(82) Secretions are collected from the vagina and external cervical orifice using wipers or cotton swabs soaked in normal saline. HPV DNA is extracted by centrifugation, washing and other methods, and then PCR amplification and gel electrophoresis are performed. Diagnosis can be made according to observation of the electrophoresis and sample comparison. Although this method is simple, it cannot locate the virus or determine whether the virus is dead or alive.

(83) (2) DNA Hybridization Method (Using PCK):

(84) HPV DNA molecular hybridization technology and CP-14 immune hybridization method are applied. The sample is considered as positive when more than 3 fluorescent spots can be seen under a fluorescence microscope at 400?. The virus can be typed, the HPV-DNA sequence can be detected by nucleic acid hybridization, and specific HPV-DNA amplification zone can be seen by PCR detection. However, this method is cumbersome and expensive. Certain equipment and conditions are required.

(85) The inventors used the lyophilized powder formulation of composition 1 prepared in Table 1 of the present invention, which was diluted with normal saline to 30 ?g/mL or 60 ?g/mL before use. Use of the composition of the present invention containing Rhodococcus ruber cell wall components in the treatment of cervical HPV infection was discussed by cervical local injection or external cervical application in subjects with cervical HPV infection.

(86) The above-mentioned cytological smear testing and TCT method were used to diagnose HPV infected patients. After signing the informed consent, the patients were treated by external application or local injection of the composition of the present invention at the infected site.

(87) The comprehensive treatment method was to administer the composition of the present invention once a day, for 20 days as a treatment cycle. The administration should be stopped during the menstrual period of the women.

(88) Results

(89) TABLE-US-00003 Active HPV type HPV type ingredient before Route of Frequency after 20 days Patient Age concentration treatment administration of treatment of treatment 1 39 30 ?g/ml 16 Cervical external Once every Negative application two days 2 49 30 ?g/ml 52, 44 Cervical external Once every Negative application two days 3 50 30 ?g/ml 51 Cervical external Once every Negative application two days 4 57 30 ?g/ml 59 Cervical external Once every Negative application two days 5 30 30 ?g/ml 52 Cervical external Once every Negative application two days 6 33 30 ?g/ml 52, 66 Cervical external Once every Negative application two days 7 35 30 ?g/ml 16, 58 Cervical external Once every Negative application two days 8 41 30 ?g/ml 58, 84 Cervical external Once every Negative application two days 9 43 30 ?g/ml 18, 11 Cervical external Once every Negative application two days 10 60 30 ?g/ml 16 Cervical external Once every Negative application two days 11 34 30 ?g/ml 16 Cervical external Once every Negative application two days 12 48 30 ?g/ml 52 Cervical external Once every Negative application two days 13 30 30 ?g/ml 68 Cervical external Once every Negative application two days 14 53 30 ?g/ml 18, 31 Cervical external Once every Negative application two days 15 33 60 ?g/ml 16 Cervical external Once every Negative application two days 16 48 60 ?g/ml 51, 52 Cervical external Once every Negative application two days 17 31 60 ?g/ml 16, 45 Cervical Once every Negative injection two days 18 37 60 ?g/ml 52 Cervical Once every Negative injection two days 19 30 60 ?g/ml 58 Cervical Once every Negative injection two days 20 32 60 ?g/ml 58 Cervical Once every Negative injection two days 21 29 60 ?g/ml 16 Cervical Once every Negative injection two days 22 37 60 ?g/ml 51 Cervical Once every Negative injection two days 23 29 60 ?g/ml 66 Cervical Once every Negative injection two days 24 37 60 ?g/ml 16, 18 Cervical Once every Negative injection two days 25 35 60 ?g/ml 58 Cervical Once every Negative injection two days

(90) During the experiment, the control group of 5 patients was treated with cervical external application or injection of dextran 40 solution without active ingredients in parallel. There was no conversion in HPV positivity before and after treatment.

(91) From the above results, it can be seen that the diluted preparation comprising the Rhodococcus ruber cell wall skeleton of the present invention can effectively treat human cervical HPV infection, and the negative conversion ratio of the virus reaches 100%.