Composition for preventing and treating HPV (human papillomavirus) as well as preparation method and application of composition
By designing multivalent HPV L1 virus-like particle immunogens and employing a multi-step chromatographic purification process, the problem of insufficient IgY titer and purity in HPV prevention and treatment has been solved, achieving efficient and safe preparation of anti-HPV specific IgY, suitable for both topical and oral dosage forms.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG ZHONGDA GENE TECHNOLOGY CO LTD
- Filing Date
- 2026-02-04
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies lack efficient immunogen design and screening systems that cover multiple HPV types. Traditional poultry immunization and antibody purification processes are crude, resulting in insufficient IgY titers, purity, and stability, which limits their application in HPV prevention and treatment.
Using multivalent HPV L1 virus-like particles as an immunogen, supplemented with a highly effective adjuvant, and combined with poultry screening and a stepwise immunization program, high-titer, high-purity anti-HPV specific IgY was prepared through PEG/sodium alginate precipitation and multi-step chromatographic purification processes.
It significantly improved the yield, purity, and in vitro neutralizing titer of anti-HPV specific IgY, ensuring the bioactivity and safety of the product and achieving the induction of broad-spectrum cross-reactive neutralizing antibodies.
Smart Images
Figure CN122005772A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of HPV prevention and control compositions, specifically to a composition for preventing and controlling HPV, its preparation method, and its application. Background Technology
[0002] Human papillomavirus (HPV) infection is a major cause of various diseases, including cervical cancer and genital warts, with a persistently high global incidence rate, posing a serious threat to public health. Current prevention and treatment methods mainly include prophylactic vaccines and local physical / chemical therapies. However, existing vaccines cannot cover all high-risk types and have limited effectiveness in already infected individuals. Local medications such as interferon have limitations, including unstable efficacy, potential side effects, and high costs. In recent years, immunizing laying hens with specific antigens and extracting high-titer specific antibodies (IgY) from their egg yolks has emerged as a novel passive immunization strategy, demonstrating unique advantages in the field of virus prevention and control. This strategy is safe and effective. The technology has the potential for high efficiency, ease of large-scale production, and design for specific pathogens. However, when applied to HPV prevention and control, it still faces a series of key bottlenecks: First, there is a lack of efficient immunogen design and screening systems covering multiple HPV types, making it difficult to induce the production of broad-spectrum, high-affinity neutralizing antibodies. Second, traditional avian immunization and antibody purification processes are crude, resulting in insufficient titers, purity, and stability of the obtained IgY, which seriously affects its biological activity and formulation feasibility. Third, existing technical solutions are mostly still in the laboratory stage, failing to form a complete and controllable industrial closed loop from immunogen design, animal immunization, antibody purification to stable formulation development, thus limiting its practical application and transformation.
[0003] Therefore, we have made improvements to this, proposing a composition for preventing and treating HPV, its preparation method, and its application. Summary of the Invention
[0004] The purpose of this invention is to provide a composition for preventing and treating HPV, a method for preparing the composition, and its application, so as to solve the problems mentioned in the background art.
[0005] To achieve the above objectives, the present invention provides the following technical solution: The active ingredient is an anti-HPV specific egg yolk immunoglobulin (IgY), which is obtained by a method comprising the following steps: Antigen preparation: Preparation of an immunogen containing L1, the major capsid protein of HPV; Animal immunization: The immunogen is used to perform stepwise immunization on selected laying poultry, including basic immunization and periodic booster immunization, to induce the production of high-titer specific antibodies. Antibody isolation and purification: Highly immune eggs produced by immunized poultry are collected, and the anti-HPV specific IgY is isolated and purified from the egg yolk; wherein, the in vitro pseudovirus neutralizing titer of the anti-HPV specific IgY in the composition is ND. No less than 1:1000.
[0006] As a preferred technical solution of this application, the immunogen is selected from one or more combinations of the following: Virus-like particles formed by the self-assembly of HPVL1 protein; The antigen and the immune adjuvant form a complex antigen, wherein the immune adjuvant is selected from oil-in-water emulsion adjuvants and TLR agonists.
[0007] As a preferred technical solution of this application, the anti-HPV specific IgY meets at least one of the following quality attributes: The purity was not less than 95% as determined by reducing SDS-PAGE, and characteristic bands appeared at 66 kDa and 25 kDa. Size exclusion chromatography was used to detect that the area of the main peak of the monomer accounted for no less than 98%; Endotoxin levels not exceeding 10 EU / mg IgY; It can specifically recognize and bind to L1 proteins of one or more HPV types.
[0008] As a preferred technical solution of this application, the composition is a dosage form suitable for local administration, selected from gels, creams, suppositories, films, sprays, foams, and rinses; or a dosage form suitable for oral administration, selected from enteric-coated capsules, enteric-coated tablets, or lozenges; wherein the local administration dosage form further comprises a pharmaceutically acceptable gel matrix, a mucosal adhesive, a penetration enhancer, and a preservative; and the anti-HPV specific IgY in the oral administration dosage form is microencapsulated.
[0009] A method for preparing a composition for preventing HPV includes the following steps: S1. Immunogen preparation: HPVL1 protein antigen is expressed and purified using genetic engineering methods, and optionally mixed with an immune adjuvant to prepare a composite antigen for immunization; S2. Screening and immunization of laying poultry: Screening: Select healthy laying poultry, conduct pre-immunization assessments, and screen out individuals with a high immune response to antigens; Immunization program: Selected poultry receive the first immunization, followed by the second and third immunizations on days 7 and 14 to complete the basic immunization; thereafter, booster immunizations are given every 4-6 weeks. S3. Collection of highly immunized eggs: Starting 20 days after the first immunization, eggs laid by poultry will be continuously collected as highly immunized eggs; S4. Extraction and purification of anti-HPV specific IgY: Egg yolk separation and pretreatment: After disinfecting and washing the highly immune eggs, separate the yolks, dilute the yolks with water in a certain proportion, adjust the pH to 5.0-6.0, let stand at 0-5℃, and take the supernatant. Preliminary purification: Polyethylene glycol or sodium alginate is added to the supernatant for precipitation treatment to remove major impurities and lipids, and the supernatant or precipitate solution rich in IgY is collected. Deep purification: The preliminarily purified IgY solution is purified by chromatography, wherein the chromatography technique is selected from one or more combinations of affinity chromatography, ion exchange chromatography, and hydrophobic interaction chromatography; Concentration and sterilization: The purified IgY solution was concentrated by ultrafiltration and then sterilized by filtration to obtain a high-purity anti-HPV specific IgY concentrate. Formulation preparation: The anti-HPV specific IgY concentrate is mixed with pharmaceutically acceptable excipients.
[0010] As a preferred technical solution of this application, the screening step S2 specifically involves: injecting candidate laying poultry with a low dose of the immunization compound antigen, detecting the level of HPV-specific antibodies in their serum 10-14 days later, and retaining individuals with antibody titers ranking in the top 20%-40% as poultry for subsequent immunization.
[0011] As a preferred technical solution of this application, in step S4, the volume dilution ratio of egg yolk to water is 1:4 to 1:8; the pH is adjusted to 5.2-5.8; and the standing time is 10-16 hours. The polyethylene glycol is PEG6000. It is added to a final concentration of 3%-4% (w / v), and the mixture is stirred at 0-4℃ for 1-3 hours before centrifugation; or sodium alginate is added to a final concentration of 0.3%-0.5% (w / v), the pH is adjusted to 4.0-4.5, and the mixture is stirred before centrifugation.
[0012] As a preferred technical solution of this application, the deep purification step S4 includes: Capture was performed using a specific affinity chromatography medium targeting the chicken IgYFc fragment, or anion exchange chromatography was used. The equilibration buffer was 20-50 mM Tris-HCl, pH 7.5-8.5. After sample loading, gradient elution or staged elution was performed by increasing the ionic strength of the buffer to collect the target protein peak. Hydrophobic interaction chromatography was used, with samples loaded in the presence of 1.0-1.5M ammonium sulfate, and gradient elution was performed by decreasing the salt concentration to collect the target protein peak.
[0013] As a preferred technical solution of this application, in step S5: when preparing oral enteric-coated capsules, the microencapsulation coating process is: spray drying microencapsulation of IgY using a pH-sensitive polymer material, wherein the polymer material is selected from acrylic resin and hydroxypropyl methylcellulose phthalate; when preparing a topical gel, the excipients include a gel matrix, a stabilizer, and a humectant, wherein polyethylene glycol 400 is added as a stabilizer, with a final concentration of 5%-10% (w / w); when preparing a lyophilized powder, after ultrafiltration concentration, a lyophilization protectant is added to the IgY solution for lyophilization, wherein the lyophilization protectant contains 5%-10% (w / v) trehalose and sucrose, and 1%-2% (w / v) glycine.
[0014] Compared with the prior art, the beneficial effects of the present invention are: By employing an immunogen design based on multivalent HPV L1 virus-like particles and supplementing it with a highly effective adjuvant, high-titer neutralizing antibodies with broad cross-reactivity were effectively induced in poultry. Secondly, by establishing a poultry culling method based on pre-immune screening, a stepwise enhanced immunization program, and a purification process combining PEG / sodium alginate precipitation and multi-step chromatography, the yield, purity, and in vitro neutralizing titer of anti-HPV specific IgY were significantly improved, while impurities such as endotoxins were greatly reduced, ensuring the bioactivity and safety of the product. Attached Figure Description
[0015] Figure 1 This is the overall flowchart of the present invention. Detailed Implementation
[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0017] This invention provides a technical solution: a composition for preventing and treating HPV, its preparation method, and its application, comprising anti-HPV specific egg yolk immunoglobulin (IgY) as an active ingredient, wherein the anti-HPV specific IgY is obtained by a method comprising the following steps: Antigen preparation: Preparation of an immunogen containing L1, the major capsid protein of HPV; Animal immunization: The immunogen is used to perform stepwise immunization on selected laying poultry, including basic immunization and periodic booster immunization, to induce the production of high-titer specific antibodies. Antibody isolation and purification: Highly immune eggs produced by immunized poultry are collected, and the anti-HPV specific IgY is isolated and purified from the egg yolk; wherein, the in vitro pseudovirus neutralizing titer of the anti-HPV specific IgY in the composition is ND. 50 No less than 1:1000.
[0018] Furthermore, the immunogen is selected from one or more combinations of the following: Virus-like particles formed by the self-assembly of HPVL1 protein; The antigen and the immune adjuvant form a complex antigen, wherein the immune adjuvant is selected from oil-in-water emulsion adjuvants and TLR agonists.
[0019] Furthermore, the anti-HPV specific IgY meets at least one of the following quality properties: The purity was not less than 95% as determined by reducing SDS-PAGE, and characteristic bands appeared at 66 kDa and 25 kDa. Size exclusion chromatography was used to detect that the area of the main peak of the monomer accounted for no less than 98%; Endotoxin levels not exceeding 10 EU / mg IgY; It can specifically recognize and bind to L1 proteins of one or more HPV types.
[0020] Furthermore, the composition is a dosage form suitable for topical administration, selected from gels, creams, suppositories, films, sprays, foams, and rinses; or a dosage form suitable for oral administration, selected from enteric-coated capsules, enteric-coated tablets, or lozenges; wherein the topical dosage form further comprises a pharmaceutically acceptable gel matrix, a mucosal adhesive, a penetration enhancer, and a preservative; and the anti-HPV specific IgY in the oral dosage form is microencapsulated.
[0021] A method for preparing a composition for preventing HPV includes the following steps: S1. Immunogen preparation: HPVL1 protein antigen is expressed and purified using genetic engineering methods, and optionally mixed with an immune adjuvant to prepare a composite antigen for immunization; S2. Screening and immunization of laying poultry: Screening: Select healthy laying poultry, conduct pre-immunization assessments, and screen out individuals with a high immune response to antigens; Immunization program: Selected poultry receive the first immunization, followed by the second and third immunizations on days 7 and 14 to complete the basic immunization; thereafter, booster immunizations are given every 4-6 weeks. S3. Collection of highly immunized eggs: Starting 20 days after the first immunization, eggs laid by poultry will be continuously collected as highly immunized eggs; S4. Extraction and purification of anti-HPV specific IgY: Egg yolk separation and pretreatment: After disinfecting and washing the highly immune eggs, separate the yolks, dilute the yolks with water in a certain proportion, adjust the pH to 5.0-6.0, let stand at 0-5℃, and take the supernatant. Preliminary purification: Polyethylene glycol or sodium alginate is added to the supernatant for precipitation treatment to remove major impurities and lipids, and the supernatant or precipitate solution rich in IgY is collected. Deep purification: The preliminarily purified IgY solution is purified by chromatography, wherein the chromatography technique is selected from one or more combinations of affinity chromatography, ion exchange chromatography, and hydrophobic interaction chromatography; Concentration and sterilization: The purified IgY solution was concentrated by ultrafiltration and then sterilized by filtration to obtain a high-purity anti-HPV specific IgY concentrate. Formulation preparation: The anti-HPV specific IgY concentrate is mixed with pharmaceutically acceptable excipients.
[0022] Furthermore, step S2 specifically involves: injecting candidate laying hens with a low dose of the aforementioned immunization complex antigen, and detecting the HPV-specific antibody level in their serum 10-14 days later, retaining individuals with antibody titers ranking in the top 20%-40% as subsequent immunization hens.
[0023] Furthermore, in step S4, the volume dilution ratio of egg yolk to water is 1:4 to 1:8; the pH is adjusted to 5.2-5.8; and the standing time is 10-16 hours. The polyethylene glycol is PEG6000. It is added to a final concentration of 3%-4% (w / v), and the mixture is stirred at 0-4℃ for 1-3 hours before centrifugation; or sodium alginate is added to a final concentration of 0.3%-0.5% (w / v), the pH is adjusted to 4.0-4.5, and the mixture is stirred before centrifugation.
[0024] Furthermore, step S4, deep purification, includes: Capture was performed using a specific affinity chromatography medium targeting the chicken IgYFc fragment, or anion exchange chromatography was used. The equilibration buffer was 20-50 mM Tris-HCl, pH 7.5-8.5. After sample loading, gradient elution or staged elution was performed by increasing the ionic strength of the buffer to collect the target protein peak. Hydrophobic interaction chromatography was used, with samples loaded in the presence of 1.0-1.5M ammonium sulfate, and gradient elution was performed by decreasing the salt concentration to collect the target protein peak.
[0025] Furthermore, in step S5: when preparing oral enteric-coated capsules, the microencapsulation coating process is as follows: IgY is spray-dried and microencapsulated using a pH-sensitive polymer material, wherein the polymer material is selected from acrylic resin and hydroxypropyl methylcellulose phthalate; when preparing a topical gel, the excipients include a gel matrix, a stabilizer, and a humectant, wherein polyethylene glycol 400 is added as a stabilizer, with a final concentration of 5%-10% (w / w); when preparing a lyophilized powder, after ultrafiltration concentration, a lyophilization protectant is added to the IgY solution for lyophilization, wherein the lyophilization protectant contains 5%-10% (w / v) trehalose and sucrose, and 1%-2% (w / v) glycine.
[0026] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.
[0027] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A composition for preventing and treating HPV, characterized in that, The active ingredient is an anti-HPV specific egg yolk immunoglobulin (IgY), which is obtained by a method comprising the following steps: Antigen preparation: Preparation of an immunogen containing L1, the major capsid protein of HPV; Animal immunization: The immunogen is used to perform stepwise immunization on selected laying poultry, including basic immunization and periodic booster immunization, to induce the production of high-titer specific antibodies. Antibody isolation and purification: Highly immune eggs produced by immunized poultry are collected, and the anti-HPV specific IgY is isolated and purified from the egg yolk; wherein, the in vitro pseudovirus neutralizing titer of the anti-HPV specific IgY in the composition is ND. 50 No less than 1:1000.
2. The composition for preventing and treating HPV according to claim 1, characterized in that: The immunogen is selected from virus-like particles formed by the self-assembly of HPVL1 protein. The antigen and the immune adjuvant form a complex antigen, wherein the immune adjuvant is selected from oil-in-water emulsion adjuvants and TLR agonists.
3. The composition for preventing and treating HPV according to claim 1, characterized in that: The anti-HPV specific IgY meets at least one of the following quality properties: The purity was not less than 95% as determined by reducing SDS-PAGE, and characteristic bands appeared at 66 kDa and 25 kDa. Size exclusion chromatography was used to detect that the area of the main peak of the monomer accounted for no less than 98%; Endotoxin levels not exceeding 10 EU / mg IgY; It can specifically recognize and bind to L1 proteins of one or more HPV types.
4. The composition for preventing and treating HPV according to claim 1, characterized in that: The composition is a dosage form suitable for local administration, selected from gels, creams, suppositories, films, sprays, foams, and rinses; wherein the local dosage form further comprises a pharmaceutically acceptable gel matrix, a mucosal adhesive, a penetration enhancer, and a preservative; and the anti-HPV specific IgY in the oral dosage form is microencapsulated.
5. A method for preparing a composition for preventing and treating HPV, using a composition for preventing and treating HPV as described in any one of claims 1-4, characterized in that: Includes the following steps: S1. Immunogen preparation: HPVL1 protein antigen is expressed and purified using genetic engineering methods, and optionally mixed with an immune adjuvant to prepare a composite antigen for immunization; S2. Screening and immunization of laying poultry: Screening: Select healthy laying poultry, conduct pre-immunization assessments, and screen out individuals with a high immune response to antigens; Immunization program: Selected poultry receive the first immunization, followed by the second and third immunizations on days 7 and 14 to complete the basic immunization; thereafter, booster immunizations are given every 4-6 weeks. S3. Collection of highly immunized eggs: Starting 20 days after the first immunization, eggs laid by poultry will be continuously collected as highly immunized eggs; S4. Extraction and purification of anti-HPV specific IgY: Egg yolk separation and pretreatment: After disinfecting and washing the highly immune eggs, separate the yolks, dilute the yolks with water in a certain proportion, adjust the pH to 5.0-6.0, let stand at 0-5℃, and take the supernatant. Preliminary purification: Polyethylene glycol or sodium alginate is added to the supernatant for precipitation treatment to remove major impurities and lipids, and the supernatant or precipitate solution rich in IgY is collected. Deep purification: The preliminarily purified IgY solution is purified by chromatography, wherein the chromatography technique is selected from one or more combinations of affinity chromatography, ion exchange chromatography, and hydrophobic interaction chromatography; Concentration and sterilization: The purified IgY solution was concentrated by ultrafiltration and then sterilized by filtration to obtain a high-purity anti-HPV specific IgY concentrate. Formulation preparation: The anti-HPV specific IgY concentrate is mixed with pharmaceutically acceptable excipients.
6. The method for preparing a composition for preventing and treating HPV according to claim 5, characterized in that: The screening process in step S2 specifically involves injecting candidate laying hens with a low dose of the aforementioned immunization complex antigen, and then detecting the level of HPV-specific antibodies in their serum 10-14 days later. Individuals with antibody titers ranking in the top 20%-40% are retained as subsequent immunization hens.
7. The method for preparing a composition for preventing and treating HPV according to claim 5, characterized in that: In step S4, the volume dilution ratio of egg yolk to water is 1:4 to 1:8; the pH is adjusted to 5.2-5.8; and the standing time is 10-16 hours. The polyethylene glycol is PEG6000, added to a final concentration of 3%-4%, and stirred at 0-4℃ for 1-3 hours before centrifugation.
8. A method for preparing a composition for preventing and treating HPV according to claim 5, characterized in that: The deep purification step S4 includes: A specific affinity chromatography medium targeting the chicken IgYFc fragment was used for capture.
9. A method for preparing a composition for preventing and treating HPV according to claim 5, characterized in that: In step S5: when preparing oral enteric-coated capsules, the microencapsulation coating process is as follows: IgY is spray-dried and microencapsulated using a pH-sensitive polymer material, wherein the polymer material is selected from acrylic resin and hydroxypropyl methylcellulose phthalate.