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CN-122005772-A - HPV prevention and treatment composition, preparation method and application thereof

CN122005772ACN 122005772 ACN122005772 ACN 122005772ACN-122005772-A

Abstract

The invention relates to the technical field of HPV control compositions, in particular to a composition for controlling HPV, a preparation method and application thereof, and the composition comprises anti-HPV specific egg yolk immunoglobulin (IgY) as an active ingredient, wherein the anti-HPV specific IgY is obtained by adopting an immunogen comprising HPV main capsid protein L1, animal immunization, step-type immunization, basic immunization and periodic booster immunization, which are performed on screened egg laying birds, are adopted to induce the generation of high-titer specific antibodies, and antibody separation and purification, namely the high-immunity eggs produced by the birds after immunization are collected, and the anti-HPV specific IgY is obtained by separating and purifying egg yolk.

Inventors

  • HE YUQIANG
  • LI ZHANG
  • LI JIAO
  • ZHANG MINGQI
  • LIU YINRONG

Assignees

  • 山东中大基因科技有限公司

Dates

Publication Date
20260512
Application Date
20260204

Claims (9)

  1. 1. A composition for controlling HPV, comprising as an active ingredient anti-HPV-specific egg yolk immunoglobulin (IgY), said anti-HPV-specific IgY being obtained by a method comprising the steps of: antigen preparation, namely preparing an immunogen containing HPV main capsid protein L1; Step-type immunity is carried out on the egg-laying poultry subjected to screening by adopting the immunogen, wherein the step-type immunity comprises basic immunity and periodic booster immunity so as to induce the generation of high-titer specific antibodies; The antibody separation and purification comprises the steps of collecting hyperimmune eggs produced by the immunized birds, and separating and purifying the hyperimmune eggs from egg yolk to obtain the anti-HPV specific IgY, wherein the in vitro pseudovirus neutralization titer ND 50 of the anti-HPV specific IgY in the composition is not lower than 1:1000.
  2. 2. A composition for preventing and treating HPV according to claim 1, characterized in that the immunogen is selected from the group consisting of virus-like particles formed by self-assembly of HPVL1 proteins; The antigen and immune adjuvant form a composite antigen, and the immune adjuvant is selected from one of an oil-in-water emulsion adjuvant and a TLR agonist.
  3. 3. A composition for the control of HPV according to claim 1 characterized in that the anti-HPV-specific IgY corresponds to at least one of the following quality attributes: Purity was not less than 95% as determined by reducing SDS-PAGE, characteristic bands at 66kDa and 25 kDa; The main peak area of the monomer is not less than 98% by size exclusion chromatography detection; Endotoxin content is not higher than 10EU/mgIgY; an L1 protein capable of specifically recognizing and binding to one or more HPV types.
  4. 4. A composition for preventing and treating HPV according to claim 1 characterized in that the composition is in a dosage form suitable for topical administration selected from the group consisting of gels, creams, suppositories, films, sprays, foams and irrigation solutions, wherein the topical administration further comprises a pharmaceutically acceptable gel matrix, mucoadhesive, penetration enhancers and preservatives, and the anti-HPV-specific IgY in the oral administration is microencapsulated.
  5. 5. A method for preparing a composition for preventing and treating HPV using the composition for preventing and treating HPV according to any one of claims 1 to 4, characterized in that: the method comprises the following steps: s1, preparing an immunogen, namely expressing and purifying an HPVL1 protein antigen by adopting a genetic engineering method, and optionally mixing the antigen with an immune adjuvant to prepare a composite antigen for immunization; S2, screening and immunizing egg laying poultry: Screening, namely selecting healthy egg laying poultry, performing pre-immune evaluation on the healthy egg laying poultry, and screening out individuals with high immune response capability to antigens; immunization program, namely performing first immunization injection on the screened poultry, performing second and third immunization injection on the 7 th day and the 14 th day to complete basic immunization, and performing booster immunization injection every 4-6 weeks; s3, collecting hyperimmune eggs, namely continuously collecting eggs laid by poultry as hyperimmune eggs from 20 days after the first immunization injection; s4, extracting and purifying the anti-HPV specific IgY: Egg yolk separation and pretreatment, namely sterilizing and cleaning the hyperimmune egg, separating the egg yolk, diluting the egg yolk with water according to a proportion, regulating the pH value to 5.0-6.0, standing at 0-5 ℃, and taking supernatant; Primary purification, namely adding polyethylene glycol or sodium alginate into the supernatant to carry out precipitation treatment, removing main hybrid proteins and lipids, and collecting supernatant or precipitation solution rich in IgY; refining the primarily purified IgY solution by adopting a chromatographic technique, wherein the chromatographic technique is selected from one or a combination of more of affinity chromatography, ion exchange chromatography and hydrophobic interaction chromatography; concentrating and sterilizing, namely performing ultrafiltration concentration on the purified IgY solution, and performing filtration sterilization to obtain high-purity anti-HPV specific IgY concentrated solution; The preparation is prepared by mixing the anti-HPV specific IgY concentrated solution with pharmaceutically acceptable auxiliary materials.
  6. 6. The method according to claim 5, wherein the step S2 is performed by injecting the candidate egg-laying fowl with a low dose of the composite antigen for immunization, detecting the HPV-specific antibody level in serum after 10-14 days, and retaining the individuals with the antibody titers ranked at the top 20% -40% as the subsequent birds for immunization.
  7. 7. The method of preparing HPV control composition according to claim 5, characterized in that the yolk to water volume dilution ratio in step S4 is 1:4 to 1:8, pH is adjusted to 5.2-5.8, and standing time is 10-16 hours; the polyethylene glycol is PEG6000, and is added with 3% -4% of final concentration, stirred and reacted for 1-3 hours at 0-4 ℃ and then centrifuged.
  8. 8. The method for preparing HPV control composition according to claim 5 characterized in that the step S4 of deep purification comprises: capturing by adopting a specific affinity chromatography medium aiming at chicken IgYFc sections.
  9. 9. The method for preparing HPV control composition according to claim 5 characterized in that in step S5 the microencapsulation coating is a spray-dried microencapsulation of IgY with a pH sensitive polymeric material selected from the group consisting of acrylic resin and hypromellose phthalate.

Description

HPV prevention and treatment composition, preparation method and application thereof Technical Field The invention relates to the technical field of HPV prevention and treatment compositions, in particular to an HPV prevention and treatment composition, a preparation method and application thereof. Background Human Papilloma Virus (HPV) infection is a main cause of various diseases such as cervical cancer and genital warts, the incidence rate in the whole world is continuously high, public health safety is seriously threatened, the current prevention and treatment means mainly comprise preventive vaccines and local physical/chemical treatment, but the existing vaccines cannot cover all high-risk types and have limited actions on infected people, local medicines such as interferon have unstable effects, potential side effects and high cost and the like, in recent years, egg laying birds are immunized by specific antigens, high-titer specific antibodies (IgY) are extracted from egg yolk of the egg laying birds and are used as an emerging passive immunization strategy, unique advantages are shown in the field of virus prevention and treatment, the method has the potential of high safety, easiness in large-scale production, capability of aiming at specific pathogen design and the like, however, the technology is applied to a series of key HPV prevention and treatment, the method is lack of a high-efficiency immunogen design and screening system which comprise multi-type HPV, wide-spectrum high-affinity neutralizing antibodies are difficult to be induced, the obtained traditional immunization and antibody process is coarse, the titer, the purity and the stability of IgY are seriously influenced by the specific antibodies are not enough, the biological antibodies are seriously influenced, and the biological activity of IgY can not be stably influenced by the traditional immunization process, and the method has the effect of the method is applied to the engineering of the preparation, and the method can not form a complete laboratory, and the complete immunization preparation can be stably applied to the laboratory, and the complete preparation can be applied to the practical, and the preparation is completely-restricted to the practical preparation, and the practical preparation can be completely purified and restricted by the preparation, and the practical preparation can be applied to the method and the method to the method can be realized. Therefore, we make improvements to this, and propose a composition for preventing and treating HPV, and a preparation method and application thereof. Disclosure of Invention The invention aims to provide a composition for preventing and treating HPV, a preparation method and application thereof, and aims to solve the problems in the background technology. In order to achieve the above purpose, the present invention provides the following technical solutions: Comprising as active ingredient anti-HPV-specific egg yolk immunoglobulins (IgY) obtained by a process comprising the steps of: antigen preparation, namely preparing an immunogen containing HPV main capsid protein L1; Step-type immunity is carried out on the egg-laying poultry subjected to screening by adopting the immunogen, wherein the step-type immunity comprises basic immunity and periodic booster immunity so as to induce the generation of high-titer specific antibodies; the antibody separation and purification comprises the steps of collecting hyperimmune eggs produced by the immunized birds, and separating and purifying the hyperimmune eggs from egg yolk to obtain the anti-HPV specific IgY, wherein the in vitro pseudovirus neutralization titer ND of the anti-HPV specific IgY in the composition is not lower than 1:1000. As a preferred embodiment of the application, the immunogen is selected from one or more of the following combinations: Virus-like particles formed by self-assembly of the HPVL1 protein; The antigen and immune adjuvant form a composite antigen, and the immune adjuvant is selected from one of an oil-in-water emulsion adjuvant and a TLR agonist. As a preferred embodiment of the application, the anti-HPV-specific IgY corresponds to at least one of the following quality attributes: Purity was not less than 95% as determined by reducing SDS-PAGE, characteristic bands at 66kDa and 25 kDa; The main peak area of the monomer is not less than 98% by size exclusion chromatography detection; Endotoxin content is not higher than 10EU/mgIgY; an L1 protein capable of specifically recognizing and binding to one or more HPV types. According to a preferred technical scheme, the composition is a dosage form suitable for local administration, selected from gel, cream, suppository, film, spray, foam and flushing liquid, or is a dosage form suitable for oral administration, selected from enteric capsules, enteric tablets or lozenges, wherein the local administration dosage form further comprises a pharmaceutically accept