Method for improving antigen immunogenicity
By covalently coupling highly homologous antigens with BCG to form an antigen-BCG complex and injecting it into animals, the problem of poor immunogenicity of highly homologous antigens was solved, and a significant improvement in immunogenicity was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHENGZHOU IMMUNO BIOTECH
- Filing Date
- 2026-02-28
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies are insufficient to effectively enhance the immunogenicity of highly homologous antigens, especially antigens with amino acid sequence homology exceeding 30% to 100%, which are difficult to induce a strong immune response when using conventional immune adjuvants.
Highly homologous antigens are covalently coupled with BCG to form antigen-BCG complexes, which are then injected into the animals to be immunized via specific routes. Specific methods include coupling with carbodiimide condensing agents and dialysis treatment before inoculation.
It significantly enhances the immunogenicity of highly homologous antigens and improves the immune response in animals.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of animal immunization, and in particular to a method for enhancing the immunogenicity of highly homologous antigens. Background Technology
[0002] An antigen (Ag) is any substance that can induce an immune response in the body. Specifically, it is a substance that can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activating T / B cells, causing them to proliferate and differentiate, producing immune response products (sensitized lymphocytes or antibodies), and specifically binding to these products both in vivo and in vitro.
[0003] Conventional adjuvants, such as Freund's adjuvant, contain liquid paraffin, lanolin, BCG, or inactivated Mycobacterium tuberculosis. Freund's adjuvant is widely used in animal immunization experiments and can induce strong immune responses against most immunogens (including soluble proteins, viruses, cells, parasites, polysaccharides, etc.). However, some protein or polypeptide antigens have very high homology (over 30%, even 90%–100%) with the corresponding proteins or polypeptides in the animal to be immunized. For these antigens with high amino acid sequence homology, conventional adjuvants are unlikely to effectively induce a strong specific immune response in immunized animals, mainly because of the poor immunogenicity of the antigen. Traditional methods to enhance antigen immunogenicity include adjuvant optimization. For example, in patent CN1343482A, BCG is injected separately into the animal to be immunized before or simultaneously with vaccine immunization, using BCG instead of traditional drug-based immunostimulants to effectively enhance the immune effect of the vaccine injection. However, this method has only been validated in animal vaccines such as swine fever vaccine and chicken coccidiosis vaccine after immunogenicity testing, on piglets aged 20-40 days or chickens aged 7-14 days. Its effectiveness in enhancing the immunogenicity of highly homologous antigens is unknown. Another approach is to conjugate antigens with poor immunogenicity to exogenous macromolecules used as carriers to improve their immunogenicity. For example, patent CN115484977A describes conjugating proteins or peptides with sugars to form sugar-protein / peptide antigen conjugates, which show improved immunogenicity compared to unconjugated protein / peptide antigens. The preferred sugar species is Streptococcus pneumoniae capsular polysaccharide. Because sugars themselves have weak immunogenicity, methods of conjugating antigens with weak immunogenicity to sugar species to enhance antigen immunogenicity are rarely reported, and their practical application requires further observation. Summary of the Invention
[0004] In view of this, the present invention provides a method for enhancing the immunogenicity of highly homologous antigens. Compared with unconjugated antigens, antigens not conjugated with BCG, or antigens injected via non-specific routes, the method provided by the present invention can significantly enhance the immunogenicity of highly homologous antigens.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides a method for enhancing the immunogenicity of highly homologous antigens, comprising covalently coupling a highly homologous antigen with BCG to obtain an antigen-BCG complex, and inoculating the animals to be immunized with the antigen-BCG complex to achieve the purpose of enhancing the immunogenicity of the highly homologous antigen.
[0007] The high homology antigen has an amino acid sequence homology of 30%, 40%, or 50% or more with the corresponding protein or polypeptide of the animal to be immunized.
[0008] In some specific embodiments of the present invention, the BCG vaccine mentioned in the above method refers to a live vaccine made from a suspension of attenuated bovine tuberculosis bacteria.
[0009] In some specific embodiments of the present invention, the covalent coupling of the above method is achieved based on a carbodiimide-type condensing agent;
[0010] The carbodiimide-type condensing agent is coupled via a carboxyl-amino group and can be at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), and N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl p-toluenesulfonate (CMC).
[0011] The carbodiimide-type condensing agent can also be coupled via an amino-thiol group, and can be 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC).
[0012] In some specific embodiments of the present invention, the mass ratio of the high homology antigen and the BCG vaccine in the above method is 1:(0.5~1.5), 1:(0.5~5), 1:(0.5~10), 1:(0.5~15), or 1:(0.5~20).
[0013] In some specific embodiments of the present invention, the above method includes the following steps:
[0014] Step (1): Mix the buffer solution containing the high homology antigen with the buffer solution containing BCG, then mix with the carbodiimide condensing agent, and react to obtain the reaction product;
[0015] Step (2): Dialyze the reaction product to obtain the antigen-BCG complex;
[0016] Step (3): Mix the antigen-BCG complex, saline and adjuvant, emulsify and inoculate the animals to be immunized to enhance the immunogenicity of the high homology antigen.
[0017] In some specific embodiments of the present invention, the carbodiimide condensing agent in the above method is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
[0018] In some specific embodiments of the present invention, the inoculation method described above is intravenous injection, which may be tail vein injection.
[0019] In some specific embodiments of the present invention, the high homology antigen in the above method is INHA-βa, and the immunized animal can be a mouse, rabbit, sheep, alpaca, cow, horse or donkey.
[0020] The mouse can be a small mouse or a large rat.
[0021] In some specific embodiments of the present invention, the amino acid sequence of INHA-βa in the above method is shown in SEQ ID NO: 1.
[0022] The present invention also provides a method for producing antibodies, comprising inoculating the animals to be immunized according to the above method to obtain immunized animals, and isolating the antibodies from the immunized animals;
[0023] Specific steps may include:
[0024] Step (a1): Mix the buffer solution containing the high homology antigen with the buffer solution containing BCG, then mix with the carbodiimide condensing agent, and react to obtain the reaction product;
[0025] Step (a2): Dialyze the reaction product to obtain the antigen-BCG complex;
[0026] Step (a3): Mix the antigen-BCG complex, physiological saline and immune adjuvant, emulsify, inoculate the animals to be immunized, and isolate the antibody from the immunized animals.
[0027] The present invention also provides a method for preparing hybridoma cells, comprising inoculating the animals to be immunized according to the above method to obtain immunized animals, and preparing the hybridoma cells from the immunized animals;
[0028] Specific steps may include:
[0029] Step (b1): Mix the buffer solution containing the high homology antigen with the buffer solution containing BCG, then mix with the carbodiimide condensing agent, and react to obtain the reaction product;
[0030] Step (b2): Dialyze the reaction product to obtain the antigen-BCG complex;
[0031] Step (b3): Mix the antigen-BCG complex, physiological saline and immune adjuvant, emulsify, inoculate the animals to be immunized, and isolate the hybridoma cells from the immunized animals.
[0032] The present invention also provides an animal immunization method, characterized in that it includes inoculating the animal to be immunized according to the above method;
[0033] Specific steps may include:
[0034] Step (c1): Mix the buffer solution containing the high homology antigen with the buffer solution containing BCG, then mix with the carbodiimide condensing agent, and react to obtain the reaction product;
[0035] Step (c2): Dialyze the reaction product to obtain the antigen-BCG complex;
[0036] Step (c3): Mix the antigen-BCG complex, saline and adjuvant, emulsify and inoculate the animals to be immunized.
[0037] The present invention has the following beneficial effects:
[0038] For highly homologous antigens, there is currently no effective method to enhance their immunogenicity. Through extensive experimental verification, this invention reveals that: after conjugating highly homologous antigens with BCG to form an antigen-BCG complex, and injecting it into animals to be immunized through a special route, the immunogenicity of the highly homologous antigen can be significantly enhanced.
[0039] The preparation method of this invention is simple, requiring only the conjugation of the antigen and BCG to form an antigen-BCG complex, which is then injected into the animal to be immunized via a special route. There is no need to fuse the protein or peptide with other proteins or conjugate a special co-stimulatory sequence; the conjugation method and injection route are mature technologies in the industry. Referring to the method of this invention, the immunogenicity of highly homologous antigens can be significantly improved. Attached Figure Description
[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.
[0041] Figure 1 Homology analysis of SEQ ID NO: 1 is shown. Detailed Implementation
[0042] This invention discloses a method for enhancing the immunogenicity of highly homologous antigens. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.
[0043] This invention covalently couples highly homologous antigens or peptides with BCG vaccine to form an antigen-BCG vaccine complex. In some specific embodiments, the highly homologous antigen peptide sequence may be:
[0044] VPTKLRPMS MLYYDDGQNI IKKDIQNMIV EECG (SEQ ID NO: 1).
[0045] It should be noted that the polypeptide shown in SEQ ID NO: 1 is a sequence on the β subunit of inhibin A protein. Inhibin A is a glycoprotein hormone derived from the testes / follicles. Before puberty in males, it is mainly secreted by Sertoli cells; after puberty, it is secreted by Sertoli cells in conjunction with spermatogenic cells at various stages, participating in the regulation of pituitary function and forming a stable negative regulatory relationship with follicle-stimulating hormone (FSH). In females, INHA is mainly synthesized by granulosa cells of medium and small antral follicles, and secretion begins in the preantral follicular phase. It enters the follicular fluid, exerting autocrine and paracrine effects locally, and enters the circulation via the ovarian vein. When the level of inhibin A in the blood of a pregnant woman is higher than expected, it may indicate that the developing infant has Down syndrome or other abnormalities. Inhibin A is a dimeric glycoprotein hormone composed of two subunits, α (18kD) and β (14kD). The mature region DNA sequence of the inhibin A-β subunit in mammals has greater than 90% homology, and the amino acid sequence homology is greater than 96% (see [link to relevant documentation]). Figure 1 Antibodies against the polypeptide epitope of SEQ ID NO: 1 were prepared, primarily for the development of a detection kit for inhibin A protein.
[0046] The preparation method of the antigen-BCG complex can be as follows: BCG and antigen are mixed at a mass ratio of (0.5:1) to (1:20), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added for cross-linking. The mixture is reacted at 2 to 8°C in the dark for 12 hours. After that, unreacted small molecules are removed by dialysis or desalting to obtain the antigen-BCG complex.
[0047] Preferably, BCG vaccine and antigen are mixed at a mass ratio of 1:1.3. The antigen-BCG complex is then injected into the animal to be immunized.
[0048] Preferably, the injection is performed via the tail vein of the animal to be immunized, administering an antigen-BCG complex.
[0049] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.
[0050] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.
[0051] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.
[0052] The use of any and all instances or exemplary language such as “e.g.” or “include” in this document is intended merely to better illustrate the application and does not constitute a limitation on the scope of the application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.
[0053] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.
[0054] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.
[0055] The present invention will be further illustrated below with reference to the embodiments.
[0056] Example 1: Preparation of antigen-BCG complex
[0057] antigen:
[0058] Select an amino acid polypeptide sequence INHA-βa from the βa subunit of human inhibin A protein, as shown in SEQ ID NO: 1.
[0059] BCG vaccine:
[0060] Therapeutic freeze-dried BCG vaccine, National Drug Approval Number S20123007.
[0061] Preparation of INHA-βa-BCG complex:
[0062] First, BCG was dissolved in 0.05 mol / L MES buffer (pH 4.7) to a concentration of 4 mg / mL; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was dissolved in 0.05 mol / L MES buffer (pH 4.7) to a concentration of 20 mg / mL; and the peptide was dissolved in 0.05 mol / L MES buffer (pH 4.7) to a concentration of 2 mg / mL.
[0063] The second step involves adding the peptide and BCG to a reaction vessel at a ratio of 1:1 (w / w), mixing at room temperature, and calculating the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride according to a ratio of 1:50 (w / w) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to peptide. The hydrochloride is then slowly added to the reaction vessel at room temperature and reacted in the dark at 2-8°C for 12 hours.
[0064] The third step involves dialyzing the reaction solution with 0.01 mol / L PBS buffer (pH=7.2) at 4°C for 12 hours to obtain the INHA-βa-BCG complex.
[0065] Example 2: Preparation of antigen-KLH or BSA conjugate complexes
[0066] First, the peptide was dissolved in 0.01 mol / L PBS to a concentration of 2 mg / mL; sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate was dissolved in DMSO to a concentration of 10 mg / mL; KLH or BSA was dissolved in 0.01 mol / L PBS to a concentration of 10 mg / mL.
[0067] The second step involves adding the peptide and KLH or BSA to a reaction vessel at a molar ratio of 500:1 and mixing at room temperature. The amount of sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate is measured according to a molar ratio of 1:2 for sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate and slowly added to the mixed solution of peptide and KLH or BSA at room temperature. The mixture is then reacted in the dark at 2-8°C for 18 hours.
[0068] The third step involves dialyzing the reaction solution with 0.01 mol / L PBS buffer (pH=7.2) at 4°C for 12 hours to obtain the INHA-βa-KLH complex or the INHA-βa-BSA complex.
[0069] Example 1: Antigen-BCG complex enhances antigen immunogenicity.
[0070] Immunogens: Four antigens were selected as immunogens for immunization: carrier-free INHA-βa, INHA-βa-BCG complex, INHA-βa-KLH conjugate complex, and carrier-free INHA-βa mixed with BCG.
[0071] Experimental animals: 4-6 week old SPF-grade female BALB / c mice were selected, and 3 mice were immunized with each antigen.
[0072] Adjuvant: SIGMA Freund's adjuvant.
[0073] Immunization dosage: 100 μg / animal for the first immunization; 50 μg / animal for the second immunization; 50 μg / animal for the third immunization;
[0074] Animal immunization: Different antigens were prepared and immunogens were added to sterile saline at a dose of 100 μg / animal. The mixture was then mixed with Freund's adjuvant at a volume ratio of 1:1, resulting in a total volume of 200 μL. The mixture was emulsified using an IKA multi-point stirrer at 2–8°C. The emulsification effect was assessed after emulsification, and immunization was performed after the formation of water-in-oil emulsions. Mice were fixed using a single-handed or two-step grasping method. The injection site was located at the intersection of the midline of the abdomen and the tibia, with a radius of 0.5 cm around the injection site. Subsequent immunizations were performed at 21-day intervals, with an immunization dose of 50 μg / animal. Blood was collected from the tail vein on days 49–52 post-immunization to determine the titer.
[0075] Potency assay: The INHA-βa-BSA complex was diluted to 0.5 μg / mL with 0.05 mol / L pH 9.6 CB buffer and coated with a 96-well ELISA plate, then blocked with casein protein. Serum from mice after three immunizations and normal mice, diluted with DMEM, was added to the INHA-βa-BSA complex-coated ELISA plate at 50 μg / well. The plate was incubated at 37°C for 30 minutes, washed 5 times with PBST, and then blotted dry. Goat anti-mouse IgG + IgM-HRP was added at 50 μL / well, and the plate was incubated at 37°C for 30 minutes. The plate was washed 5 times with PBST, blotted dry, and then the ELISA substrate was added. The OD was measured using an ELISA reader. 450 value.
[0076] Mice were immunized with carrier-free INHA-βa antigen (numbers 1-3); mice were immunized with INHA-βa-BCG complex (numbers 4-6); mice were immunized with INHA-βa-KLH conjugate complex (numbers 7-9); mice were immunized with carrier-free INHA-βa and BCG mixture (numbers 10-12).
[0077] Table 1
[0078]
[0079] As shown in Table 1, the titer obtained by using the INHA-βa-BCG complex was significantly higher than that of the carrier-free INHA-βa antigen, the INHA-βa-KLH conjugate complex, and the carrier-free INHA-βa and BCG mixture. This indicates that BCG has a clear advantage as an antigen carrier when the homology between the antigen and the target immunized organism is high.
[0080] Example 2: Comparison of Immune Pathways
[0081] Preparation of antigen-BCG complex: INHA-βa-BCG complex was prepared according to Example 1.
[0082] Experimental animals: 4-6 week old SPF-grade female BALB / c mice were selected, and 3 mice were immunized by each immunization route.
[0083] Adjuvant: SIGMA Freund's adjuvant.
[0084] Immunogen: INHA-βa-BCG complex.
[0085] Immunization dosage: 100 μg / animal for the first immunization; 50 μg / animal for the second immunization; 50 μg / animal for the third immunization; booster immunization dose is the same as the first immunization dose, 100 μg / animal.
[0086] Animal immunization: Comparing the immunization effects of different immunization routes, experimental mice were randomly selected and immunized according to different immunization routes.
[0087] Intraperitoneal immunization: Same as the immunization regimen for the animal in Example 1.
[0088] Tail vein immunization: 100 μg / mouse of immunogen was prepared in sterile saline. The volume of the antigen and saline mixture was 50 μL. 50 μL / mouse was injected into the tail vein. The mice were removed and placed above the cage. One mouse was selected and secured in the tube. The mouse tail was disinfected by wiping it with alcohol. The distal end of the lateral tail vein was selected for injection. Subsequent immunizations were performed at intervals of 7-10 days, with an immunization dose of 50 μg / mouse. Blood was collected from the tail vein on days 49-52 post-immunization to detect the titer.
[0089] Subcutaneous immunization: 100 μg / mouse of immunogen was mixed with Freund's adjuvant in sterile saline at a volume ratio of 1:1. The volume of the mixture of antigen, saline, and adjuvant was 200 μL. The mixture was emulsified using an IKA multi-point stirrer at 2-8°C. The emulsification effect was tested after emulsification. Immunization was performed after water-in-oil emulsion was formed. Mice were fixed using a single-handed or two-step grasping method, and the lymph nodes of the limbs and neck were selected as injection sites. Subsequent immunizations were performed 14 days apart, with an immunization dose of 50 μg / mouse. Blood was collected from the tail vein on days 49-52 post-immunization to detect the titer.
[0090] Potency test: Same as Example 1.
[0091] Comparison of carrier complexes
[0092] Mice 1#~3# were immunized via tail vein; mice 4#~6# were immunized via intraperitoneal injection; and mice 7#~9# were immunized via subcutaneous injection. Serum was collected from the tail vein of each mouse and coated with INHA-βa antigen. The titer was then measured.
[0093] Table 2
[0094]
[0095] As shown in Table 2, the titer obtained by tail vein immunization was significantly higher than that obtained by intraperitoneal immunization and subcutaneous immunization, indicating that intravenous immunization has a significant advantage.
[0096] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for enhancing the immunogenicity of highly homologous antigens for non-diagnostic purposes, characterized in that, This includes covalently coupling a highly homologous antigen with BCG to obtain an antigen-BCG complex, and then inoculating the animals to be immunized with the antigen-BCG complex to obtain an antigen with enhanced immunogenicity. The high homology antigen shares more than 30% amino acid sequence homology with the corresponding protein or polypeptide of the animal to be immunized.
2. The method as described in claim 1, characterized in that, The covalent coupling is achieved based on a carbodiimide-type condensing agent.
3. The method as described in claim 2, characterized in that, Includes the following steps: Step (1): Mix the buffer solution containing the high homology antigen with the buffer solution containing BCG, then mix with the carbodiimide condensing agent, and react to obtain the reaction product; Step (2): Dialyze the reaction product to obtain the antigen-BCG complex; Step (3): Mix the antigen-BCG complex, saline and adjuvant, emulsify, and inoculate the animals to be immunized to obtain an antigen with enhanced immunogenicity.
4. The method as described in claim 2 or 3, characterized in that, The carbodiimide condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
5. The method according to any one of claims 1 to 4, characterized in that, The method of inoculation is intravenous injection.
6. The method as described in claim 5, characterized in that, The intravenous injection was administered via the tail vein.
7. The method according to any one of claims 1 to 6, characterized in that, The highly homologous antigen is INHA-βa, and the animal to be immunized is a mouse.
8. The method as described in claim 7, characterized in that, The amino acid sequence of INHA-βa is shown in SEQ ID NO:
1.
9. A method for preparing antibodies, characterized in that, The method includes inoculating the animal to be immunized according to any one of claims 1 to 8, obtaining an immunized animal, and isolating the antibody from the immunized animal.
10. A method for preparing hybridoma cells, characterized in that, The method includes inoculating the animal to be immunized according to any one of claims 1 to 8, obtaining an immunized animal, and preparing the hybridoma cells from the immunized animal.