Application of pantothenic acid in regulating activation of dendritic cells and preparing immunologic adjuvant

Abstract

The invention discloses application of pantothenic acid in regulating activation of dendritic cells and preparing an immunologic adjuvant. Experiments prove that the pantothenic acid has a remarkable promoting effect on related innate immunity and adaptive immunity of the dendritic cells; various vaccine models find that pantothenic acid can remarkably promote vaccine reactivity and is more efficient than traditional adjuvants, new application of pantothenic acid is provided, the application range of pantothenic acid is expanded, and the pantothenic acid has important economic benefits and social benefits.

Description

Technical Field

[0001] This invention belongs to the field of immunomodulation and immune adjuvant technology, specifically relating to the application of pantothenic acid in regulating dendritic cell activation and preparing immune adjuvants. Background Technology

[0002] Dendritic cells (DCs), named for their dendritic shape, are responsible for initiating adaptive immune responses, thus acting as "sentinels" of the immune system. DCs are leukocytes derived from bone marrow (BM) and are professional and highly efficient antigen-presenting cell types, responsible for initiating all antigen-specific immune responses. Therefore, they are key regulators of the immune response by linking the microbiome-sensing characteristics of the innate immune system with the fine-grained specificity of adaptive responses. Thus, vaccines, by activating DCs and inducing antigen-specific T and B cell responses, are a crucial step in achieving protective immunity. Regulating DC activation and enhancing their function is an effective means of improving vaccine immunogenicity.

[0003] In recent years, although new vaccine technologies have continuously advanced globally, and traditional vaccines (such as inactivated vaccines and subunit vaccines), as well as mRNA vaccines and viral vector vaccines, are widely used, the protective efficacy after vaccination varies significantly among populations. Numerous studies have shown that vaccine-induced immune responses exhibit significant inter-individual differences, a phenomenon known as "vaccine responsiveness" or "low responsiveness." In some high-risk groups (such as the elderly, diabetic patients, patients undergoing chemotherapy for cancer, individuals with chronic infections, and organ transplant recipients), vaccines fail to induce sufficiently strong immune responses, resulting in significantly reduced protection rates. For example, in influenza vaccination, the antibody response rate in people over 60 years of age is significantly lower than in younger populations; after COVID-19 vaccination, some immunosuppressed patients fail to develop effective neutralizing antibodies. The reasons for vaccine responsiveness are complex, mainly including decreased host immune system function, reduced dendritic cell numbers or weakened activation capacity, decreased antigen presentation efficiency, and insufficient T-cell response activation. Therefore, enhancing the immunogenicity of vaccines in low-responsive populations has become a significant challenge in current vaccinology research and application.

[0004] One of the mainstream strategies for improving vaccine responsiveness is the use of adjuvants. Adjuvants are exogenous substances that can enhance antigen immunogenicity and improve vaccine efficacy. Commonly used adjuvants include aluminum salt adjuvants (such as aluminum hydroxide), lipopolysaccharide-based monophospholipase A (MPLA), Toll-like receptor agonists (such as CpG oligonucleotides, Poly(I:C)), and water-in-oil adjuvants (MF59, AS03). These adjuvants enhance vaccine-induced T / B cell responses by activating dendritic cells and enhancing their chemotaxis, antigen uptake, and presentation capabilities. However, existing adjuvants still have certain limitations, such as the possibility of excessive stimulation inducing an overactive inflammatory response, poor tolerability of some adjuvants in certain populations, and the lack of highly effective and safe adjuvants for some vaccines. In addition, adjuvants are mostly added to vaccine formulations as exogenous components, and the complex formulation and regulatory standards also limit their promotion in various vaccine types.

[0005] Pantothenic acid, also known as vitamin B5, is a water-soluble vitamin and a precursor to coenzyme A (CoA) synthesis. It is widely involved in fatty acid metabolism, energy metabolism, and redox reactions. In recent years, research on the role of pantothenic acid in immune regulation and metabolic control has increased, and it has gradually shown its potential for functional regulation in the immune system and tumor microenvironment. Regarding anti-tumor immunity, studies have shown that pantothenic acid enhances anti-tumor immune responses by increasing coenzyme A synthesis. However, it is noteworthy that pantothenic acid has also been found to promote the growth of certain types of tumor cells, possibly indirectly supporting tumor development by accelerating energy metabolism or providing synthetic substrates, suggesting a "double-edged sword" effect in its role within the tumor environment. Currently, there are no studies or reports on pantothenic acid as an immune adjuvant. Summary of the Invention

[0006] In order to overcome the technical problems existing in the prior art, the purpose of this invention is to provide the art with the application of pantothenic acid in regulating dendritic cell activation and preparing immune adjuvants.

[0007] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0008] A first aspect of the present invention provides the use of pantothenic acid in the preparation of immune adjuvants.

[0009] Furthermore, the immune adjuvant is an adjuvant that promotes vaccine responsiveness;

[0010] Optionally, the enhancement of vaccine responsiveness manifests in at least one of the following ways:

[0011] (1) Enhance the titer of specific antibodies induced by the vaccine;

[0012] (2) Increase the number of B cells in the germinal center induced by the vaccine;

[0013] (3) Enhance vaccine-induced cellular and humoral immune responses;

[0014] (4) Promote the activation of T cells by dendritic cells during vaccine action;

[0015] Optionally, the specific antibody includes one or more of total IgG, IgG1, IgG2a, IgG2b, IgG2c, and IgG3;

[0016] Optionally, the enhanced vaccine-induced cellular immune response includes promoting the secretion of one or more of the cytokines IFN-γ, IL-4, and IL-17A by T cells;

[0017] Optionally, the enhancement of the vaccine-induced humoral immune response includes one or more of the following: increasing the titer of specific antibodies and increasing the number of germinal center B cells.

[0018] Furthermore, the vaccine includes one or more of the following: ovalbumin vaccine, hepatitis B vaccine, and HPV vaccine.

[0019] In this invention, pantothenic acid is a water-soluble vitamin with the chemical formula C9H. 17 NO5, with a molecular weight of 219.23 and CAS number 79-83-4, is a precursor for coenzyme A (CoA) synthesis and is widely involved in fatty acid metabolism, energy metabolism, and redox reactions. Currently, there are no studies or reports on the application of pantothenic acid in promoting vaccine responsiveness or regulating dendritic cell activation.

[0020] In a specific embodiment of this invention, mouse primary dendritic cells (DCs) were used as the research object to investigate the effects of pantothenic acid on the antigen processing and presentation capacity of DCs stimulated by EαEGFP, the maturation of DCs stimulated by LPS, and the activation of T cells under co-culture conditions of DCs and T cells. The experimental results showed that after EαEGFP stimulation of DCs, pantothenic acid significantly promoted the antigen processing and presentation capacity of DCs; under LPS stimulation or no stimulation of DCs, pantothenic acid affected the maturation of DCs, significantly promoting the expression of co-stimulatory molecules; under co-culture conditions of DCs and T cells, pantothenic acid affected the ability of DCs to activate T cells, significantly promoting T cell proliferation and cytokine secretion. These results indicate that pantothenic acid has a significant promoting effect on both DC-related innate and adaptive immunity.

[0021] Furthermore, this invention investigated the effect of pantothenic acid on vaccine responsiveness using various vaccine models, specifically an ovalbumin (OVA)-induced vaccine model, a hepatitis B vaccine model, and an HPV vaccine model. The results showed that, compared to mice immunized with the antigen alone, mice immunized with alum adjuvant along with the antigen exhibited significantly increased specific antibody titers, germinal center (GC) B cell content, and IL-4 cytokine levels, but without affecting IFN-γ cytokine expression. Subsequently, immunization of mice with a mixture of 500 μg / mouse and 5 mg / mouse of pantothenic acid and the antigen via intramuscular injection showed a dose-dependent effect in promoting specific antibody titers, GC B cell content, and the expression of IFN-γ and IL-4 cytokines. This invention is the first to discover the role of pantothenic acid in regulating dendritic cell activation and promoting vaccine responsiveness, providing a new use for pantothenic acid, expanding its application scope, and possessing significant economic and social benefits.

[0022] In this invention, promoting vaccine responsiveness refers to enhancing the immune response induced by the vaccine in the body through the action of specific substances. This is specifically manifested in the improvement of multi-dimensional immune indicators, including humoral immunity, such as increasing the titer of vaccine-specific antibodies and increasing the number of germinal center B cells (a key site for B cell proliferation and differentiation into antibody-secreting cells); and cellular immunity, such as promoting T cell proliferation and cytokine secretion. Moreover, its mechanism of action is closely related to enhancing the function of dendritic cells (DCs), that is, by promoting the antigen processing and presentation capacity of DCs (such as increasing the expression of Eα antigen and MHC II complex) and increasing the expression of co-stimulatory factors on the surface of DCs, sufficient signals are provided for subsequent immune cell activation, ultimately enhancing the immune protection effect of the vaccine, and even showing better immune enhancement efficiency and more comprehensive immune activation (simultaneously activating humoral and cellular immunity) in some cases (such as compared with traditional aluminum adjuvants).

[0023] In a specific embodiment of the present invention, promoting vaccine responsiveness refers to increasing the titer of specific antibodies induced by the vaccine, increasing the number of germinal center B cells induced by the vaccine, enhancing the cellular and humoral immune responses induced by the vaccine, and / or promoting the activation of T cells by dendritic cells during the vaccine's action.

[0024] In this invention, the vaccine is not limited to one or more of the above-mentioned ovalbumin vaccine, hepatitis B vaccine, and HPV vaccine, but also includes all types of vaccines that require activation of dendritic cells (DCs) to mediate an immune response in order to achieve immune protection.

[0025] In some implementations, the vaccines include, but are not limited to: viral vaccines (such as influenza virus vaccine, COVID-19 vaccine, varicella-zoster virus vaccine, polio virus vaccine, measles virus vaccine, rubella virus vaccine, mumps virus vaccine, Japanese encephalitis virus vaccine, rabies virus vaccine, etc.), bacterial vaccines (such as BCG vaccine, pneumococcal vaccine, Neisseria meningitidis vaccine, Haemophilus influenzae vaccine, Bordetella pertussis vaccine, Corynebacterium diphtheriae vaccine, Clostridium tetani vaccine, Vibrio cholerae vaccine, Salmonella typhi vaccine, etc.), parasitic vaccines (such as malaria vaccine, Toxoplasma gondii vaccine, Schistosomiasis vaccine, etc.), and cancer therapeutic vaccines (such as vaccines prepared based on tumor-associated antigens or tumor-specific antigens, such as melanoma-associated antigen vaccine, colorectal cancer-associated antigen vaccine, etc.), and autoimmune disease-related regulatory vaccines, etc.

[0026] In some implementation schemes, regardless of the type of vaccine technology, such as inactivated vaccines, live attenuated vaccines, subunit vaccines, recombinant protein vaccines, viral vector vaccines, mRNA vaccines, DNA vaccines, or tumor vaccines, as long as the initiation of the immune response depends on the antigen processing and presentation of dendritic cells, the expression of co-stimulatory molecules, and the activation of T / B cells, pantothenic acid can be used as an adjuvant or immunomodulator in this invention to promote vaccine responsiveness, thereby enhancing the humoral and cellular immune effects induced by the vaccine, expanding the scope of application of the vaccine, and enhancing its immune protection efficacy in different populations (including normal populations and high-risk populations with weakened immune function).

[0027] A second aspect of the invention provides the use of pantothenic acid in the preparation of products that regulate the activation function of dendritic cells.

[0028] Furthermore, the regulation of dendritic cell activation function manifests itself in at least one of the following ways:

[0029] (1) Promotes the antigen processing and presentation capabilities of dendritic cells;

[0030] (2) Promotes the expression of co-stimulatory factors associated with dendritic cells;

[0031] (3) Enhance the ability of dendritic cells to activate T cells;

[0032] (4) Promotes dendritic cell activation, leading to enhanced cellular and humoral immunity;

[0033] Optionally, the co-stimulatory factors include one or more of CD80, CD86, MHC I, and MHC II;

[0034] Optionally, the ability of dendritic cells to activate T cells includes promoting T cell proliferation and secretion of one or more of the cytokines IFN-γ, IL-4, and IL-17A.

[0035] A third aspect of the invention provides an adjuvant that promotes vaccine responsiveness.

[0036] Furthermore, the active ingredient of the adjuvant is pantothenic acid;

[0037] Optionally, the adjuvant may further comprise pharmaceutically acceptable excipients;

[0038] Optionally, the effective concentration of pantothenic acid in the adjuvant is 5-20 mM;

[0039] Optionally, the effective concentration of pantothenic acid in the adjuvant is 10 mM;

[0040] Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-250 mg / kg;

[0041] Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-25 mg / kg;

[0042] Optionally, the effective concentration of pantothenic acid in the adjuvant is 200-250 mg / kg.

[0043] In some implementations, the pharmaceutically acceptable excipients include, but are not limited to, various excipients suitable for the formulation of immune adjuvants, improvement of stability, optimization of administration convenience, and assurance of biocompatibility.

[0044] In some implementations, the specific pharmaceutically acceptable excipients include, but are not limited to: diluents and solvents (such as water for injection, physiological saline (0.9% sodium chloride solution), phosphate buffered saline (PBS), etc., which can be used to adjust the concentration of pantothenic acid so that the adjuvant and vaccine are mixed to achieve an appropriate dosing volume and osmotic pressure, ensuring safety and comfort during injection, while maintaining the stable solubility of pantothenic acid in the formulation), and stabilizers (such as sugar excipients such as sucrose, trehalose, and mannitol, or polyol excipients such as glycerol and propylene glycol, which can extend the shelf life of the adjuvant formulation by forming hydrogen bonds, inhibiting protein denaturation, or preventing the degradation and aggregation of pantothenic acid during storage, especially under low-temperature storage or repeated freeze-thaw conditions). Adjuvants include: adjuvants that maintain the immune activity of pantothenic acid; suspending and thickening agents (such as sodium carboxymethyl cellulose, povidone (PVP), xanthan gum, etc.; if pantothenic acid is mixed with the vaccine in solid powder form or the preparation is a suspension, these excipients can increase the viscosity of the preparation, prevent pantothenic acid particles from settling, ensure the uniformity of the preparation, and ensure accurate dosage of pantothenic acid for each administration); and pH adjusters (such as hydrochloric acid, sodium hydroxide, citrate-sodium citrate buffer pairs, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer pairs, etc.; these can adjust the pH value of the adjuvant preparation to be close to the physiological pH range of the human body). (6.5-7.5), to avoid irritating the injection site tissue due to excessively high or low pH, reduce local inflammatory response, and maintain the chemical stability of pantothenic acid to prevent structural changes due to improper pH; antioxidants (such as sodium sulfite, sodium bisulfite, vitamin E, etc., which can inhibit the oxidation reaction of pantothenic acid during storage or exposure to air, avoid oxidation products affecting the immune-enhancing effect of adjuvants, and prevent quality problems such as discoloration and off-odors in the preparation); and local analgesics (such as benzyl alcohol, etc., if the adjuvant needs to be administered by intramuscular injection, such excipients can reduce the pain of local tissues during and after injection, improve the recipient's medication experience, and are especially suitable for vaccine scenarios that require multiple immunizations).

[0045] In some implementation schemes, the selection and dosage of the above-mentioned excipients need to be determined based on the physicochemical properties of pantothenic acid, the formulation of the adjuvant (such as liquid dosage form, lyophilized powder injection), and compatibility with the vaccine, to ensure that the excipients do not interact with pantothenic acid or vaccine antigens, do not affect the core function of pantothenic acid in promoting vaccine responsiveness, and at the same time meet the safety standards for pharmaceutical excipients.

[0046] A fourth aspect of the present invention provides a vaccine composition.

[0047] Furthermore, the active ingredients of the vaccine composition are pantothenic acid and vaccine antigen;

[0048] Optionally, the vaccine composition may further comprise pharmaceutically acceptable excipients;

[0049] Optionally, the effective concentration of pantothenic acid in the vaccine composition is 5-20 mM;

[0050] Optionally, the effective concentration of pantothenic acid in the vaccine composition is 10 mM;

[0051] Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-250 mg / kg;

[0052] Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-25 mg / kg;

[0053] Optionally, the effective concentration of pantothenic acid in the adjuvant is 200-250 mg / kg.

[0054] In some implementations, the pharmaceutically acceptable excipients include, but are not limited to, various excipients that adapt to the formulation characteristics of the vaccine composition, ensure the synergistic stability and immunomodulatory activity of pantothenic acid and vaccine antigens, and optimize the safety and convenience of administration.

[0055] In some implementations, the specific pharmaceutically acceptable excipients include, but are not limited to: solvents and diluents (such as water for injection, physiological saline (0.9% sodium chloride solution), phosphate buffer (PBS, pH 6.8-7.4), etc., which can adjust pantothenic acid and vaccine antigen to appropriate concentrations and dosage volumes, ensuring that the osmotic pressure of the formulation conforms to the physiological range of the human body and avoiding local tissue osmotic pressure disorders after injection; at the same time, providing a stable dissolution or suspension environment for pantothenic acid and vaccine antigen, preventing aggregation or precipitation due to excessive concentration, and ensuring the accuracy of pantothenic acid and antigen dosages for each administration), stabilizers (including sugar stabilizers (such as sucrose, trehalose, mannitol), polyol stabilizers (such as glycerol, propylene glycol) and protein protectants (such as bovine serum albumin, casein hydrolysate), suspending and thickening agents (if the vaccine composition is a suspension formulation (such as containing particulate antigen or pantothenic acid solid dispersion), sodium carboxymethyl cellulose, povidone (PVP) can be selected). Excipients such as K30 and xanthan gum. These excipients increase the viscosity of the formulation, slow down the sedimentation rate of pantothenic acid and antigen particles, ensure the uniformity of the formulation, avoid dosage deviation due to particle stratification, and improve the fluidity of the formulation during injection, reducing the difficulty of injection operation. pH adjusters (such as citrate-sodium citrate buffer pairs, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer pairs, hydrochloric acid or sodium hydroxide solutions, etc.). These can adjust the pH of the vaccine composition to a suitable range of 6.0-7.8, which matches the chemical environment in which pantothenic acid exists stably (avoiding structural damage due to excessively high or low pH), maintains the immunogenicity of vaccine antigens (such as viral antigens, recombinant protein antigens), and reduces local irritation at the injection site (such as redness, swelling, and pain) caused by pH incompatibility. Antioxidants. Dosage agents (such as sodium bisulfite, vitamin C, sodium thiosulfate, etc., can inhibit the oxidation reaction between pantothenic acid and antigens (especially antigen molecules containing thiol and amino groups) in the vaccine composition during contact with air or storage, avoid the generation of oxidation products that affect the immune enhancement effect and antigen activity, and at the same time prevent the preparation from discoloration, off-odor and other quality problems, and extend the shelf life of the composition), local analgesics and absorption aids (such as benzyl alcohol can reduce local pain during intramuscular injection and improve recipient compliance; while nonionic surfactants such as polysorbate 80 and Tween 20 can moderately improve the compatibility of pantothenic acid with lipid-soluble vaccine antigens (such as viral envelope antigens), promote the uniform diffusion of pantothenic acid at the injection site, assist its contact with dendritic cells, and indirectly ensure the immune enhancement effect).

[0056] In some implementation schemes, the selection of the above-mentioned excipients needs to be based on the physicochemical properties of pantothenic acid and specific vaccine antigens (such as OVA antigen, HBsAg, E7 antigen, etc.) and the formulation form of the composition (liquid, lyophilized powder for injection), to ensure that the excipients do not interact with pantothenic acid or antigens, do not interfere with the core function of pantothenic acid in promoting vaccine reactivity (such as enhancing DC antigen presentation, activating humoral and cellular immunity), and at the same time meet the safety standards for pharmaceutical excipients and the regulatory requirements for vaccine formulations.

[0057] A fifth aspect of the present invention provides a composition for regulating the activation function of dendritic cells.

[0058] Furthermore, the active ingredient in the composition is pantothenic acid;

[0059] Optionally, the composition further comprises cell culture grade excipients or pharmaceutically acceptable excipients;

[0060] Optionally, the effective concentration of pantothenic acid in the composition is 5-20 mM;

[0061] Optionally, the effective concentration of pantothenic acid in the composition is 10 mM;

[0062] Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-250 mg / kg;

[0063] Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-25 mg / kg;

[0064] Optionally, the effective concentration of pantothenic acid in the adjuvant is 200-250 mg / kg.

[0065] In some implementations, the cell culture-grade excipients include, but are not limited to, various substances that provide basic nutritional support for the in vitro culture of dendritic cells, maintain the stability of the cell growth microenvironment, ensure pantothenic acid activity, and meet cell culture-grade purity standards (such as being free of mycoplasma, endotoxins, and low in endogenous contaminants).

[0066] In some embodiments, the pharmaceutically acceptable excipients are as described in the fourth aspect of the invention.

[0067] The sixth aspect of the invention provides the use of pantothenic acid in in vitro cultured dendritic cells.

[0068] Furthermore, the application is selected from any one or more of the following:

[0069] (1) Promotes the antigen processing and presentation capabilities of dendritic cells;

[0070] (2) Promotes the expression of co-stimulatory factors associated with dendritic cells;

[0071] (3) Enhance the ability of dendritic cells to activate T cells;

[0072] (4) Promotes dendritic cell activation, leading to enhanced cellular and humoral immunity;

[0073] Optionally, the co-stimulatory factors include one or more of CD80, CD86, MHC I, and MHC II;

[0074] Optionally, the ability of dendritic cells to activate T cells includes promoting T cell proliferation and secretion of one or more of the cytokines IFN-γ, IL-4, and IL-17A;

[0075] Optionally, the effective concentration of pantothenic acid is 5-20 mM;

[0076] Optionally, the effective concentration of pantothenic acid is 10 mM;

[0077] Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-250 mg / kg;

[0078] Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-25 mg / kg;

[0079] Optionally, the effective concentration of pantothenic acid in the adjuvant is 200-250 mg / kg.

[0080] A seventh aspect of the present invention provides a method for producing therapeutic dendritic cells.

[0081] Furthermore, the method includes: treating dendritic cells with pantothenic acid to obtain therapeutic dendritic cells;

[0082] Optionally, the effective concentration of pantothenic acid is 5-20 mM;

[0083] Optionally, the effective concentration of pantothenic acid is 10 mM;

[0084] Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-250 mg / kg;

[0085] Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-25 mg / kg;

[0086] Optionally, the effective concentration of pantothenic acid in the adjuvant is 200-250 mg / kg.

[0087] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0088] (1) This invention proposes a novel application of pantothenic acid in regulating dendritic cell activation and promoting vaccine responsiveness. Using mouse primary dendritic cells (DCs) as the research object, this invention investigated the effects of pantothenic acid on the antigen processing and presentation capacity of DCs stimulated by EαEGFP, the maturation of DCs stimulated by LPS, and the activation of T cells under co-culture conditions of DCs and T cells. The experimental results showed that after EαEGFP stimulation of DCs, pantothenic acid significantly promoted the antigen processing and presentation capacity of DCs; under LPS stimulation or no stimulation of DCs, pantothenic acid affected the maturation of DCs and significantly promoted the expression of co-stimulatory molecules in DCs; under co-culture conditions of DCs and T cells, pantothenic acid affected the ability of DCs to activate T cells and significantly promoted T cell proliferation and cytokine secretion. These results indicate that pantothenic acid has a significant promoting effect on both DC-related innate and adaptive immunity.

[0089] (2) This invention investigated the effect of pantothenic acid on vaccine reactivity using various vaccine models, including an ovalbumin (OVA)-induced vaccine model, a hepatitis B vaccine model, and an HPV vaccine model. The experimental results showed that, compared with mice immunized with the antigen alone, mice immunized with alum adjuvant along with the antigen showed significantly increased specific antibody titers, germinal center (GC) B cell content, and IL-4 cytokine levels, but did not affect IFN-γ cytokine expression. Subsequently, immunizing mice with a mixture of 500 μg / mouse and 5 mg / mouse of pantothenic acid and the antigen via intramuscular injection showed a dose-dependent effect on promoting specific antibody titers, germinal center (GC) B cell content, and IFN-γ and IL-4 cytokine expression. This invention is the first to discover the role of pantothenic acid in regulating dendritic cell activation and promoting vaccine responsiveness. Through comparative experiments, it has been demonstrated that, compared with traditional adjuvants, the pantothenic acid provided by this invention exhibits superior immune enhancement efficiency and more comprehensive immune activation (simultaneously activating humoral and cellular immunity). This provides a new use for pantothenic acid in the field, expands the application scope of pantothenic acid, and has significant economic and social benefits. Attached Figure Description

[0090] Figure 1 The effect of pantothenic acid on antigen protein processing and presentation after stimulation with EαEGFP protein in BMDC cells was investigated. Values ​​are expressed as mean ± SD; no significant difference was found in ns; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. t-tests were used for statistical analysis.

[0091] Figure 2The effect of pantothenic acid on the production of co-stimulatory factors in BMDC cells under LPS stimulation or non-stimulation. Values ​​are expressed as mean ± SD; no significant difference was found in ns; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. t-tests were used for statistical analysis.

[0092] Figure 3 Effects of pantothenic acid on T cell proliferation and cytokine secretion after co-culture of BMDC and OT II T cells. Values ​​are expressed as mean ± SD; no statistically significant difference was found in ns; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Statistical analysis was performed using the t-test method.

[0093] Figure 4 The effect of pantothenic acid on the OVA-induced mouse vaccine model was investigated. Values ​​are expressed as mean ± SD; no significant difference was found in ns; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. t-tests were used for statistical analysis.

[0094] Figure 5 The effect of pantothenic acid on a mouse hepatitis B vaccine model was investigated. Values ​​are expressed as mean ± SD; no significant difference was found in ns; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. t-tests were used for statistical analysis.

[0095] Figure 6 The effect of pantothenic acid on a mouse HPV vaccine model was investigated. Values ​​are expressed as mean ± SD; no significant difference was found in ns; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. t-tests were used for statistical analysis. Detailed Implementation

[0096] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0097] The reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit, the invention as described in the claims.

[0098] In the following examples, primary mononuclear dendritic cells (BMDCs) of mice were obtained from the femoral bone marrow of 6-8 week old male C57BL / 6 mice; pantothenic acid was purchased from Sigma-Aldrich; GM-CSF and IL-4 were purchased from Peprotech; mouse CD4+ T cell separation magnetic bead kit was purchased from Miltenyi Biotechnology; MACS auto-running buffer was purchased from Miltenyi Biotechnology; LS sorting column was purchased from Miltenyi Biotechnology; magnetic rack was purchased from Miltenyi Biotechnology; Eα-EGFP protein was prepared and purified in our laboratory; lipopolysaccharide (LPS) was purchased from Sigma-Aldrich; ovalbumin (OVA) was purchased from Sigma-Aldrich; OVA 323-339 peptide was purchased from Sangon Biotech Co., Ltd.; OTII transgenic mice (Jackson Laboratory) were purchased from Shanghai Southern Model Biotechnology Co., Ltd.; HBsAg was purchased from Genscript Biotech Co., Ltd.; E7 was purchased from Genscript Biotech Co., Ltd.; CellTrace™ CFSE cell proliferation kit was purchased from Invitrogen; Brefeldin was also used. Solution A (1000X) was purchased from Thermo Fisher Scientific; APC-IFNγ flow cytometry antibody was purchased from Thermo Fisher Scientific; APC-IL4 flow cytometry antibody was purchased from Biolegend; PE-Cy7-IL-17A flow cytometry antibody was purchased from Biolegend; saponin permeabilizing solution was purchased from Thermo Fisher Scientific; 7-AAD was purchased from BD Biosciences; PMA / ion stimulant was purchased from Hangzhou Lianke Biotechnology Co., Ltd.; APC-CD11c flow cytometry antibody was purchased from Thermo Fisher Scientific; FITC-MHC II flow cytometry antibody was purchased from Thermo Fisher Scientific; PE-CD86 flow cytometry antibody was purchased from Biolegend; PE-CD80 flow cytometry antibody was purchased from Biolegend; Biotin-Ea52-68 peptide bound to I-Ab Monoclonal Antibody (eBioY-Ae (YAe, Y-Ae)) antibody was purchased from eBioscience; APC Streptavidin was purchased from Biolegend; PE-CD4 flow cytometry antibody was purchased from Biolegend; APC anti-MU / HU GL7 Antigen (T / B Cell Act)The marker flow cytometry antibody was purchased from Biolegend; the CD95 (APO-1 / Fas) Monoclonal Antibody (15A7) PE flow cytometry antibody was purchased from Thermo Fisher Scientific; the CD45R (B220) Monoclonal Antibody (RA3-6B2) FITC flow cytometry antibody was purchased from Thermo Fisher Scientific; 4% paraformaldehyde was purchased from Promega; the mouse IFNγ ELISA kit, mouse IL-4 ELISA kit, and mouse IL-17A ELISA kit were all purchased from Biolegend; HRP conjugated Goat anti-Mouse IgG2a was purchased from FinBio; Goat Anti-Mouse IgG1, Human ads-HRP, Goat Anti-Mouse IgG2c-HRP, Goat Anti-Mouse IgG2b-HRP, and Goat Anti-Mouse IgG3-HRP were all purchased from Southern Biotech; Peroxidase AffiniPure Goat Anti-Mouse IgG... (H+L) was purchased from Jackson ImmunoResearch.

[0099] Example 1: Isolation and Differentiation of Primary Mouse Dendritic Cells (BMDCs)

[0100] 1. Isolation of mouse femur

[0101] (1) Place an absorbent paper or a layer of plastic wrap on the foam board.

[0102] (2) The mice were euthanized by cervical dislocation and placed on absorbent paper or plastic wrap covered with foam board. The mice were disinfected with alcohol and fixed with their ventral side facing upward.

[0103] (3) Hold the skin of the mouse's hind leg with tweezers, cut upwards to the root of the mouse's thigh with scissors, and cut downwards to about 1 cm below the mouse's knee. Hold the cut leg with tweezers and peel it upwards until the mouse's hip joint is exposed, revealing the white ball-shaped bone joint. Remove the entire femur from this point, remove excess tissue, and immerse the femur in PBS.

[0104] (4) Take the femur from the other side, using the same method as above.

[0105] 2. Isolation of mononuclear cells from mouse femur

[0106] (1) Preparation of materials: alcohol lamp, 50 mL centrifuge tube (for holding culture medium and PBS), sterile hemostat, sterile gauze, 1 mL syringe, 1640 + 10% serum + 1% penicillin antibody.

[0107] (2) Using sterilized hemostatic forceps, grasp a femur from PBS, place it on sterile gauze, fold and wrap it, hold it in place with both hands, place your thumbs behind the knee joint and forcefully break it upwards, creating a quincunx pattern on the fracture surface. Insert the needle of a syringe filled with culture medium into the center of the quincunx pattern and push the needle downwards until it can't go any further. Place the syringe along with the femur obliquely against the inner wall of a 50 mL centrifuge tube, and slowly push the syringe downwards to inject the culture medium into the bone marrow cavity, flushing out the cells from the bone marrow, which will gradually turn white. (Monocytes from the other side of the bone marrow are obtained using the same method.)

[0108] (3) After counting the extracted cells, press 2×10 6 Cells were seeded at a density of [number] cells / mL into 6-well plates for differentiation, with 2 mL of solution added to each well, along with 10 ng / mL GM-CSF and 5 ng / mL IL-4. The plates were incubated at 37°C for 6 days. On the third day of culture, 2 mL of 1640 medium containing 10 ng / mL GM-CSF and 5 ng / mL IL-4 was added to each well.

[0109] Example 2: Detection of the effect of pantothenic acid on BMDC antigen processing and presentation

[0110] 1. Cell inoculation

[0111] (1) Collect the supernatant from the 6-well plate into a 50 mL centrifuge tube and centrifuge (1500 rpm, 5 min); wash the cells with PBS and centrifuge (1500 rpm, 5 min).

[0112] (2) After centrifugation, discard the supernatant, resuspend the cells in fresh culture medium, mix well, and pipette 10 μL for cell counting. Finally, count the cells at a concentration of 1 × 10⁻⁶ cells per well. 5 Cells were seeded in 24-well plates.

[0113] 2. Drug pretreatment

[0114] Prepare a 200 mM stock solution by dissolving pantothenic acid in PBS. Add a certain volume of pantothenic acid to each well of a 24-well plate to achieve a final concentration of 10 mM, ensuring a consistent volume of PBS added to all wells. Incubate at 37°C for 2 hours.

[0115] 3. EαEGFP stimulates BMDCs

[0116] After pretreating BMDC cells with pantothenic acid, 50 μg / mL of EαEGFP was added, and the cells were incubated at 37°C for 24 h.

[0117] 4. Flow cytometry detection of BMDC antigen processing and presentation capabilities

[0118] Stimulated BMDCs were collected into 1.5 mL centrifuge tubes, washed with PBS, centrifuged at 1500 rpm for 5 min to collect the cell pellet, washed again with PBS, and resuspended in 100 μL. Flow cytometry antibody was added according to the instructions, and the mixture was incubated on ice or at 4°C for 30 min. Flow cytometry was then performed to detect the antigen presentation ability, and the fluorescence intensity of EGFP and YAe was used to evaluate the antigen presentation ability.

[0119] Example 3: Detection of the effect of pantothenic acid on BMDC maturity

[0120] 1. Cell inoculation

[0121] (1) Collect the supernatant from the 6-well plate into a 50 mL centrifuge tube and centrifuge (1500 rpm, 5 min); wash the cells with PBS and centrifuge (1500 rpm, 5 min).

[0122] (2) After centrifugation, discard the supernatant, resuspend the cells in fresh culture medium, mix well, and pipette 10 μL for cell counting. Finally, count the cells at a ratio of 1 × 10⁻⁶ cells per well. 5 Cells were seeded in 24-well plates with a solution volume of 500 μL per well.

[0123] 2. Drug pretreatment

[0124] Prepare a 200 mM stock solution by dissolving pantothenic acid in PBS. Add a certain volume of pantothenic acid to each well of a 24-well plate to achieve a final concentration of 10 mM, ensuring a consistent volume of PBS added to all wells. Incubate at 37°C for 2 hours.

[0125] 3. LPS stimulation of BMDC

[0126] After pretreating BMDC cells with pantothenic acid, 1 μg / mL of LPS was added, and the cells were incubated at 37°C for 24 h.

[0127] 4. Flow cytometry detection of BMDC maturity

[0128] Stimulated BMDCs were collected into 1.5 mL centrifuge tubes, washed with PBS, centrifuged at 1500 rpm for 5 min to collect the cell pellet, washed again with PBS, and resuspended in 100 μL. Flow cytometry antibody was added according to the instructions, and the mixture was incubated on ice or at 4°C for 30 min. The expression levels of CD80, CD86, and MHC II were detected by flow cytometry.

[0129] Example 4: Detection of the effect of pantothenic acid on BMDC-activated T cells

[0130] 1. BMDC cell seeding

[0131] (1) Collect the supernatant from the 6-well plate into a 50 mL centrifuge tube and centrifuge (1500 rpm, 5 min); wash the cells with PBS and centrifuge (1500 rpm, 5 min).

[0132] (2) After centrifugation, discard the supernatant, resuspend the cells in fresh culture medium, mix well, and pipette 10 μL for cell counting. Finally, count the cells at a ratio of 1 × 10⁻⁶ cells per well. 5 Cells were seeded in 24-well plates.

[0133] 2. Drug pretreatment

[0134] Prepare a 200 mM stock solution by dissolving pantothenic acid in PBS. Add a certain volume of pantothenic acid to each well of a 24-well plate to achieve a final concentration of 10 mM, ensuring a consistent volume of PBS added to all wells. Incubate at 37°C for 12–16 hours.

[0135] 3. OVA323-339 peptide stimulates BMDC

[0136] The following day, 10 μg / mL of OVA323-339 peptide was added to BMDC cells in 24-well plates and incubated at 37°C for 2 h.

[0137] 4. Isolation of OT II T cells

[0138] (1) Isolation of spleen cells from OT II mice: Mice were euthanized by cervical dislocation and placed on absorbent paper or plastic wrap lined with foam. The mice were disinfected with alcohol and fixed ventrally upwards. The fur on the abdomen was grasped with tweezers in the left hand and cut open from bottom to top along the midline with scissors in the right hand. The spleen (about 1 cm in size, blood red, and easy to separate) was located in the lower right corner of the abdomen with tweezers in both hands. (2) The spleen was removed and placed on a 50 mL centrifuge tube with a 70 μm filter. A small amount of MACS auto buffer was added to moisten the filter. The piston end of a syringe was used to grind the filter until the red area was no longer visible (during which time MACS auto buffer was added to rinse). The filtrate was collected and centrifuged at 1500 rpm for 5 min. (3) After centrifugation, the supernatant was discarded. The cell pellet was resuspended in 1 mL of gibco ACK lysing buffer for lysis of red blood cells. The cells were lysed at room temperature for 3-5 min. (4) Add 10 mL of MACS autobuffer to terminate lysis, mix well, filter through a new 70 μm filter (placed on a new 50 mL centrifuge tube), centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend the cell pellet in 1 mL of MACS auto buffer. (5) Cell counting: Mix 10 μL of suspension with 10 μL of trypan blue, add 10 μL to a cell counting chamber, and count the cells using a cell counter. (6) Incubate to separate CD4 + Antibodies against T cells: mouse CD4 + Antibody standard for T cell separation magnetic bead kit: 2~3×10 7 Cells / mL, total solution volume is 100 μL, corresponding to an antibody volume of 10 μL. Add antibody volume according to the above ratio, mix well, and incubate at 4℃ for 5-10 min by rotation. (7) Incubation to separate CD4 + T cell magnetic beads: Take 20 μL / tube and add it to the suspension containing antibody and cells (antibody: magnetic bead volume ratio = 1:2), rotate and incubate at 4℃ for 10-15 min; take out the magnetic rack and place it on a sterile table, place the LS sorting column on the magnetic rack, use buffer (3 mL / column) to wash the column, and then place a 50 mL centrifuge tube below (to prepare for T cell separation). (8) After incubating the magnetic beads, add 500 μL of MACS auto buffer to each tube, mix well and pass through the column, then add 3 mL buffer to wash the column, wash 3 times, 3 mL each time, collect T cell fluid in a 50 mL centrifuge tube, centrifuge at 1500 rpm for 3-5 min, and discard the supernatant; add 1 mL buffer to resuspend the precipitate, take 10 μL of the suspension and mix with 10 μL of trypan blue, take 10 μL and add it to the cell counting plate, and count using a cell counter.

[0139] 5. T cell marker CFSE

[0140] After sorting, the OT II T cells were washed once with PBS at room temperature, centrifuged at 1500 rpm for 3-5 min, the supernatant was discarded, and the serum in the MACS auto buffer was removed. The cells were resuspended in 1 mL PBS, 5 μM CFSE was added, and the cells were incubated at 37°C in the dark for 20 min. Five times the volume of the original chromosomes in culture medium (containing at least 1% FBS) was added to the cells and incubated for 5 min.

[0141] Cells were precipitated by centrifugation and resuspended in fresh, preheated complete culture medium;

[0142] With 1×10 5 cells / well, 100 μL / well, i.e., 1×10 6 cells / mL, seeded into 96-well plates.

[0143] 6. Co-culture of DCs and T cells

[0144] Pretreated DC cells were collected into 1.5 mL centrifuge tubes, and DC cells and CD4+ were separated. + T cell co-culture ratio based on cell mass ratio: DC:CD4 + T cells were seeded at a ratio of 1:2 into 96-well plates, resulting in 2.5 × 10⁶ DC cells. 4 Cell, 100 μL / well: 5 × 10 T cells 4 Cells, 100 μL / well, 37℃, 72 h. Approximately 5 h before cell collection, 50 μL of supernatant was collected from each well and frozen at -80℃ for subsequent ELISA detection; afterwards, a certain proportion of protein transport inhibitor and PMA / ion mixed stimulant was added to each well to amplify detectable intracellular cytokine signals. After incubation for 4-5 h, cells were collected for subsequent flow cytometry analysis.

[0145] 7. Flow cytometry detection of T cell proliferation capacity

[0146] CFSE-labeled T cells spontaneously emit 488 fluorescence. As the cell proliferation generation increases, the fluorescence intensity gradually decreases. Analyzing the degree of fluorescence attenuation can help analyze cell proliferation.

[0147] 8. Flow cytometry detection of T cell cytokine secretion ability

[0148] After co-culturing, cells were collected, washed once with PBS, and labeled with CD4 surface flow cytometry antibody. After labeling, cells were fixed with 200 μL / tube of 4% paraformaldehyde at room temperature for 15-20 min. Then, the cells were washed with permeabilization buffer. The flow cytometry antibody required for intracellular staining was prepared using the permeabilization buffer. After adding the antibody according to the ratio, the cells were incubated at room temperature for 45 min-1 h for intracellular staining. After washing with permeabilization buffer, the expression of cytokines IFNγ, IL-4, and IL-17A was detected by flow cytometry.

[0149] 9. ELISA detection of cytokines in co-culture supernatant

[0150] Thaw the cell supernatant stored at -80℃ on ice. After thawing, use an ELISA kit to detect the levels of cytokines IFNγ, IL-4, and IL-17A. Refer to the kit instructions for specific procedures.

[0151] Example 5: Effects of pantothenic acid on a chicken ovalbumin (OVA)-induced mouse vaccine model

[0152] 1. Establishment of a mouse vaccine model induced by chicken ovalbumin (OVA)

[0153] Mice (20-25 g) were vaccinated using a prime-boost immunization protocol: Primary immunization was performed on day 0 by injection of antigen (OVA, 100 μg), either alone or mixed with different adjuvants, and co-suspended in PBS, with a total injection volume of 100 μL. Seven days later, mice received a booster immunization, administered via the same method. Unless otherwise specified, all injections were performed intramuscularly (IM). Sample collection began on day 14, including the collection of mouse serum, isolation of draining lymph nodes, and preparation of single-cell suspensions.

[0154] 2. Effects of pantothenic acid on ovalbumin (OVA)-induced mouse vaccine model

[0155] The experiment was divided into five groups: ① negative control group, which received 100 μL PBS intramuscularly on day 0 and day 7; ② antigen group, which received 100 μg OVA antigen intramuscularly on day 0 and day 7; ③ alum adjuvant (i.e. aluminum adjuvant) group, which received 100 μg OVA antigen + 2 mg alum adjuvant (volume ratio 1:1) mixture intramuscularly on day 0 and day 7; ④ low-dose pantothenic acid group, which received 100 μg OVA antigen + 500 μg pantothenic acid mixture intramuscularly on day 0 and day 7; and ⑤ high-dose pantothenic acid group, which received 100 μg OVA antigen + 5 mg pantothenic acid mixture intramuscularly on day 0 and day 7.

[0156] 3. ELISA detection of serum antibody titer

[0157] (1) Blood collection and serum separation from mouse tail: Cut off about 3 mm of mouse tail and collect about 100 μL-500 μL of blood. Let stand overnight at 4℃. (2) The next day, centrifuge at 3000 rpm for 10 min at 4℃. If the serum separation can be clearly seen, directly aspirate the supernatant and dispense (10-20 μL / tube) and freeze at -80℃. (3) Preparation of serum for ELISA detection: ① Coating diluent (0.05mol / L Na2CO3-NaHCO3 buffer, pH 9.6): 2.9 g NaHCO3, 1.5 g Na2CO3, 0.2 g NaN3, add double-distilled water to 1000 mL and adjust to pH 9.6. ② Blocking solution (2% BSA / PBS solution): 1 g BSA + 50 mL PBS sample and universal diluent for secondary antibody (2% BSA, PBS, pH 7.4). ③ OVA stock solution: Concentration 1 mg / mL, working concentration for coated plates is 5 μg / mL, diluted 200 times, 50 μL required for a 10 mL plate. ④ Stop solution: 2N H2SO4 (1 mol / L) + 100 mL of 1 mol / L sulfuric acid: Dilute according to calculation. Measure 50 mL of distilled water into a beaker using a graduated cylinder, then slowly add 5.4 mL of 98% concentrated sulfuric acid to the 50 mL of water using a pipette. Rinse the pipette three times with a small amount of distilled water and add it to the beaker. Gently stir with a glass rod and let stand until the heat is released. Make up to volume. Transfer the diluted sulfuric acid solution from the beaker to a volumetric flask using a glass rod. Rinse the beaker and glass rod with an appropriate amount of distilled water at least three times until 2 cm from the mark on the volumetric flask. Add distilled water dropwise using a dropper until the volume is level with the mark. Tightly cap the volumetric flask and invert it repeatedly to ensure the solution is homogeneous. 100 mL of 1 mol / L sulfuric acid solution was prepared. (4) Dilute OVA protein to 5 μg / mL with coating buffer, add 100 μL / well to the corresponding well, and incubate overnight at 4°C. (5) Empty the liquid and pat dry the remaining liquid on a towel, wash with washing buffer for 3 min, pat dry, and repeat three times. (6) Add 200 μL / well of 5% BSA (PBS) blocking buffer to the corresponding well and incubate at 37°C for 2 h. (7) Empty the liquid and pat dry the remaining liquid on a towel, add 100 μL of washing buffer and wash three times, pat dry. (8) Serially dilute the primary antibody to 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400, and add 100 μL to each well (note the need to set up positive / negative control wells: wells without blocking solution, wells without primary antibody, and blank water control wells), and incubate at 37°C for 2 h. (9) Empty the liquid and pat dry any remaining liquid on a towel. Fill each well with washing buffer and repeat the washing process three times.(10) Dilute the enzyme-labeled secondary antibody 5000 times according to the instructions, add 100 μL / well, and incubate at 37℃ for 1 h. (11) Empty the liquid and pat dry any remaining liquid. Fill each well with washing buffer and let stand for 5 min. Empty the liquid and pat dry any remaining liquid. Repeat 3 times. (12) Add 100 μL of single-component TMB colorimetric solution to each well and place in a 37℃ incubator in the dark for color development. (13) Add stop solution 50 μL / well after 15-20 min of color development. (14) Measure the absorbance using an ELISA reader within 15 min after termination. Measure the absorbance at 450 nm. (15) The formula for judging antibody titer by performing the above indirect ELISA test is: when P / N = (sample absorbance value, blank absorbance value) / (negative absorbance value - blank absorbance value) > 2.1 (Formula 3-1), the antibody titer is considered to be positive. The antibody titer is the highest dilution factor in the positive group, that is, the antibody still has specificity at the highest dilution factor.

[0158] 4. Flow cytometry detection of GC B cells (germinal center B cells) in mouse draining lymph nodes

[0159] Draining lymph nodes were isolated at the model endpoint. Single-cell suspensions were prepared by gently grinding the tissue with MACS auto-running buffer. Cells were then filtered through a nylon filter to remove cell clumps. Cells were stained using the following fluorescently labeled antibodies, incubated on ice for 30 minutes: FITC-labeled anti-mouse B220 antibody, PE-labeled anti-mouse Fas antibody, and APC-labeled anti-mouse GL7 antibody. After staining, cells were washed with PBS and analyzed by flow cytometry.

[0160] 5. DLN cell restimulation and proliferation

[0161] Dissection of draining lymph nodes was performed at the end of the model. 2 × 10⁻⁶ lymph nodes were collected. 5DLN cells were cultured in 96-well plates (200 μL per well) for 72 hours with or without OVA antigen (100 μg / mL). Approximately 5 hours before sample collection, 50 μL of supernatant was aspirated for ELISA detection of cytokines, following the manufacturer's instructions. For the remaining cells, a specific ratio of protein transport inhibitor and PMA / ion mixed stimulant was added to each well to amplify detectable intracellular cytokine signals. After incubation for 4-5 hours, cells were collected for subsequent flow cytometry analysis. Collected cells were washed once with PBS, labeled with CD4 surface flow cytometry antibody, and then fixed with 200 μL / tube of 4% paraformaldehyde at room temperature for 15-20 minutes. Cells were then washed with permeabilization buffer, and the required flow cytometry antibody for intracellular staining was prepared using the permeabilization buffer. After adding the antibody according to the specified ratio, intracellular staining was performed at room temperature for 45 minutes to 1 hour. Cells were then washed with permeabilization buffer, and the expression of cytokines IFNγ, IL-4, and IL-17A was detected by flow cytometry.

[0162] Example 6: Effects of pantothenic acid on a mouse hepatitis B vaccine model

[0163] 1. Establishment of HBsAg-induced mouse hepatitis B vaccine model

[0164] Mice were vaccinated using a primary immunization followed by two booster immunizations: The primary immunization was performed on day 0 by injection of the antigen (HBsAg, 10 μg), either alone or mixed with different adjuvants, and co-suspended in PBS, with a total injection volume of 100 μL. Seven days later, mice received the first booster immunization, following the same procedure. Fourteen days later, mice received the second booster immunization, again following the same procedure. Unless otherwise specified, all injections were administered intramuscularly (im). Sample collection began on day 21, including the collection of mouse serum, the isolation of draining lymph nodes, and the preparation of single-cell suspensions.

[0165] 2. Effects of pantothenic acid on HBsAg-induced mouse hepatitis B vaccine model

[0166] The experiment was divided into four groups: ① negative control group, which received 100 μL PBS intramuscularly on day 0 and day 7; ② antigen group, which received 10 μg HBsAg antigen intramuscularly on day 0 and day 7; ③ alum adjuvant group, which received 10 μg HBsAg antigen + 2 mg alum adjuvant (volume ratio 1:1) mixture intramuscularly on day 0 and day 7; ④ high-dose pantothenic acid group, which received 10 μg HBsAg antigen + 5 mg pantothenic acid mixture intramuscularly on day 0 and day 7.

[0167] 3. ELISA detection of serum antibody titer

[0168] (1) Blood collection and serum separation from mouse tail: Cut off about 3 mm of mouse tail and collect about 100 μL-500 μL of blood. Let stand overnight at 4℃. (2) The next day, centrifuge at 3000 rpm for 10 min at 4℃. If the serum separation can be clearly seen, directly aspirate the supernatant and dispense (10-20 μL / tube) and freeze at -80℃. (3) Preparation of serum for ELISA detection: ① Coating diluent (0.05mol / L Na2CO3-NaHCO3 buffer, pH 9.6): 2.9 g NaHCO3, 1.5 g Na2CO3, 0.2 g NaN3, add double-distilled water to 1000 mL and adjust to pH 9.6. ② Blocking solution (2% BSA / PBS solution): 1 g BSA + 50 mL PBS sample and universal diluent for secondary antibody (2% BSA, PBS, pH 7.4). ③ HBsAg stock solution: The working concentration for coated plates is 5 μg / mL, diluted 200 times. One 10 mL plate requires 50 μL. ④ Stop solution: 2N H2SO4 (1 mol / L) + 100 mL of 1 mol / L sulfuric acid: Dilute according to calculation. Measure 50 mL of distilled water into a beaker using a graduated cylinder. Then, slowly add 5.4 mL of 98% concentrated sulfuric acid to the 50 mL of water using a pipette. Rinse the pipette three times with a small amount of distilled water and add it to the beaker. Gently stir with a glass rod and let stand until the heat is released. Make up to volume: Transfer the diluted sulfuric acid solution from the beaker to a volumetric flask using a glass rod. Rinse the beaker and glass rod with an appropriate amount of distilled water at least three times until there are 2 cm of water left to the mark on the volumetric flask. Add distilled water dropwise using a dropper until the volume is level with the mark. Tightly cap the volumetric flask and invert it repeatedly to ensure the solution is homogeneous. 100 mL of 1 mol / L sulfuric acid solution is now prepared. (4) Dilute HBsAg protein to 5 μg / mL with coating buffer, add 100 μL / well to the corresponding well, and incubate overnight at 4°C. (5) Empty the liquid and pat dry with a towel, wash with washing buffer for 3 min, pat dry, and repeat three times. (6) Add 200 μL / well of 5% BSA (PBS) blocking buffer to the corresponding well and incubate at 37°C for 2 h. (7) Empty the liquid and pat dry with a towel, add 100 μL of washing buffer and wash three times, pat dry. (8) Perform serial dilutions of the primary antibody at dilution ratios of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400, and add 100 μL / well to the corresponding well (note the setting of positive / negative control wells: wells without blocking buffer, wells without primary antibody, and blank water control wells), and incubate at 37°C for 2 h. (9) Empty the liquid and pat dry the remaining liquid on a towel. Fill each well with washing solution and repeat the washing process three times. (10) Dilute the enzyme-labeled secondary antibody 5000 times according to the instructions and add 100 μL / well. Incubate at 37°C for 1 h.(11) Empty the liquid and pat dry any remaining liquid. Fill each well with washing buffer and let stand for 5 min. Empty the liquid and pat dry any remaining liquid. Repeat 3 times. (12) Add 100 μL of single-component TMB colorimetric solution to each well and place in a 37℃ incubator in the dark for color development. (13) Add stop solution 50 μL / well after 15-20 min of color development. (14) Measure the absorbance using an ELISA reader within 15 min after the stop solution is applied. Measure the absorbance at 450 nm. (15) Perform the above indirect ELISA test. The formula for determining the antibody titer is: when P / N = (sample absorbance - blank absorbance) / (negative absorbance - blank absorbance) > 2.1 (Formula 3-1), the antibody titer is considered positive. The antibody titer is the highest dilution factor in the positive group, meaning that the antibody still has specificity at the highest dilution factor.

[0169] Example 7: Effects of pantothenic acid on a mouse HPV vaccine model

[0170] 1. Establishment of an E7 antigen-induced mouse HPV vaccine model

[0171] Mice were vaccinated using a primary immunization-two booster immunization regimen: Primary immunization was performed on day 0 by injection of antigen (E7, 20 μg), either alone or mixed with different adjuvants, and co-suspended in PBS, with a total injection volume of 100 μL. Seven days later, mice received their first booster immunization, following the same procedure. Fourteen days later, mice received their second booster immunization, also following the same procedure. Unless otherwise specified, all injections were administered intramuscularly (im). Sample collection began on day 21, including the collection of mouse serum, the isolation of draining lymph nodes, and the preparation of single-cell suspensions.

[0172] 2. Effects of pantothenic acid on E7-induced mouse HPV vaccine model

[0173] The experiment was divided into four groups: ① negative control group, which received 100 μL PBS intramuscularly on day 0 and day 7; ② antigen group, which received 20 μg E7 antigen intramuscularly on day 0 and day 7; ③ alum adjuvant group, which received 20 μg E7 antigen + 2 mg alum adjuvant (volume ratio 1:1) mixture intramuscularly on day 0 and day 7; ④ high-dose pantothenic acid group, which received 20 μg E7 antigen + 5 mg pantothenic acid mixture intramuscularly on day 0 and day 7.

[0174] 3. ELISA detection of serum antibody titer

[0175] (1) Blood collection and serum separation from mouse tail: Cut off about 3 mm of mouse tail and collect about 100 μL-500 μL of blood. Let stand overnight at 4℃. (2) The next day, centrifuge at 3000 rpm for 10 min at 4℃. If the serum separation can be clearly seen, directly aspirate the supernatant and dispense (10-20 μL / tube) and freeze at -80℃. (3) Preparation of serum for ELISA detection: ① Coating diluent (0.05mol / L Na2CO3-NaHCO3 buffer, pH 9.6): 2.9 g NaHCO3, 1.5 g Na2CO3, 0.2 g NaN3, add double-distilled water to 1000 mL and adjust to pH 9.6. ② Blocking solution (2% BSA / PBS solution): 1 g BSA + 50 mL PBS sample and universal diluent for secondary antibody (2% BSA, PBS, pH 7.4). ③ E7 stock solution: The working concentration for coated plates is 5 μg / mL, diluted 200 times. One 10 mL plate requires 50 μL. ④ Stop solution: 2N H2SO4 (1 mol / L) + 100 mL of 1 mol / L sulfuric acid: Dilute according to calculation. Measure 50 mL of distilled water into a beaker using a graduated cylinder. Then, slowly add 5.4 mL of 98% concentrated sulfuric acid to the 50 mL of water using a pipette. Rinse the pipette three times with a small amount of distilled water and add it to the beaker. Gently stir with a glass rod and let stand until the heat is released. Make up to volume: Transfer the diluted sulfuric acid solution from the beaker to a volumetric flask using a glass rod. Rinse the beaker and glass rod with an appropriate amount of distilled water at least three times until there are 2 cm of water left to the mark on the volumetric flask. Add distilled water dropwise using a dropper until the volume is level with the mark. Tightly cap the volumetric flask and invert it repeatedly to ensure the solution is homogeneous. 100 mL of 1 mol / L sulfuric acid solution is now prepared. (4) Dilute E7 protein to 5 μg / mL with coating buffer, add 100 μL / well to the corresponding well, and incubate overnight at 4°C. (5) Empty the liquid and pat dry with a towel, wash with washing buffer for 3 min, pat dry, and repeat three times. (6) Add 200 μL / well of 5% BSA (PBS) blocking buffer to the corresponding well and incubate at 37°C for 2 h. (7) Empty the liquid and pat dry with a towel, add 100 μL of washing buffer and wash three times, pat dry. (8) Perform serial dilutions of the primary antibody at dilution ratios of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400, and add 100 μL / well to each well (note the need to set up positive / negative control wells: wells without blocking buffer, wells without primary antibody, and blank water control wells), and incubate at 37°C for 2 h. (9) Empty the liquid and pat dry the remaining liquid on a towel. Fill each well with washing solution and repeat the washing process three times. (10) Dilute the enzyme-labeled secondary antibody 5000 times according to the instructions and add 100 μL / well. Incubate at 37°C for 1 h.(11) Empty the liquid and pat dry any remaining liquid. Fill each well with washing buffer and let stand for 5 min. Empty the liquid and pat dry any remaining liquid. Repeat 3 times. (12) Add 100 μL of single-component TMB colorimetric solution to each well and place in a 37℃ incubator in the dark for color development. (13) Add stop solution 50 μL / well after 15-20 min of color development. (14) Measure the absorbance using an ELISA reader within 15 min after the stop solution is applied. Measure the absorbance at 450 nm. (15) Perform the above indirect ELISA test. The formula for determining the antibody titer is: when P / N = (sample absorbance - blank absorbance) / (negative absorbance - blank absorbance) > 2.1 (Formula 3-1), the antibody titer is considered positive. The antibody titer is the highest dilution factor in the positive group, meaning that the antibody still has specificity at the highest dilution factor.

[0176] Experimental results

[0177] 1. Effect of pantothenic acid on BMDC antigen processing and presentation under EαEGFP stimulation

[0178] This invention determined the effect of pantothenic acid on the Eα-EGFP protein stimulation in mouse primary BMDCs, and the effect of BMDC treatment on Eα presentation capacity. The results are as follows: Figure 1 As shown.

[0179] Depend on Figure 1 It can be seen that BMDC cells can process and present EαEGFP protein 24 h after stimulation, and the Eα presentation capacity of BMDC cells is significantly increased compared with the control group. At the same time, 10 mM pantothenic acid treatment can significantly promote the Eα presentation capacity of BMDC cells (p<0.01), suggesting that pantothenic acid has a significant promoting effect on the antigen processing and presentation capacity of BMDC cells.

[0180] 2. Effects of pantothenic acid on the maturity of BMDCs stimulated by LPS

[0181] To further investigate the effect of pantothenic acid on the subsequent activation and maturation capacity of BMDCs, this invention determined the effect of pantothenic acid on the expression of co-stimulatory factors CD80, CD86, and MHC II in primary mouse BMDCs after lipopolysaccharide (LPS) stimulation. Subsequently, we also examined the effect of pantothenic acid treatment alone on the expression of co-stimulatory factors in BMDCs, and the results are as follows. Figure 2 As shown.

[0182] Depend on Figure 2As can be seen, after LPS stimulation, flow cytometry analysis showed a significant increase in the expression of CD80, CD86, and MHC II on the cell surface of BMDCs compared to the unstimulated group. Meanwhile, 10 mM pantothenic acid further promoted the expression of these co-stimulatory factors (p<0.01). Furthermore, we also observed that the addition of pantothenic acid alone significantly promoted the expression of BMDC co-stimulatory factors, suggesting that pantothenic acid has a promoting effect on BMDC activation.

[0183] 3. Effects of pantothenic acid on T cell activation under DC-T cell co-culture conditions

[0184] Based on the previous results of DC antigen presentation and maturity detection, in order to further clarify the effect of pantothenic acid on DC function, a co-culture system of DC and OT II was used to study the effect of pantothenic acid on the subsequent activation of CD4 by DC. + The effect of T cells. Results as follows: Figure 3 As shown, by Figure 3 It can be seen that, compared with untreated BMDCs, co-culturing DCs treated with OVA peptides with T cells can promote T cell proliferation and the secretion of related cytokines IFNγ, IL-17A, and IL-4. Co-culturing DCs treated with LPS and OVA peptides with T cells can more significantly promote T cell proliferation and related cytokine secretion. Compared with treatment with 10 mM pantothenic acid, BMDCs have a significantly enhanced ability to activate T cells, specifically manifested in significantly improved proliferation and cytokine secretion.

[0185] 4. Effects of pantothenic acid on OVA-induced mouse vaccine model

[0186] This invention uses an OVA vaccine model associated with DC and T cell activation as an in vivo model to explore the effect of pantothenic acid on vaccine responsiveness. Results are as follows: Figure 4 As shown.

[0187] Depend on Figure 4It can be seen that, compared with the antigen group mice, the total specific antibody and the titers of multiple subtype antibodies (IgG, IgG1, IgG2a, IgG2b, IgG2c, IgG3) in the serum of mice in the alum adjuvant group were significantly increased. Flow cytometry analysis also showed a significant increase in the content of GC B cells in the draining lymph nodes. In addition, both flow cytometry and ELISA detection showed an increase in the secretion of the cytokine IL-4 by T cells in the draining lymph nodes, but IFN-γ had no significant effect. This indicates that conventional alum adjuvant can significantly enhance the humoral immune response in mice, but has no significant effect on cellular immunity. However, using pantothenic acid as an adjuvant can enhance the titers of total specific antibodies and multiple subtype antibodies (IgG, IgG1, IgG2a, IgG2b, IgG2c, IgG3) in mouse serum in a dose-dependent manner, as well as the content of GC B cells in draining lymph nodes and the secretion of cytokines IFN-γ and IL-4 by T cells in draining lymph nodes. This suggests that pantothenic acid has a significant promoting effect on vaccine responsiveness in mice. Moreover, compared with alum adjuvant, pantothenic acid can not only enhance humoral immune response but also significantly enhance cellular immune response. That is, compared with traditional adjuvants, the pantothenic acid provided by this invention exhibits superior immune enhancement efficiency and more comprehensive immune activation (simultaneously activating humoral and cellular immunity). This result is a technical effect that those skilled in the art could not have anticipated based on the prior art.

[0188] 5. Effects of pantothenic acid on a mouse hepatitis B vaccine model

[0189] This invention uses a hepatitis B vaccine model associated with DC and T cell activation as an in vivo model to explore the effect of pantothenic acid on vaccine responsiveness. Results are as follows: Figure 5 As shown. By Figure 5 It can be seen that, compared with the antigen group mice, the total specific antibody (IgG) titer in the serum of mice in the alum adjuvant group was significantly increased. Using pantothenic acid as an adjuvant can also enhance the total specific antibody titer in mouse serum, and the enhancement is greater than that of alum. This suggests that pantothenic acid has a significant promoting effect on vaccine responsiveness in mice, and the effect of pantothenic acid is better than that of alum adjuvant. This comparative result is also a technical effect that was not expected by the prior art in this field.

[0190] 6. Effects of pantothenic acid on a mouse HPV vaccine model

[0191] This invention uses an HPV vaccine model associated with DC and T cell activation as an in vivo model to explore the effect of pantothenic acid on vaccine responsiveness. Results are as follows: Figure 6 As shown. By Figure 6It can be seen that, compared with the antigen group mice, the total specific antibody (IgG) titer in the serum of mice in the alum adjuvant group was significantly increased. Using pantothenic acid as an adjuvant can also enhance the total specific antibody titer in mouse serum, and the enhancement is greater than that of alum. This suggests that pantothenic acid has a significant promoting effect on vaccine responsiveness in mice, and the effect of pantothenic acid is better than that of alum adjuvant. This comparative result is also a technical effect that was not expected by the prior art in this field.

Claims

1. Application of pantothenic acid in the preparation of immune adjuvants.

2. The application according to claim 1, characterized in that, The immune adjuvant is an adjuvant that promotes vaccine responsiveness; Optionally, the enhancement of vaccine responsiveness manifests in at least one of the following ways: (1) Enhance the titer of specific antibodies induced by the vaccine; (2) Increase the number of B cells in the germinal center induced by the vaccine; (3) Enhance vaccine-induced cellular and humoral immune responses; (4) Promote the activation of T cells by dendritic cells during vaccine action; Optionally, the specific antibody includes one or more of total IgG, IgG1, IgG2a, IgG2b, IgG2c, and IgG3; Optionally, the enhanced vaccine-induced cellular immune response includes promoting the secretion of one or more of the cytokines IFN-γ, IL-4, and IL-17A by T cells; Optionally, the enhancement of the vaccine-induced humoral immune response includes one or more of the following: increasing the titer of specific antibodies and increasing the number of germinal center B cells.

3. The application according to claim 1, characterized in that, The vaccines include one or more of the following: ovalbumin vaccine, hepatitis B vaccine, and HPV vaccine.

4. Application of pantothenic acid in the preparation of products that regulate the activation function of dendritic cells.

5. The application according to claim 4, characterized in that, The regulation of dendritic cell activation function manifests itself in at least one of the following ways: (1) Promotes the antigen processing and presentation capabilities of dendritic cells; (2) Promotes the expression of co-stimulatory factors associated with dendritic cells; (3) Enhance the ability of dendritic cells to activate T cells; (4) Promotes dendritic cell activation, leading to enhanced cellular and humoral immunity; Optionally, the co-stimulatory factors include one or more of CD80, CD86, MHC I, and MHC II; Optionally, the ability of dendritic cells to activate T cells includes promoting T cell proliferation and secretion of one or more of the cytokines IFN-γ, IL-4, and IL-17A.

6. An adjuvant that promotes vaccine responsiveness, characterized in that, The active ingredient of the adjuvant is pantothenic acid; Optionally, the adjuvant may further comprise pharmaceutically acceptable excipients; Optionally, the effective concentration of pantothenic acid in the adjuvant is 5-20 mM; Optionally, the effective concentration of pantothenic acid in the adjuvant is 10 mM; Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-250 mg / kg; Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-25 mg / kg; Optionally, the effective concentration of pantothenic acid in the adjuvant is 200-250 mg / kg.

7. A vaccine composition, characterized in that, The active ingredients of the vaccine composition are pantothenic acid and vaccine antigen; Optionally, the vaccine composition may further comprise pharmaceutically acceptable excipients; Optionally, the effective concentration of pantothenic acid in the vaccine composition is 5-20 mM; Optionally, the effective concentration of pantothenic acid in the vaccine composition is 10 mM; Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-250 mg / kg; Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-25 mg / kg; Optionally, the effective concentration of pantothenic acid in the adjuvant is 200-250 mg / kg.

8. A composition for regulating dendritic cell activation, characterized in that, The active ingredient in the composition is pantothenic acid; Optionally, the composition further comprises cell culture grade excipients or pharmaceutically acceptable excipients; Optionally, the effective concentration of pantothenic acid in the composition is 5-20 mM; Optionally, the effective concentration of pantothenic acid in the composition is 10 mM; Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-250 mg / kg; Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-25 mg / kg; Optionally, the effective concentration of pantothenic acid in the adjuvant is 200-250 mg / kg.

9. The application of pantothenic acid in in vitro cultured dendritic cells, characterized in that, The application is selected from any one or more of the following: (1) Promotes the antigen processing and presentation capabilities of dendritic cells; (2) Promotes the expression of co-stimulatory factors associated with dendritic cells; (3) Enhance the ability of dendritic cells to activate T cells; (4) Promotes dendritic cell activation, leading to enhanced cellular and humoral immunity; Optionally, the co-stimulatory factors include one or more of CD80, CD86, MHC I, and MHC II; Optionally, the ability of dendritic cells to activate T cells includes promoting T cell proliferation and secretion of one or more of the cytokines IFN-γ, IL-4, and IL-17A; Optionally, the effective concentration of pantothenic acid is 5-20 mM; Optionally, the effective concentration of pantothenic acid is 10 mM; Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-250 mg / kg; Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-25 mg / kg; Optionally, the effective concentration of pantothenic acid in the adjuvant is 200-250 mg / kg.

10. A method for producing therapeutic dendritic cells, characterized in that, The method includes: treating dendritic cells with pantothenic acid to obtain therapeutic dendritic cells; Optionally, the effective concentration of pantothenic acid is 5-20 mM; Optionally, the effective concentration of pantothenic acid is 10 mM; Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-250 mg / kg; Optionally, the effective concentration of pantothenic acid in the adjuvant is 20-25 mg / kg; Optionally, the effective concentration of pantothenic acid in the adjuvant is 200-250 mg / kg.

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