WO2024160088A1 - Use of aescin and/or salt compound thereof as adjuvant in vaccine - Google Patents

Abstract

The present invention provides a use of aescin and/or an aescin salt compound as an adjuvant in a vaccine. The results of animal experiments show that the combination of aescin and an antigen can increase the antibody levels of sera IgG, IgG1, and IgG2a, i.e., enhance both humoral and cellular immune responses, and can be used as an adjuvant in a vaccine.

Description

Application of aescin and/or its salt compounds as adjuvant in vaccines

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of Chinese patent application filed with the China Patent Office on January 31, 2023, with application number 202310047342.8 and titled “Application of aescin and/or its salt compounds as adjuvants in vaccines”, the entire contents of which are incorporated by reference into this application.

Technical Field

The present application relates to the field of biomedicine technology, and in particular to the application of aescin compounds in the preparation of vaccine adjuvants and products.

Background Art

Vaccination is one of the most remarkable health achievements in human history and one of the most cost-effective ways to prevent and control infectious diseases. Vaccines have eradicated smallpox, eliminated polio in most parts of the world, and reduced the mortality and incidence of many infectious diseases worldwide. Vaccines refer to immune preparations made from pathogenic microorganisms (such as bacteria, viruses, etc.) and their metabolites for the prevention of infectious diseases. Vaccines retain the characteristics of pathogenic microorganisms to stimulate the body's immune system, allowing the body's immune system to produce certain protective substances to prevent the harm of related pathogens. The immunogenicity of antigens alone is usually low, and after injection, they often cannot induce an effective immune response to fight the invasion of pathogens. Adjuvants are usually added to induce a protective immune response of sufficient strength. Therefore, the efficacy of vaccines depends not only on the antigen component, but also on the type of adjuvant that stimulates the immune system. Adjuvants refer to a class of substances that can increase the body's immune response to antigens or change the type of response after being injected before, mixed with, or simultaneously with antigens.

So far, the number of vaccine adjuvants approved for use in humans is very limited, with a total of eight types, including aluminum adjuvants, MF59, viral particles, AS04, AS03, AS01, CpG 1018 and Matrix-M. The types of immune responses induced by different adjuvants vary greatly. Different diseases also have very different requirements for the type of immune response. For example, common bacterial infectious diseases mostly require humoral immune responses to participate in the elimination of pathogens, while diseases such as intracellular bacteria, viruses and tumors require cellular immune responses to kill infected cells. Taking the most commonly used traditional aluminum adjuvant as an example, it can effectively stimulate the body to produce IgG1 antibodies and induce a strong humoral immune response. However, aluminum adjuvants cannot induce cellular immune responses, so they are very effective in treating diseases such as intracellular bacteria, viruses and tumors that rely on cytotoxic T cells to kill. The efficacy of aluminum adjuvants is limited, and the human body may experience adverse reactions such as fever and local redness and swelling after injection. In order to improve the effectiveness of vaccines and broaden the application scope of adjuvants, the development of new, safe and effective vaccine adjuvants has become an important strategic goal of the country.

Therefore, the development of novel vaccine adjuvants that can induce both humoral and cellular immune responses is very important for human health.

Summary of the invention

The present application found that aescin and its salt compounds can significantly improve humoral immunity and cellular immune response, have obvious immunoenhancing effects, and have vaccine adjuvant effects, which can solve the problems of the small number of existing vaccine adjuvants and the inability of aluminum adjuvants to induce cellular immune response.

One of the purposes of the present application is to provide an application of an aescin compound in the preparation of a vaccine adjuvant, wherein the aescin compound is aescin or aescin salt compound; aescin is a seed extract of the Aesculus plant Thunbergia paniculata, which has pharmacological effects such as increasing venous tension, improving blood circulation, and correcting brain dysfunction. It is currently mainly used in clinical practice in the form of tablets and injections for the treatment of brain diseases such as cerebral edema.

The inventors of the present application have found that aescin and/or its salt compounds can enhance humoral and cellular immune responses and can be used as vaccine adjuvants.

Optionally, the structure of the aescin salt compound is as shown in formula (I):

Wherein, R in formula (I) includes glycine, alanine, valine, leucine, isoleucine, One or more of methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, wherein R in formula (I) further comprises a metal salt, and the metal salt comprises any one of sodium, potassium, zinc, aluminum, iron, zirconium, calcium, manganese and magnesium.

One of the purposes of the present application is to provide an immune complex, wherein the immune complex comprises an antigen and an aescin compound, wherein the aescin compound is aescin and/or an aescin salt compound.

Optionally, the antigen includes: one or more of proteins, polypeptides, nucleic acids, tumor cell lysates, viral lysates, bacterial lysates, bacterial cell membranes, mycoplasma cell membranes, viral envelopes, exosomes, bacterial antigens, tumor cell lysate antigens, tumor cell membrane vesicle antigens, tumor cell exosome antigens, and the model antigen chicken ovalbumin OVA.

Optionally, the weight ratio of the antigen to the aescin compound is 1:0.01-100.

Preferably, the weight ratio is 1:1-50, and further preferably, the mass ratio is 1:2-10.

Optionally, the antigen and the aescin compound are co-loaded in a carrier, and the carrier includes one or more of organic particles, inorganic particles and bionic particles. The organic particles include: microspheres and microcapsules, emulsions, liposomes, metal-organic framework compounds, gels, polystyrene particles, dendritic molecular compounds and high molecular polymer particles; the inorganic particles include: gold nanoparticles, iron oxide particles, mesoporous silica particles, aluminum salt particles, calcium phosphate particles and calcium carbonate particles; the bionic particles include: cell carriers, bacterial carriers, exosomes, viral particles and virus-like particles.

Preferably, the co-carrier is selected from one or more of the following: microemulsion, liposome and aluminum salt microparticles.

Further preferably, the microemulsion comprises squalene, Tween 80 and egg yolk phosphatidylcholine; the aluminum salt microparticles comprise chondroitin sulfate, aluminum sulfate and/or aluminum hydroxide; and the liposomes comprise DOPC, cholesterol and DSPE-PEG.

Optionally, the antigen and the aescin compound are respectively loaded in a carrier and then mixed for administration. The carrier includes one or more of organic particles, inorganic particles and bionic particles. The organic particles include: microspheres and microcapsules, emulsions, liposomes, metal-organic framework compounds, gels, polystyrene particles, dendritic molecular compounds and high molecular polymer particles; the inorganic particles include: gold nanoparticles, iron oxide particles, mesoporous silica particles, aluminum salt particles, calcium phosphate particles, calcium carbonate particles; the bionic particles include: cell carriers, bacterial carriers, exosomes, viral particles, virus-like particles.

Preferably, the carrier for mixed administration is selected from one or more of the following: microemulsion, liposome and aluminum salt microemulsion. grain.

Further preferably, the microemulsion comprises squalene, Tween 80 and egg yolk phosphatidylcholine; the aluminum salt microparticles comprise chondroitin sulfate, aluminum sulfate and/or aluminum hydroxide; and the liposomes comprise DOPC, cholesterol and DSPE-PEG.

Optionally, the immune complex further comprises an additional adjuvant, and the additional adjuvant comprises: one or more of one or more complexes of aluminum adjuvant, antigen-associated molecular pattern adjuvant, bacterial toxin and its derivatives, saponins, cytokines, heat shock proteins, A151, GTP-GDP, dimethyl dioctadecyl quaternary ammonium bromide DDA;

The antigen-associated molecular pattern adjuvants include: Toll-like receptor agonists, NOD-like receptor agonists, NOD-like receptor agonists, C-type lectin receptors, and STING agonists; the Toll-like receptor agonists include: peptidoglycan, lipoteichoic acid, MPLA, imiquimod, resiquimod, CpG-ODN, bacterial flagellin, and Poly I:C; the RIG-I-like receptor agonists include: 3pRNA, short double-stranded RNA; the NOD-like receptor agonists include: muramyl dipeptide MDP, N-acetylglucosamine; the C-type lectin receptors include: β-glucan, trehalose diborate; the bacterial toxins and their derivatives include: cholera toxin CT, Escherichia coli heat-labile enterotoxin LT, and cholera toxin B subunit; the saponins include: QS21, tomatin, and Quil-A; the cytokines include: GM-CSF, IL-2, IL-12, IL-6, IFN-γ, and lymphocyte chemotactic factor.

Preferably, the additional adjuvant is selected from one or more of the following: aluminum adjuvant, CpG-OND and MPLA.

One of the purposes of the present application is to provide the use of any of the above-mentioned immune complexes in the preparation of vaccines.

One of the purposes of the present application is to provide a vaccine comprising any of the immune complexes described above.

The present application found that aescin compounds have immune adjuvant effects and proposed their use as vaccine adjuvants. The types of adjuvants in this field are very limited. The most commonly used aluminum adjuvant can only induce humoral immunity. CpG-ODN has more advantages in inducing cellular immunity, but there is still a lack of adjuvants that can simultaneously improve humoral immunity and cellular immune responses. The present application found that aescin compounds can effectively induce the production of IgG1 and IgG2a antibodies, play an adjuvant role, and induce humoral immunity and cellular immune responses at the same time. Compared with conventional aluminum adjuvants and CpG-ODN, the adjuvant effect is more comprehensive.

The present application also found that aescin compounds can be applied to a variety of carriers, and can synergistically play an adjuvant role in a variety of vaccine carriers with different properties such as microemulsions, aluminum salt nanoparticles and liposomes, thereby enhancing their adjuvant properties. The present application also found that the aescin compound, when used in combination with adjuvants such as CpG-ODN and MPLA, can further enhance the ability of aescin to induce cellular immune response and effectively induce the production of cytotoxic T cells. It can be seen that aescin compounds have a synergistic effect with conventional immune adjuvants.

The only saponin adjuvant reported so far is QS-21, which is mainly used as one of the components of AS01 adjuvant and is used in combination with MPLA to make liposome adjuvants. It is currently mainly used in herpes zoster and malaria vaccines, and has a narrow range of applications. Although QS-21 can induce humoral and cellular immune responses, QS-21 can only be extracted from the bark of the soap bark tree in South America, and there are problems such as low purity, low yield and poor stability, which greatly limits the use of adjuvants in my country's vaccines. Secondly, there are many types of saponins, but this application only finds that aescin compounds can enhance the body's humoral and cellular immune responses and can be used as vaccine adjuvants. At the same time, compared with QS-21, aescin compounds are easy to obtain in my country, and purity and yield are well guaranteed. At the same time, aescin compounds can also improve humoral immunity and cellular immune responses at the same time in terms of effect, and can be used in combination with a variety of adjuvants including MPLA to play a synergistic effect, which can be used as a better saponin adjuvant than QS-21. The research results of this application provide a new adjuvant for the vaccine field that can induce both humoral immunity and cellular immunity, solving the problems of existing adjuvants such as lack of variety, difficulty in obtaining, and large side effects.

The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present application or the related technologies, the following is a brief introduction to the drawings required for use in the embodiments or the related technical descriptions. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

Figure 1 shows the results of specific antibodies IgG, IgG1 and IgG2a in the serum of mice immunized by subcutaneous injection at the base of the tail after administration of a mixture of five saponins and OVA.

Figure 2 shows the results of specific antibodies IgG, IgG1 and IgG2a in the serum of mice immunized with aescin and OVA loaded into microemulsion and injected subcutaneously at the base of the tail.

Figure 3 shows aescin loaded into liposomes and mixed with OVA and injected subcutaneously at the base of the tail to immunize mice The results of specific antibodies IgG, IgG1 and IgG2a in the serum were analyzed.

Figure 4 shows the results of specific antibodies IgG, IgG1 and IgG2a in the serum of mice immunized with aluminum salt nanoparticles loaded with aescin and OVA and injected subcutaneously at the base of the tail.

Figure 5 shows the results of specific antibodies IgG, IgG1 and IgG2a in the serum of mice immunized with a mixture of aescin and OVA and aluminum gel and injected subcutaneously at the base of the tail.

Figure 6 shows the results of specific antibodies IgG, IgG1 and IgG2a in the serum of mice immunized by subcutaneous injection at the base of the tail after loading aescin and OVA into microemulsion and adding adjuvant MPLA.

Figure 7 shows the results of specific antibodies IgG, IgG1 and IgG2a in the serum of mice immunized by subcutaneous injection at the base of the tail after sodium aescinate was loaded into liposomes and mixed with OVA solution and added with adjuvant MPLA.

Figure 8 shows the results of specific antibodies IgG, IgG1 and IgG2a in the serum of mice immunized by subcutaneous injection at the base of the tail after loading aescin and OVA into microemulsion and adding adjuvant CpG.

FIG. 9 shows the results of in vivo CTL detection in mice after subcutaneous injection at the base of the tail after loading aescin and OVA into microemulsion and adding adjuvant MPLA.

FIG. 10 shows the results of in vivo CTL detection in mice after subcutaneous injection at the base of the tail after loading aescin and OVA into microemulsion and adding adjuvant CpG.

Specific embodiments

In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

Example 1

First, using ovalbumin (OVA) as an antigen, the immunostimulatory effects of five saponins (ginsenoside, saikosaponin, notoginsenoside, soybean saponin, and aescin) were systematically evaluated after subcutaneous injection at the base of the tail. Aescin (Esc), ginsenoside (Gin), saikosaponin (Sai), and notoginsenoside (Not) were dissolved in DMSO to prepare a 2 mg/ml stock solution, and soybean saponin (Soy) and ovalbumin (OVA) were dissolved in water for injection to prepare a 2 mg/ml stock solution. 10 μl of the saponin solution and 5 μl of the OVA solution were mixed, and 85 μl of water for injection were added. C57BL/6 mice were subcutaneously injected with 100 μl of the mixed solution containing 20 μg of saponin and 10 μg of OVA at the base of the tail on days 0, 14, and 21, respectively. On the 27th day, blood was collected from the orbits, and the supernatant was taken after centrifugation at 8000rpm for 10min. The OVA-specific antibodies IgG, IgG1 and IgG2a were determined by ELISA. The results are shown in Figure 1. Figure 1 shows that the immunogenicity of free OVA is low, and the IgG and IgG1 in the mouse serum are only slightly increased after immunization, but there is no significant difference with the PBS group. In the mixed solution of five saponins and OVA, the antibody levels of ginsenoside, bupleurum saponin, notoginseng saponin and soybean saponin groups are consistent with free OVA. It can be seen that the above saponins cannot induce immune response and have no immune adjuvant effect. Only aescin can significantly increase the levels of IgG, IgG1 and IgG2a antibodies (****, p<0.0001). The results show that only aescin has a significant immune enhancement effect and can enhance both humoral and cellular immune responses. In Figure 1 , PBS was used as the blank group, OVA was the free OVA group, OVA+Gin represented the mixed solution group of OVA and ginsenoside, OVA+Sai represented the mixed solution group of OVA and saikosaponin, OVA+Not represented the mixed solution group of OVA and notoginseng saponin, OVA+Soy represented the mixed solution group of OVA and soybean saponin, and OVA+Esc represented the mixed solution group of OVA and aescin.

Example 2

The preparation method of microemulsion (NE) is as follows: 0.2g of egg yolk lecithin is dissolved in 0.5g of squalene, 0.2g of Tween 80 is dissolved in water for injection, and the mixture is mixed to form colostrum. The colostrum is homogenized by high pressure to form 40ml of microemulsion (NE), 312.5μl of 37.5mM aluminum sulfate aqueous solution is added, and the mixture is vortexed to obtain the microemulsion (NE).

The preparation method of microemulsion (NE-Esc) loaded with OVA and aescin is as follows: dissolve aescin in methanol to prepare a stock solution of a certain concentration, take an appropriate amount of aescin methanol solution and remove the methanol by rotary evaporation, add the above-prepared 400μl NE and 210μl Hepes buffer (100mM, pH 8.0), vortex and water bath sonicate for 3min, then add the prescribed amount of OVA solution, vortex to mix, and then add 40μl Al 2(SO4)3 solution (0.075mol/L) dropwise to obtain.

C57BL/6 mice were subcutaneously injected with 100μl of a mixture containing 20μg saponin and 10μg OVA at the base of the tail on days 0, 14, and 21, respectively. Blood was collected from the eye sockets on day 27, and the supernatant was taken after centrifugation at 8000rpm for 10min. OVA-specific antibodies IgG, IgG1, and IgG2a were determined by ELISA. The results are shown in Figure 2. Figure 2 shows that the IgG and IgG1 antibodies of the microemulsion loaded with OVA were significantly increased compared with the PBS group (**, p<0.01), while there was no significant difference in IgG2a, indicating that it mainly induces humoral immunity and has poor effects in cellular immunity. After co-loading aescin and OVA in the emulsion, IgG and IgG1 antibodies were significantly increased compared with free OVA (****, p<0.0001), and were also significantly increased compared with the microemulsion loaded with OVA (**, p<0.01). It can be seen that aescin has the effect of inducing humoral immune response. From the results of IgG2a antibody, OVA loading into microemulsion had no significant effect on IgG2a antibody, while microemulsion co-loading OVA and aescin had a significant increase compared with OVA and microemulsion loaded with OVA (****, p<0.0001), indicating that aescin Aescin effectively improves the ability of microemulsion to induce cellular immune response. The above results show that aescin can not only synergistically improve the adjuvant efficacy of microemulsion, but also effectively promote humoral immunity and cellular immune response at the same time, especially significantly improving the immune adjuvant effect of microemulsion in cellular immunity. In the figure, PBS is the blank group, OVA is the free OVA group, NE represents the microemulsion group loaded with OVA, and NE-Esc represents the microemulsion group co-loaded with OVA and aescin.

Example 3

The preparation method of liposomes (Lip) is as follows: weigh 6 mg DOPC, 2 mg cholesterol and 1.5 mg DSPE-PEG and dissolve them in chloroform, remove the organic solvent by rotary evaporation, add 2 ml of aqueous solution containing 200 μg OVA to hydrate the film, and use probe ultrasound at 150w for 5 minutes under ice bath conditions.

The preparation method of liposomes loaded with aescin (Lip-Esc) is as follows: weigh 6 mg DOPC, 2 mg cholesterol and 1.5 mg DSPE-PEG and dissolve them in chloroform, add methanol solution containing 400 μg aescin, remove the organic solvent by rotary evaporation, add appropriate amount of water for injection to hydrate the film, ultrasonicate the probe at 150w for 5min under ice bath conditions, and add OVA aqueous solution to make its final concentration 0.1 mg/ml.

C57BL/6 mice were subcutaneously injected with 100μl Lip and Lip-Esc at the base of the tail on days 0, 14, and 21, respectively, containing 20μg saponin and 10μg OVA. Blood was collected from the eye sockets on day 27, and the supernatant was taken after centrifugation at 8000rpm for 10min. OVA-specific antibodies IgG, IgG1, and IgG2a were determined by ELISA. The results are shown in Figure 3. Figure 3 shows that compared with free OVA, the Lip group and OVA only had a certain increase in IgG (*, p<0.05), but did not increase IgG1 and IgG2a antibodies, indicating that liposome-encapsulated OVA has only a weak immunostimulatory effect and can only induce a weak humoral immune response. Compared with OVA and Lip, Lip-Esc had a significant increase in IgG, IgG1, and IgG2a. (****, p<0.0001). The above results show that loading aescin into liposomes can not only synergistically improve the level of humoral immune response, but also induce cellular immunity, and the adjuvant effect is further enhanced, and it is reflected in both humoral immune response and cellular immune response. It can be seen that aescin can effectively promote humoral immunity and cellular immunity at the same time. In Figure 3, PBS is the blank group, OVA is the free OVA group, Lip represents the liposome group with OVA added, and Lip-Esc represents the liposome group with both OVA and aescin added.

Example 4

The preparation method of OVA-loaded aluminum salt nanoparticles (ACN) is as follows: mix 80 μl Hepes solution (10 mM, pH 6.8), 75 μl chondroitin sulfate solution (10 mg/ml), and 10 μl OVA (10 mg/ml), and add 110 μl Al 2 (SO 4) 3 solution while vortexing.

The preparation method of aluminum salt nanoparticles (ACN-Esc) loaded with sodium aescinate and OVA is as follows: Mix 80 μl Hepes solution (10 mM, pH 6.8), 75 μl chondroitin sulfate solution (10 mg/ml), 10 μl OVA (10 mg/ml) and 20 μl sodium aescinate (10 mg/ml), and add 110 μl Al 2(SO 4 ) 3 solution dropwise while vortexing.

C57BL/6 mice were subcutaneously injected with 100 μl ACN and ACN-Esc at the base of the tail on days 0, 14, and 21, respectively, containing 20 μg saponin and 10 μg OVA. Blood was collected from the eye sockets on day 27, and the supernatant was collected after centrifugation at 8000 rpm for 10 min. OVA-specific antibodies IgG, IgG1, and IgG2a were measured by ELISA. The results are shown in Figure 4. Figure 4 shows that compared with the OVA group, the IgG and IgG1 antibodies of the aluminum salt nanoparticles loaded with OVA were significantly increased (**, p<0.01), while there was no significant difference in IgG2a. After sodium aescinate and OVA were co-loaded into aluminum nanoparticles, IgG and IgG1 antibodies were significantly increased compared with free OVA (****, p<0.0001), and compared with aluminum nanoparticles loaded with OVA (**, p<0.01), and its IgG2a antibody was also significantly increased compared with free OVA and aluminum nanoparticles (**, p<0.01). The above results show that after sodium aescinate is loaded into aluminum nanoparticles, its adjuvant effect will be further enhanced, which can be manifested in both humoral and cellular immunity. It can be seen that aescinate can effectively promote humoral immunity and cellular immunity at the same time, especially in humoral immunity. The immune adjuvant effect of aluminum nanoparticles is significantly improved. In Figure 4, PBS is the blank group, OVA is the free OVA group, ACN represents the aluminum nanoparticle group with OVA added, and ACN-Esc represents the aluminum nanoparticle group with OVA and aescin added at the same time.

Example 5

The preparation method of aluminum gel (Algel) for adsorbing OVA is as follows: take 20μl of aluminum gel and mix it with an appropriate amount of OVA solution to make a solution with a final concentration of 0.05mg/ml OVA.

The preparation method of aluminum gel (Algel) for adsorbing OVA and sodium aescinate is as follows: take 20μl of aluminum gel and mix it with appropriate amounts of OVA and sodium aescinate solution to prepare a solution with a final concentration of 0.05mg/ml OVA and 0.5mg/ml sodium aescinate.

C57BL/6 mice were subcutaneously injected with 100μl Algel and Algel-Esc at the base of the tail on days 0, 14, and 21, respectively, containing 50μg saponin and 5μg OVA. On day 27, blood was collected from the eye sockets and centrifuged at 8000rpm for 10min before the supernatant was taken. OVA-specific antibodies IgG, IgG1, and IgG2a were determined by ELISA. The results are shown in Figure 5. The results showed that IgG and IgG1 antibodies were significantly increased after OVA was mixed with aluminum gel (**, p<0.01), while IgG2a had no significant difference, indicating that aluminum gel can only produce humoral immune response, but lacks cellular immune response. After sodium aescinate and OVA were co-adsorbed on aluminum gel, IgG and IgG1 antibodies were significantly increased compared with free OVA (****, p<0.0001), and were also significantly increased compared with aluminum gel adsorbed with OVA (**, p<0.01). Its IgG2a antibody was also significantly increased compared with free OVA and aluminum gel adsorbed with OVA. The above results show that the adjuvant effect of sodium aescinate loaded into aluminum gel will be further enhanced, which can be manifested in both humoral and cellular immunity. It can be seen that aescinate can effectively promote humoral immunity and cellular immunity at the same time, especially in humoral immunity. The immune adjuvant effect of aluminum gel is significantly improved. In Figure 5, PBS is the blank group, OVA is the free OVA group, Algel represents the aluminum gel group adsorbing OVA, and Algel-Esc represents the aluminum gel group adsorbing OVA and aescinate at the same time.

Example 6

The preparation method of NE-Esc is as described in Example 2

The preparation method of NE-MEsc is as follows: dissolve aescin and MPLA in methanol and ethanol respectively to prepare a certain concentration of stock solution, take an appropriate amount of aescin methanol solution and MPLA ethanol solution and remove the organic solvent by rotary evaporation, add 400μl NE and 210μl Hepes buffer (100mM, pH 8.0), vortex and water bath sonicate for 3min, then add the prescribed amount of OVA solution, vortex mix, and add 40μl Al 2(SO4)3 solution (0.075mol/L) dropwise to obtain NE-MEsc.

C57BL/6 mice were subcutaneously injected with 100 μl of the above preparation at the base of the tail on days 0, 14, and 21, respectively, which contained 20 μg of saponin, 10 μg of OVA, and 5 μg of MPLA. Blood was collected from the eye sockets on day 27, and the supernatant was taken after centrifugation at 8000 rpm for 10 min. OVA-specific antibodies IgG, IgG1, and IgG2a were determined by ELISA. The results are shown in Figure 6. The results showed that the IgG, IgG1, and IgG2a antibodies of NE-MEsc were significantly increased compared with the PBS group. At the same time, after adding MPLA adjuvant, the IgG, IgG1, and IgG2a antibodies of NE-MEsc were significantly higher than those of NE-Esc (****, p<0.0001). The above results show that the combined use of aescin and MPLA can further enhance the immune enhancement effect. It can be seen that aescin and MPLA have a synergistic effect, which can greatly improve the immune effect of the immune complex. In Figure 6 , PBS was used as the blank group, NE-Esc represented the microemulsion loaded with OVA and aescin, and NE-MEsc represented the microemulsion co-loaded with OVA, aescin and MPLA.

Example 7

The preparation method of EsM, i.e., a mixed solution of sodium aescinate, MPLA and OVA, is as follows: prepare 1 mg/ml sodium aescinate aqueous solution, 1 mg/ml MPLA ethanol solution and 1 mg/ml OVA aqueous solution, mix to obtain a mixed solution, and add water for injection to 1 ml.

The preparation method of Lip-M, i.e., the MPLA-loaded liposome and OVA mixed group, is as follows: 6 mg DOPC, 2 mg cholesterol and 1.5 mg DSPE-PEG are weighed and dissolved in chloroform, an appropriate amount of ethanol solution containing MPLA is added, the organic solvent is removed by rotary evaporation, an appropriate amount of water for injection is added to hydrate the film, the probe is ultrasonicated at 150w for 5min under ice bath conditions, and an OVA aqueous solution is added to make its final concentration 0.1 mg/ml.

The preparation method of Lip-MEs, i.e., liposomes co-loaded with sodium aescinate and MPLA and a mixed group with OVA, is as follows: 6 mg DOPC, 2 mg cholesterol and 1.5 mg DSPE-PEG are weighed and dissolved in chloroform, an appropriate amount of ethanol solution containing MPLA is added, the organic solvent is removed by rotary evaporation, an appropriate amount of aqueous solution containing sodium aescinate is added to hydrate the film, the probe is ultrasonicated at 150w for 5min under ice bath conditions, and an OVA aqueous solution is added to make its final concentration 0.1 mg/ml.

C57BL/6 mice were subcutaneously injected with 100 μl of sodium aescinate 20 μg, OVA 10 μg and MPLA 5 μg at the base of the tail on days 0, 14 and 21, respectively. Blood was collected from the eye sockets on day 27, and the supernatant was collected after centrifugation at 8000 rpm for 10 min. OVA-specific antibodies IgG, IgG1 and IgG2a were determined by ELISA. The results are shown in Figure 7. The results showed that the antibody levels of IgG, IgG1 and IgG2a in Lip-MEs were significantly increased compared with the mixed solution group of free OVA, MPLA and sodium aescinate (****, p<0.0001). At the same time, after adding MPLA adjuvant, Lip-MEs significantly increased the antibody levels of IgG, IgG1 and IgG2a compared with the Lip-M group loaded with MPLA alone (*, p<0.05). The above results show that sodium aescinate can significantly enhance the cellular and humoral immune responses, and its immune enhancement effect is further enhanced after being used in combination with MPLA. It can be seen that aescinate and MPLA have a synergistic effect and can greatly enhance the immune effect of the immune complex. In Figure 7, EsM represents a mixed solution of sodium aescinate, MPLA and OVA, Lip-M represents a mixture of MPLA-loaded liposomes and OVA, and Lip-MEs represents a mixed group of MPLA-loaded liposomes and sodium aescinate and OVA.

Example 8

The preparation method of NE-Esc is as described in Example 2

The preparation method of NE-CEsc is as follows: dissolve aescin in methanol to prepare a stock solution of a certain concentration, take an appropriate amount of aescin methanol solution and remove the organic solvent by rotary evaporation, add 400μl NE and 210μl Hepes buffer (100mM, pH 8.0), vortex and ultrasonicate in a water bath for 3min, then add the prescribed amount of OVA solution, vortex to mix, add 40μl Al 2(SO4)3 solution (0.075mol/L) dropwise, and then add CpG solution dropwise under vortexing.

C57BL/6 mice were subcutaneously injected with 100 μl of the above preparation at the base of the tail on days 0, 14, and 21, respectively. The preparation contained 20 μg of saponin, 10 μg of OVA, and 1 μg of CpG. Blood was collected from the eye sockets on day 27, and the supernatant was collected after centrifugation at 8000 rpm for 10 min. The OVA-specific antibodies IgG, IgG1, and IgG2a were measured by ELISA. The results are shown in Figure 8. The results showed that the antibody levels of IgG, IgG1, and IgG2a in NE-CEsc were significantly increased compared with those in the PBS group. At the same time, after adding CpG adjuvant, the antibodies of IgG, IgG1, and IgG2a in NE-CEsc were significantly higher than those in NE-Esc (****, p<0.0001). The above results indicate that the combination of aescin and CpG The combined use can further improve the immune enhancement effect. It can be seen that aescin and CpG have a synergistic effect and can greatly improve the immune effect of the immune complex. In Figure 8, PBS is used as a blank group, NE-Esc represents the microemulsion loaded with OVA and aescin, and NE-CEsc represents the microemulsion co-loaded with OVA, aescin and CpG.

Embodiment 9

NE and NE-MEsc were prepared as described in Example 6, and NE-Esc was prepared as described in Example 2. C57BL/6 mice were subcutaneously injected with 100 μl of the above preparation at the base of the tail on days 0, 14, and 21, respectively, containing 20 μg of saponin, 10 μg of OVA, and 5 μg of MPLA. Splenocytes of blank C57BL/6 mice were obtained after lysing red blood cells on day 27, and divided into two and incubated with PBS and SIINFEKL respectively, and then treated with low and high concentrations of CFSE respectively. Then, the two cells were evenly mixed and injected into the test mice by tail vein injection. The injection volume for each mouse was 200 μl, containing 1×10 7 target cells. After 16-24 hours, the mice were killed by cervical dislocation, and spleen cells were obtained after grinding and lysing red blood cells. The killing rate was detected and calculated by flow cytometry. CTL results are shown in Figure 9. The results showed that NE had almost no killing effect on target cells, NE-Esc could improve the killing efficiency of CTL (*, p<0.05), and NE-MEsc could greatly improve the killing efficiency, which was 5.2 times and 4.3 times of NE and NE-Esc groups respectively (****, p<0.0001). The above results show that aescin can significantly induce the production of cytotoxic T cells and significantly improve the cellular immune response. After being used in combination with MPLA, its immune enhancement effect is further enhanced. It can be seen that aescin and MPLA have a synergistic effect and can greatly improve the immune effect of the immune complex. In Figure 9, NE represents the microemulsion loaded with OVA, NE-Esc represents the microemulsion co-loaded with OVA and aescin, and NE-MEsc represents the microemulsion co-loaded with OVA, aescin and MPLA.

Example 10

NE and NE-CEsc were prepared as described in Example 8, and NE-Esc was prepared as described in Example 2. C57BL/6 mice were subcutaneously injected with 100 μl of saponin 20 μg, OVA 10 μg and CpG 1 μg at the base of the tail on days 0, 14 and 21, respectively. Target cells were prepared as described in Example 8 and infused back into the mice to be tested for flow cytometry. CTL results are shown in Figure 10. The results showed that NE had almost no killing effect on target cells, NE-Esc could improve the killing efficiency of CTL (*, p<0.05), and the killing efficiency of NE-CEsc was the highest, which was significantly different from the other groups (****, p<0.0001), which was 5.4 times and 4.6 times that of the NE and NE-Esc groups, respectively. The above results show that aescin can significantly induce the production of cytotoxic T cells and significantly improve cellular immune response. When used in combination with CpG, its immune enhancement effect is further enhanced. It can be seen that aescin and CpG have a synergistic effect and can greatly improve the immune effect of the immune complex. Figure 10 In the figure, NE represents microemulsion loaded with OVA, NE-Esc represents microemulsion co-loaded with OVA and aescin, and NE-CEsc represents microemulsion co-loaded with OVA, aescin and CpG.

In summary, aescin compounds have immune adjuvant effects, while other saponins do not have immune adjuvant effects. Meanwhile, aescin and/or its salt compounds, relative to conventional aluminum adjuvants, can not only induce the body to produce humoral immunity, but also produce cellular immunity, and the immune adjuvant effect is more comprehensive. Secondly, aescin and/or its salt compounds can not only be used alone to exert immune adjuvant effects, but also can be used in conjunction with other adjuvants (such as MPLA, CpG), further enhance the immune effects of other adjuvants, i.e., produce synergistic effects with other adjuvants. In addition, aescin and/or its salt compounds are convenient to use, not only can be used as a preparation, but also can be contained in carriers of different properties and functions, and play a synergistic effect with a variety of carriers, further improve the immune adjuvant effect. It can be seen that the application has found that aescin compounds can be used as immune adjuvants, and aescin compounds are used as immunopotentiators, which can significantly enhance antigen immunogenicity and can be used as vaccine adjuvants. The results of animal experiments show that the combination of aescin and antigen can significantly increase serum IgG, IgG1 and IgG2a antibodies, and significantly induce the production of cytotoxic T cells, that is, simultaneously enhance humoral and cellular immune responses, and can be used as a vaccine adjuvant in vaccines.

The term "one embodiment", "embodiment" or "one or more embodiments" herein means that a particular feature, structure or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present application. In addition, please note that the examples of the term "in one embodiment" here do not necessarily all refer to the same embodiment.

In the description provided herein, a large number of specific details are described. However, it is understood that the embodiments of the present application can be practiced without these specific details. In some instances, well-known methods, structures and techniques are not shown in detail so as not to obscure the understanding of this description.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims (10)

The use of an aescin compound in the preparation of a vaccine adjuvant is characterized in that the aescin compound is an aescin and/or an aescin salt compound. The use according to claim 1, characterized in that the structure of the aescin salt compound is as shown in formula (I):
Wherein, R in formula (I) includes one or more of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, and R in formula (I) also includes a metal salt, and the metal salt includes any one of sodium, potassium, zinc, aluminum, iron, zirconium, calcium, manganese and magnesium. An immune complex, characterized in that the immune complex comprises an antigen and an aescin compound, wherein the aescin compound is aescin and/or an aescin salt compound. The immune complex according to claim 3, characterized in that the antigen comprises: one or more of proteins, polypeptides, nucleic acids, tumor cell lysates, viral lysates, bacterial lysates, bacterial cell membranes, mycoplasma cell membranes, viral envelopes, exosomes, bacterial antigens, tumor cell lysate antigens, tumor cell membrane vesicle antigens, tumor cell exosome antigens, and model antigen chicken ovalbumin OVA. The immune complex according to any one of claims 3 to 4, characterized in that the weight ratio of the antigen to the aescin compound is 1:0.01-100. The immune complex according to any one of claims 3 to 4, characterized in that the antigen and the aescin compound are co-loaded in a carrier, the carrier comprising one or more of organic particles, inorganic particles and bionic particles, the organic particles comprising: microspheres and microcapsules, emulsions, liposomes, metal-organic framework compounds, gels, polystyrene particles, dendritic molecular compounds and polymer particles; the inorganic particles comprising: gold nanoparticles, iron oxide particles, mesoporous silica particles, aluminum salt particles, calcium phosphate particles and calcium carbonate particles; the bionic particles comprising: cell carriers, bacterial carriers, exosomes, viral particles and virus-like particles. The immune complex according to any one of claims 3 to 4, characterized in that the antigen and the aescin compound are respectively contained in a carrier and then mixed for administration, wherein the carrier comprises one or more of organic particles, inorganic particles and bionic particles, wherein the organic particles comprise microspheres and microcapsules, emulsions, liposomes, metal-organic framework compounds, gels, polystyrene particles, dendritic molecular compounds and polymer particles; the inorganic particles comprise gold nanoparticles, iron oxide particles, mesoporous silica particles, aluminum salt particles, calcium phosphate particles and calcium carbonate particles; the bionic particles comprise cell carriers, bacterial carriers, exosomes, viral particles and virus-like particles. The immune complex according to any one of claims 3 to 4, characterized in that the immune complex further comprises an additional adjuvant, wherein the additional adjuvant comprises: one or more of one or more complexes of aluminum adjuvant, antigen-associated molecular pattern adjuvant, bacterial toxin and its derivatives, saponins, cytokines, heat shock proteins, A151, GTP-GDP, dimethyl dioctadecyl quaternary ammonium bromide DDA; The antigen-associated molecular pattern adjuvants include: Toll-like receptor agonists, RIG-I-like receptor agonists, NOD-like receptor agonists, NOD-like receptor agonists, C-type lectin receptors, and STING agonists; The Toll-like receptor agonists include: peptidoglycan, lipoteichoic acid, MPLA, imiquimod, resiquimod, CpG-ODN, bacterial flagellin, Poly I:C; the RIG-I-like receptor agonists include: 3pRNA, short double-stranded RNA; the NOD-like receptor agonists include: muramyl dipeptide MDP, N-acetylglucosamine; the C-type lectin receptors include: β-glucan, trehalose diborate; the bacterial toxins and their derivatives include: cholera toxin CT, Escherichia coli heat-labile enterotoxin LT, cholera toxin B subunit; the saponins include: QS21, tomatidine, Quil-A; the cytokines include: GM-CSF, IL-2, IL-12, IL-6, IFN-γ, lymphocyte chemotactic factor. Use of the immune complex according to any one of claims 3 to 8 in the preparation of a vaccine. A vaccine, characterized in that it comprises the immune complex described in any one of claims 3 to 8.