MPLA-QS-21-loaded composite nano adjuvant as well as preparation method and application of MPLA-QS-21-loaded composite nano adjuvant

Abstract

The invention relates to an MPLA-QS-21 loaded polymer solid lipid composite nano adjuvant as well as a preparation method and application thereof in prevention of virus infection. The MPLA-QS-21-loaded polymer lipid composite nano adjuvant is solid lipid spherical nano particles which entrap MPLA and QS-21 and are formed by taking a polylactic acid degradable polymer, cholesterol and cationic lipid as materials in an O / W mode, and an antigen is carried on the surface of the nano particles in an electrostatic adsorption mode to form a nano adjuvant vaccine. The MPLA-QS-21 loaded polymer solid lipid composite nano adjuvant vaccine can promote the activation of antigen presenting cells, quickly activate cellular immunity and humoral immunity and secrete high-level antibodies and cytokines, so that the purpose of effectively preventing virus infectious diseases is achieved, and the MPLA-QS-21 loaded polymer solid lipid composite nano adjuvant vaccine is an effective delivery system for enhancing antigen immunogenicity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology. Specifically, it relates to a preparation process for a biodegradable, solid lipid composite nanoadjuvant loaded with MPLA and QS-21, and its application in vaccines for the prevention of viral infections. Background Technology

[0002] As vaccine research deepens, increasing research indicates that inactivated vaccines have weak antigenic immunogenicity, often failing to achieve effective immune response when used alone, necessitating the addition of adjuvants to enhance immunogenicity. Currently, commonly used adjuvants in clinical practice include aluminum adjuvants, polysaccharide adjuvants, cytokine adjuvants, and saponin adjuvants. However, single adjuvants typically fail to meet immunization needs due to their low induced immune response intensity and biased immune type. Therefore, recent adjuvant development has shown a trend towards compound adjuvants. Compound adjuvants can exert synergistic effects through different pathways, modulating immune response types and significantly enhancing immune strength.

[0003] Currently, marketed adjuvants for combined vaccines include AS01, AS02, AS04, AS15, and GLA-SE. Among them, the AS01 combined adjuvant contains two independent immunostimulatory molecules with adjuvant properties: QS-21 and MPLA. QS-21 is a triterpenoid saponin obtained by isolating and purifying saponin from the bark of the South American soapberry tree, which can effectively enhance antigen-specific antibodies. MPLA is a detoxification derivative of Salmonella endotoxin lipopolysaccharide, capable of activating Toll-like receptor 4 (TLR4), thereby activating antigen-presenting cells and producing cytokines to induce cellular immunity. Studies have shown that the AS01 combined adjuvant can increase the number of dendritic cells (DCs) and monocytes in draining lymph nodes, significantly increase the expression level of the T cell stimulating molecule CD40, and enhance cellular immune responses. Currently, two vaccines using AS01 as an adjuvant are marketed: the malaria vaccine RTS, used in humans, and the varicella-zoster virus glycoprotein E (gE) subunit vaccine Shingrix. However, liposomes as delivery carriers have disadvantages such as poor membrane stability, complicated preparation process, and expensive excipients. Summary of the Invention

[0004] To address at least some of the technical problems in existing technologies, this invention combines the advantages of MPLA adjuvant, QS-21 adjuvant, and biodegradable polymer nanoadjuvants. Using polylactic acid-based biodegradable polymers, cholesterol, and cationic lipids as materials, a safer and more stable novel multifunctional nanocomposite adjuvant system has been developed. This system can also efficiently carry viral antigen proteins on its surface, thus realizing a nanovaccine system that co-delivers viral antigen proteins and adjuvants. This system can effectively exert the immunogenicity of the antigen, thereby effectively promoting the body's immune response and achieving the goal of disease prevention.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In one aspect, the present invention provides a polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21, wherein the nanoadjuvant comprises MPLA, QS-21, polylactic acid biodegradable polymer, cholesterol, and cationic lipids.

[0007] In some embodiments, the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 of the present invention, wherein the polylactic acid-based biodegradable polymer is selected from at least one of polylactic acid, polylactic acid-polyglycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, and polylactic acid-polyglycolic acid copolymer.

[0008] In some embodiments, the MPLA-QS-21-loaded polymeric solid lipid composite nanoadjuvant of the present invention, wherein the cationic lipid is selected from at least one of bis(octadecyldimethylammonium bromide), trimethyl-2,3-dioleoyloxypropylammonium bromide, trimethyl-2,3-dioleenoyloxypropylammonium chloride, and 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl]cholesterol.

[0009] In some embodiments, the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 of the present invention has a particle size of 50 to 200 nm, a polydispersity index of 0.1 to 0.4, and a zeta potential of +25 to +60 mV.

[0010] In another aspect, the present invention provides a method for preparing a polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21, comprising the following steps:

[0011] (1) Dissolve MPLA, QS-21, cholesterol, polylactic acid polymers and cationic lipids in organic solvents respectively, mix them evenly to obtain a mixed solution;

[0012] (2) The mixed solution is added to the aqueous phase to prepare a nano-adjuvant loaded with MPLA-QS-21. Preferably, the nano-adjuvant loaded with MPLA-QS-21 is prepared by nano-precipitation method.

[0013] In some embodiments, the method for preparing the MPLA-QS-21-loaded polymer solid lipid composite nanoadjuvant of the present invention, wherein the cholesterol accounts for 3%-40% of the total mass percentage of the cholesterol and polylactic acid polymer, preferably, the cholesterol accounts for 5-35% of the total mass percentage of the cholesterol and biodegradable polymer, and preferably, the total concentration of the cholesterol and polylactic acid polymer in step (1) is 2-20 mg / mL.

[0014] In some embodiments, the preparation method of the MPLA-QS-21-loaded polymer solid lipid composite nanoadjuvant of the present invention, wherein the mass ratio of the cationic lipid to the biodegradable polymer is 1:2 to 1:12.

[0015] Preferably, the concentration of the cationic lipid in step (1) is 1-10 mg / mL.

[0016] In some embodiments, the preparation method of the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 according to the present invention, wherein the mass ratio of QS-21 to the biodegradable polymer is 1:10-1:100, and the mass ratio of MPLA to the biodegradable polymer is 1:10-1:100.

[0017] Preferably, the concentration of QS-21 in step (1) is 0.1-1 mg / mL;

[0018] Preferably, the concentration of MPLA in step (1) is 0.1-1 mg / mL.

[0019] In some embodiments, the method for preparing the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 according to the present invention is characterized in that, in step (1), the organic solvent is acetone and anhydrous ethanol, preferably, the acetone accounts for 20%-80% of the total volume of the solvent.

[0020] In another aspect, the present invention provides a vaccine composition comprising a polymer solid lipid composite nanoadjuvant according to any one of claims 1-9 and an antigen component, preferably wherein the nanoparticle adjuvant and the antigen component are bound together by electrostatic adsorption.

[0021] In some embodiments, preferably, the antigen includes infectious disease-associated antigens, such as at least one of herpes zoster virus glycoprotein E, the receptor-binding region of the novel coronavirus S protein, hepatitis B surface antigen, human papillomavirus L1 protein, and the model antigen Ovalbumin.

[0022] The MPLA-QS-21-loaded polymer solid lipid composite nanoadjuvant provided by this invention can significantly enhance the activation of T cells and B cells, increase antibody immunotiter, promote antibody production, and simultaneously promote cytokine secretion. This MPLA-QS-21-loaded polymer solid lipid composite nanoadjuvant can effectively enhance cellular and humoral immunity, playing a vital role in maintaining immune homeostasis. Attached Figure Description

[0023] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0024] Figure 1 The effect of cholesterol content on the prepared QS-21 loaded nanoparticles in Example 1 is shown. A is the hemolysis rate result graph, and B is the particle size and PDI result graph.

[0025] Figure 2 SEM images showing the effect of cholesterol percentage on the prepared QS-21 nanoparticles in Example 1 are shown, with A, G representing SEM images of cholesterol percentages of 0%, 10%, 20%, 30%, 40%, 50%, and 60%, respectively.

[0026] Figure 3 The diagram shows the cytotoxicity analysis of BMDCs by the QS-21 nanoparticles prepared in Example 2.

[0027] Figure 4 The figure shows the characterization results of the various nano-adjuvants prepared by the present invention. The left figure is the potential diagram and the right figure is the particle size and PDI diagram.

[0028] Figure 5 The diagram shows a comparison of the effects of pure antigen group, aluminum adjuvant group, QS-21 adjuvant group, nanoparticle group, QS-21 nanoadjuvant-loaded group, MPLA-QS-21 nanoadjuvant-loaded group and control nanoadjuvant group AS01b adjuvant group on the in vitro induction of DC maturation and activation. AD are statistical graphs of the expression levels of DC cell surface marker molecules MHCII, co-stimulatory molecules CD40, CD80 and CD86, respectively.

[0029] Figure 6 The graphs show the in vivo induced antigen-specific antibody levels in the pure antigen group, QS-21 adjuvant group, nanoparticle group, QS-21 nanoadjuvant-loaded group, MPLA-QS-21 nanoadjuvant-loaded group, and AS01b adjuvant group. A represents the IgG antibody titer, B represents the IgG1 antibody titer, C represents the IgG2a antibody titer, and D represents the IgG2a / IgG1 ratio.

[0030] Figure 7 The study demonstrated the in vivo induction of spleen cell proliferation and CD4 levels in the pure antigen group, QS-21 adjuvant group, nanoparticle group, QS-21 nanoadjuvant-loaded group, MPLA-QS-21 nanoadjuvant-loaded group, and AS01b adjuvant group. + CD8 + Statistical graph of T and B cell activation levels, Treg cell activation, and FasL expression.

[0031] Figure 8The following graphs show the in vivo induced cytokine secretion levels in the PBS group, pure antigen group, QS-21 adjuvant group, nanoparticle group, QS-21 nanoadjuvant-loaded group, MPLA-QS-21 nanoadjuvant-loaded group, and AS01b adjuvant group. A represents IL-2, B represents IL-4, C represents IL-6, D represents IFN-γ, E represents TNF-α, and F represents IFN-γ / IL-4. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] In one aspect, the present invention provides a polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21, wherein the nanoadjuvant comprises MPLA, QS-21, polylactic acid biodegradable polymer, cholesterol, and cationic lipids.

[0036] In some embodiments, according to the present invention, the polymer solid lipid nanoadjuvant loaded with MPLA and QS-21 is wherein the nanovaccine adjuvant system comprises a nanoparticle structure composed of polylactic acid-based biodegradable polymers and cholesterol, embedded with cationic lipids, and loaded with MPLA and QS-21 adjuvants.

[0037] In another aspect, the present invention provides a method for preparing a polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21, comprising the following steps:

[0038] (1) Dissolve MPLA, QS-21, cholesterol, polylactic acid polymers and cationic lipids in organic solvents respectively, mix them evenly to obtain a mixed solution;

[0039] (2) The mixed solution is added to the aqueous phase to prepare a nano-adjuvant loaded with MPLA-QS-21. Preferably, the nano-adjuvant loaded with MPLA-QS-21 is prepared by nano-precipitation method.

[0040] In some embodiments, step (1) of the present invention is to dissolve QS-21, cholesterol, biodegradable polymer and cationic lipid in an organic solvent, mix them evenly, and obtain an oil phase.

[0041] In a preferred embodiment of the present invention, the biodegradable polymer is selected from at least one of polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA), polylactic acid-polyethylene glycol copolymer (PLA-PEG), and polylactic acid-polyglycolic acid copolymer (PLGA-PEG). In one preferred embodiment, the biodegradable polymer is PLA. In another preferred embodiment, the biodegradable polymer is PLGA. The molecular weight and viscosity of PLA and PLGA are not particularly limited and can be adjusted according to actual needs. Preferably, the molecular weight of PLA is 50kDa-250kDa, for example 80kDa-120kDa; the molecular weight of PLGA is 10000Da-20000Da, preferably 13000-17000Da.

[0042] In a preferred embodiment of the present invention, the cationic lipid is selected from at least one of bis(octadecyldimethylammonium bromide) (DDAB), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), trimethyl-2,3-dioleenoyloxypropylammonium chloride (DOTMA), and 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl]cholesterol (DC-Chol). In a preferred embodiment, the cationic lipid is bis(octadecyldimethylammonium bromide).

[0043] In a preferred embodiment of the present invention, in step (1) of the preparation method of the present invention, the concentration of the cationic lipid is 1-10 mg / mL, for example, it can be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0044] In a preferred embodiment of the present invention, in the preparation method of the present invention, the mass ratio of the cationic lipid to the biodegradable polymer is 1:2-1:12, preferably 1:4-1:10, for example, it can be 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc., but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0045] In a preferred embodiment of the present invention, in the preparation method of the present invention, the mass ratio of QS-21 to the biodegradable polymer is 1:10-1:100, for example, 1:20-1:80.

[0046] In a preferred embodiment of the present invention, in step (1), the concentration of QS-21 is 0.1-1 mg / mL, for example, it can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0047] In a preferred embodiment of the present invention, in the preparation method of the present invention, the mass ratio of MPLA to the biodegradable polymer is 1:10-1:100, for example, 1:20-1:80.

[0048] In a preferred embodiment of the present invention, in step (1), the MPLA concentration is 0.1-1 mg / mL, for example, it can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0049] In a preferred embodiment of the present invention, in step (1), the total concentration of the biodegradable polymer and cholesterol is 2-20 mg / mL, for example, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, etc., but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0050] Through extensive research, the inventors have discovered that in order to obtain nano-adjuvants with good particle size dispersibility and low cytotoxicity, the amount of cholesterol added should not be too high or too low. Too high or too low cholesterol will result in the final adjuvant having excessive cytotoxicity and adverse effects. At the same time, too high cholesterol will cause the final nanoparticles to fail to maintain their normal morphology and have poor dispersibility, making them unsuitable for use as adjuvants.

[0051] In a preferred embodiment of the present invention, in the preparation method of the present invention, the percentage of cholesterol in the total mass of cholesterol and polylactic acid biodegradable polymer is 3%-40%, preferably 5-35%, for example 7-15%, such as 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 35%, etc., but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0052] In a preferred embodiment of the present invention, in step (1), the organic solvent is selected from ethanol and acetone, and the acetone accounts for 20%-80% of the total volume of the solvent, for example, it can be 20%, 30%, 40%, 50%, 60%, 70%, 80%, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0053] In a preferred embodiment of the present invention, step (2) involves adding the oil phase to the aqueous phase, preferably by nanoprecipitation to form nanoparticles. In a preferred embodiment, the curing speed is 500-600 rpm, for example, 500 rpm, 510 rpm, 520 rpm, 530 rpm, 540 rpm, 550 rpm, 560 rpm, 570 rpm, 580 rpm, 590 rpm, 600 rpm, etc.; the curing time is 0.5-5 h, for example, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, etc.; the curing temperature is 10-35℃, preferably 10-30℃, for example, 10, 15, 20, 25, 30℃, but not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0054] In a preferred embodiment, the preparation method of the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 of the present invention includes the following steps:

[0055] (a) QS-21, MPLA, cholesterol and polylactic acid biodegradable polymers and cationic lipid materials were dissolved separately in organic solvents, and the two solutions were mixed evenly to form the oil phase;

[0056] (b) The oil phase solution prepared in (a) is slowly added to the aqueous phase under stirring conditions;

[0057] (c) Continue stirring and solidifying for a period of time, then centrifuge and wash the suspension, and resuspend it in water or PBS buffer to obtain the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21.

[0058] In a preferred embodiment of the present invention, in step (b), the stirring speed is 400-800 rpm, for example, it can be 400 rpm, 450 rpm, 500 rpm, 550 rpm, 600 rpm, 650 rpm, 700 rpm, 750 rpm, 800 rpm, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0059] In a preferred embodiment of the present invention, in step (b), the rate at which the oil phase is added to the water phase is 0.05-0.3 mL / min, for example, it can be 0.05 mL / min, 0.1 mL / min, 0.15 mL / min, 0.2 mL / min, 0.25 mL / min, 0.3 mL / min, etc., but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0060] In a preferred embodiment of the present invention, in step (b), the volume of the aqueous phase is generally larger than the volume of the oil phase. In a preferred embodiment, the volume ratio of the aqueous phase to the oil phase is (5-50):1, preferably (10-50):1, and even more preferably (15-50):1, for example 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0061] In a preferred embodiment, the centrifugal washing uses an ultrafiltration tube with a molecular weight cutoff of 3-100 kD. Within this range, the nano-adjuvant can be retained while washing to rapidly remove organic solvents. The number of washing cycles is not particularly limited and can be 1-5 times, for example, 1 time, 2 times, 3 times, 4 times, 5 times, etc.

[0062] In a preferred embodiment, the pH of the PBS buffer is 8.2-9.3, for example, it can be 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0063] Nano adjuvants

[0064] In one aspect, the present invention provides a polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21, which is prepared by the preparation method described in the present invention. Preferably, the nanoadjuvant has a particle size of 50-200 nm, a dispersion coefficient (PDI value) range of 0.05-0.4, is positively charged, and has a zeta potential range of +25 mV to +60 mV.

[0065] In a preferred embodiment of the present invention, the nano-adjuvant is spherical with a smooth surface, good dispersibility, uniform distribution, and a positively charged surface, capable of adsorbing negatively charged viral proteins to prepare a nano-adjuvant vaccine. The nanoparticles of the present invention have an activating effect on antigen-presenting cells, enabling effective prevention against infectious disease-related viruses.

[0066] In a preferred embodiment of the present invention, the particle size of the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 is 50-200 nm, for example, it can be 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, etc., but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0067] When the particle size of the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 is within the aforementioned range, it can better mimic the size of pathogens. This allows the nanoadjuvant to be easily taken up by antigen-presenting cells and transported to secondary lymphoid organs, and also to be actively transported to secondary lymphoid organs via draining lymph nodes. Furthermore, particles smaller than 200 nm are more prone to lysosomal escape, promoting cross-presentation and thus inducing a rapid and sustained immune response.

[0068] In a preferred embodiment, the dispersion coefficient of the MPLA-QS-21-loaded polymer solid lipid composite nanoadjuvant ranges from 0.05 to 0.4, for example, it can be 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable. When the dispersion coefficient of the MPLA-QS-21-loaded polymer solid lipid composite nanoadjuvant is as described above, the nanoparticles have a better morphology and the system is more stable, which is beneficial for promoting cellular uptake and thus generating an immune response.

[0069] In a preferred embodiment, the potential of the MPLA-QS-21-loaded polymer solid lipid composite nanoadjuvant is from +25mV to +60mV, for example, it can be +25mV, +30mV, +35mV, +40mV, +45mV, +50mV, +55mV, +60mV, but is not limited to the listed values. Other unlisted values ​​within this range are also applicable. When the potential of the MPLA-QS-21-loaded polymer solid lipid composite nanoadjuvant is at the above values, it can better adsorb negatively charged antigen proteins, making the prepared QS-21-loaded polymer nanoparticle vaccine more stable.

[0070] In a preferred embodiment, the nanoparticles of the present invention are of stable quality, which can not only effectively reduce the hemolytic activity of QS-21, but also enhance the immunogenicity of the antigen.

[0071] In a preferred embodiment, the nanoparticles of the present invention can induce CD4 in vivo. + T cells, CD8 + It can inhibit the expression of FasL ligands on T cells and the surface of T cells, and at the same time inhibit the activation level of Treg cells.

[0072] vaccine composition

[0073] In one aspect, the present invention also provides a nanoadjuvant vaccine or vaccine composition comprising a polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 as described above and an antigen component, preferably wherein the nanoadjuvant and the antigen component are bound together by electrostatic adsorption.

[0074] In this invention, the antigens include, but are not limited to, herpes zoster virus glycoprotein E (gE), novel coronavirus S protein receptor binding domain (RBD), hepatitis B surface antigen (HBsAg), human papillomavirus (HPV) L1 protein, and model antigen Ovalbumin (OVA).

[0075] In a preferred embodiment of the vaccine composition, the particle size of the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 is 80-200 nm, for example, it can be 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, etc., but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0076] In a preferred embodiment of the vaccine composition, the dispersion coefficient of the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 is in the range of 0.1-0.4, for example, it can be 0.1, 0.2, 0.3, 0.4, etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0077] In a preferred embodiment of the vaccine composition, the potential of the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 is from +25mV to +50mV, for example, it can be +25mV, +30mV, +35mV, +40mV, +45mV, +50mV, etc., but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0078] All reagent kit materials used in the embodiments of the present invention are commercially available. For devices, conditions (temperature, time, etc.), substances, dosages, methods, etc. not specifically described in the present invention, those known in the art or those who are skilled in the art can determine them according to conventional techniques.

[0079] Example 1

[0080] The following shows the effect of cholesterol content on polymer solid lipid composite nanoadjuvants.

[0081] 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, and 4 mg of PLGA (purchased from Beijing Huanotai Biomedical Technology Co., Ltd.) with a molecular weight of 15000 Da were dissolved in 1 mL of acetone and anhydrous ethanol (1:1, v / v) with 0 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, and 6 mg of CHO-HP (purchased from Shanghai Aivito Pharmaceutical Technology Co., Ltd.), respectively, to obtain 10 mg / mL CHO-HP at a concentration of 10 mg / mL of CHO-HP and PLGA. Solutions with total GA concentrations of 0%, 10%, 20%, 30%, 40%, 50%, and 60% were prepared. 10 mg of DOTAP (purchased from Shanghai Aivito Pharmaceutical Technology Co., Ltd.) was dissolved in 2 mL of acetone and anhydrous ethanol (1:1, v / v) to obtain a 5 mg / mL DOTAP solution. 3 mg of QS-21 (purchased from Beijing Huanotai Biomedical Technology Co., Ltd.) was dissolved in 20 mL of acetone and anhydrous ethanol (1:1, v / v) to obtain a 0.15 mg / mL QS-21 solution. 100 μL of CHO-HP and PLGA solution, 50 μL of DOTAP solution, and 100 μL of QS-21 solution were used as the oil phase. After mixing, the mixture was slowly added to 4 mL of deionized water under magnetic stirring (700 rpm), and cured at 500 rpm for 3 h at room temperature. Finally, after washing twice with a 100kD ultrafiltration tube at 5000r / min for 10min, 1mL of ultrapure water was added to the nanoparticles, and they were stored at 4℃.

[0082] 300 μL of nanoparticles were mixed with 2 × 10 7 Red blood cells were incubated at room temperature on a suspension apparatus for 30 min, centrifuged at 800g for 6 min, and 100 μL of the supernatant was transferred to a 96-well plate. The absorbance was read at 541 nm. Free QS-21 was used as a positive control and PBS as a negative control, and the hemolysis rate was calculated. Different proportions of CHO-HP had a significant impact on the hemolysis rate. The nanoparticles without CHO-HP had the highest hemolysis rate, which was 77.6 ± 0.1%. The addition of CHO-HP effectively reduced the hemolysis rate of QS-21, but the hemolysis rate of nanoparticles prepared with different proportions of CHO-HP varied. The nanoparticles with a CHO-HP content of 40% had the lowest hemolysis rate, which was 12.3 ± 0.1%, followed by the group with a CHO-HP content of 10%, which was 15.5 ± 0.1%. Figure 1 ).

[0083] Take 1 mL of the nanoparticle suspension and add it to a quartz cuvette. Set the parameters for materials, solvent, and number of detections. Place the cuvette in the sample well for particle size analysis. The results show that the nanoparticle size is between 50-150 nm, and the nanoparticle size decreases with increasing CHO-HP content. Figure 150 μL of nanoparticle suspension was dropped onto the surface of tin foil and allowed to evaporate in a fume hood. The foil was then fixed flat onto the sample stage with conductive adhesive. Under vacuum, the sample was sputtered with gold using an ion sputtering apparatus. The sample was then placed in the sample chamber of a scanning electron microscope. Observation revealed that as the proportion of CHO-HP gradually increased, excessive aggregation between nanoparticles occurred. Furthermore, the high proportion of CHO-HP caused the nanoparticles to lose their normal morphology, resulting in numerous large sheet-like materials. Figure 2 Therefore, considering both the hemolysis rate and the morphology of the nanoparticles, the appropriate CHO-HP content is 3-40%, preferably 5-35%.

[0084] Example 2

[0085] The following demonstrates the cytotoxicity of the polymer solid lipid composite nanoadjuvant prepared in Example 1.

[0086] Male C57BL / 6 mice aged 4-6 weeks were euthanized by cervical dislocation. After separating the two leg bones and removing leg muscle tissue, culture medium was drawn into the bone marrow using a 1 mL syringe. The bone marrow was washed with RPMI 1640 medium, lysed with 3 mL of erythrocyte lysis buffer for 1 min, and centrifuged (1500 rpm, 5 min). The supernatant was discarded, and the cells were collected. The cells were washed once with 5 mL of RPMI 1640 medium under the same centrifugation conditions. The cells were resuspended in complete RPMI 1640 medium containing 10 ng / mL rmGM-CSF, 50 ng / mL IL-4, and 10% penicillin-streptomycin antibiotic solution. The cells were then evenly seeded into 24-well plates (5 × 10⁻⁶). 5 -1×10 6 (cells / well) were placed in a 37℃, 5% CO2 cell culture incubator and cultured for 48 h. 800 μL of culture medium was aspirated and discarded, and 900 μL of fresh complete RPMI 1640 culture medium was added. The cells were cultured for another 5 days. 500 μL of culture medium was aspirated and discarded, and 600 μL of fresh complete RPMI 1640 culture medium was added. The cells were cultured for another 7 days, and myeloid-derived dendritic cells (BMDCs) were collected.

[0087] The collected BMDCs were dispersed using a pipette, centrifuged at 1500 rpm for 5 min, and resuspended in 1 mL of complete RPMI 1640 medium. The cells were then cultured at 1 × 10⁻⁶ cells / mL. 5Cells were seeded at a density of [number] cells / well in 96-well plates, and the volume of each well was brought up to 150 μL with complete RPMI 1640 medium. The cell culture plates were incubated at 37°C in a cell culture incubator containing 5% CO2 for 4 h. After incubation, 50 μL of QS-21 nano-adjuvant at concentrations of 10, 30, 50, 75, 100, 200, 300, and 400 μg / mL were added sequentially, and the cell culture plates were incubated at 37°C in a cell culture incubator containing 5% CO2 for another 18 h. 20 μL of CCK-8 solution was added to each well of the cell culture plate, and the plates were incubated at 37°C in a cell culture incubator (5% CO2) for another 4 h. The absorbance of each well was then measured at 450 nm using a microplate reader. It was found that cytotoxicity gradually increased with increasing concentration of QS-21 nanoparticles. At a nanoparticle concentration of 75 μg / mL, the viability of BMDCs remained greater than 80%, demonstrating its good biocompatibility. Figure 3 ).

[0088] Example 3

[0089] The following illustrates the preparation of a polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21.

[0090] In this embodiment, MPLA-QS-21 nano-adjuvants were prepared using a nanoprecipitation method. 1 mg of cholesterol (CHO-HP, purchased from Shanghai Aivito Pharmaceutical Technology Co., Ltd.) and 9 mg of polylactic acid (PLA, purchased from Jinan Jufukai Biotechnology Co., Ltd.) were accurately weighed and dissolved in 1 mL of acetone and anhydrous ethanol (1:1, v / v) to obtain a 10 mg / mL cholesterol and PLA solution. 10 mg of bis(octadecyldimethylammonium bromide) (DDAB, purchased from Sinopharm Chemical Reagent Co., Ltd.) was weighed and dissolved in 2 mL of acetone and anhydrous ethanol (1:1, v / v) to obtain a 5 mg / mL DDAB solution. 100 μL of cholesterol and PLA solution, 40 μL of DDAB solution, 50 μL of 0.3 mg / mL QS-21 solution (purchased from Beijing Huanotai Biomedical Technology Co., Ltd.) in acetone and anhydrous ethanol (1:1, v / v) and 50 μL of 0.3 mg / mL MPLA solution (purchased from Beijing Huanotai Biomedical Technology Co., Ltd.) in acetone and anhydrous ethanol (1:1, v / v) were mixed and slowly added to 5 mL of deionized water under magnetic stirring (750 rpm). The mixture was then cured at 550 rpm for 1 h to remove the organic solvent. After washing three times with a 100 kD ultrafiltration tube at 4000 rpm for 10 min, the nano-adjuvant was harvested. The nano-adjuvant had a particle size of 141.07±0.85 nm, a PDI of 0.15±0.02, and a potential of 47.7±0.82 mV. Figure 4 ).

[0091] Example 4

[0092] The following illustrates the preparation of a vaccine loaded with MPLA-QS-21 nano-adjuvant.

[0093] Add 800 μL of the 3.75 mg / mL MPLA-QS-21 nano-adjuvant prepared in Example 1 to a 1.5 mL EP tube. Place a rotor in the centrifuge tube containing the nano-adjuvant and place it on a magnetic stirrer. Slowly add 200 μL of 0.5 mg / mL OVA antigen solution (purchased from Sigma) dropwise below the liquid surface, changing the pipette tip position continuously, taking care not to add too much antigen to the same position. After the addition is complete, place the centrifuge tube on a suspension apparatus and allow it to adsorb at room temperature for 2 hours or at 4°C for 4 hours to obtain the MPLA-QS-21 nano-vaccine adjuvant.

[0094] The obtained nano-adjuvant vaccine exhibits a spherical morphology with small and uniform particle size and a potential of approximately +35 mV. The antigen adsorption rate was determined using the subtraction method, specifically by measuring the antigen concentration in the ultrafiltration filtrate using the MicroBCA method, and then calculating the antigen adsorption rate of the nano-adjuvant vaccine to be 84%.

[0095] Example 5

[0096] The following illustrates the preparation of a vaccine loaded with MPLA-QS-21 nano-adjuvant.

[0097] Add 800 μL of the 3.75 mg / mL MPLA-QS-21 nano-adjuvant prepared in Example 3 to a 1.5 mL EP tube. Place a rotor in the centrifuge tube containing the nano-adjuvant and place it on a magnetic stirrer. Slowly add 200 μL of a 0.5 mg / mL gE antigen solution (purchased from Sinopharm Chemical Reagent Co., Ltd.) dropwise below the liquid surface, changing the pipette tip position continuously, taking care not to add too much antigen to the same position. After the addition is complete, place the centrifuge tube on a suspension apparatus and allow it to adsorb at room temperature for 2 hours or at 4°C for 4 hours to obtain the MPLA-QS-21 nano-vaccine adjuvant.

[0098] The obtained nano-adjuvant vaccine exhibits a spherical morphology with small and uniform particle size and a potential of approximately +35 mV. The antigen adsorption rate was determined using the subtraction method, specifically by measuring the antigen concentration in the filtrate after ultrafiltration using the MicroBCA method, and then calculating the antigen adsorption rate of the nano-adjuvant vaccine to be 82%.

[0099] Example 6

[0100] The following shows the in vitro induction of BMDC maturation and activation experiments of the MPLA-QS-21 nano-adjuvant vaccine.

[0101] The MPLA-QS-21 nanoadjuvant vaccine prepared in Example 4 was used to study the in vitro induction of BMDC maturation and activation. 75 μg / mL of the nanoadjuvant vaccine was weighed and suspended in RPMI 1640 complete medium (Thermo Fisher Scientific), and co-incubated with extracted BMDCs. OVA, OVA + aluminum adjuvant OVA + QS-21 adjuvant, OVA + PLA NPs, OVA + QS-21 NPs, OVA + MPLA-QS-21 NPs, and OVA + AS01b (purchased from Beijing Huanotai Biomedical Technology Co., Ltd.) vaccines were used as controls. Each dose contained 10 μg of gE antigen, 10 μg of QS-21, 10 μg of MPLA, and 3 mg / mL of nanoparticles. The mixture was cultured at 37°C and 5% CO2 for 24 h, and the expression of related molecules on the surface of BMDCs was measured by flow cytometry.

[0102] The results showed that the MPLA-QS-21 nanoadjuvant vaccine group induced the highest levels of MHC II, CD40, CD80, and CD86 expression in BMDCs. The percentages of each activated molecule to the total cell number were 1.42, 2.13, 1.32, and 1.55 times that of the pure antigen group, and 1.03, 1.43, 1.10, and 1.09 times that of the QS-21 nanoadjuvant vaccine group, respectively. Figure 5 ).

[0103] Example 7

[0104] The following shows the detection of specific antibody levels in vivo for the MPLA-QS-21 nano-adjuvant vaccine.

[0105] The MPLA-QS-21 nanoadjuvant vaccine prepared in Example 5 was used to study the secretion of specific antibodies in mice. Forty-two Balb / c female mice aged 4-6 weeks (n=6 per group) were used to evaluate the immunization effect. Immunization was performed three times via intramuscular injection into the inner thighs on days 0, 14, and 28. Each injection consisted of 100 μL of pure antigen, gE+QS-21, gE+NPs, gE+QS-21NPs, gE+MPLA-QS-21NPs, and gE+AS01b (each dose contained 10 μg of gE antigen, 10 μg of QS-21, 10 μg of MPLA, and 3 mg / mL of nanoparticles). Blood was collected from the retro-orbital venous plexus of mice on days 14, 21, 28 and 35 post-immunization. The collected orbital blood was centrifuged and serum was collected from the blood. The levels of specific IgG, IgG1 and IgG2a antibodies against gE antigen were measured by indirect ELISA. The results are expressed as log10.

[0106] The results showed that, compared with the AS01b group, the MPLA-QS-21 nano-adjuvant vaccine group could rapidly induce the production of antigen-specific IgG, IgG1, and IgG2a antibodies. Compared with the single QS-21 nano-adjuvant vaccine group, the MPLA-QS-21 nano-adjuvant vaccine group could induce higher levels of antigen-specific IgG, IgG1, and IgG2a antibodies. Furthermore, the IgG2a / IgG1 ratio indicates that the MPLA-QS-21 nano-adjuvant vaccine can better maintain immune homeostasis, showing a slight bias towards Th1-type immunity. Figure 6 ).

[0107] Example 8

[0108] The following shows a study on the effect of a vaccine loaded with MPLA-QS-21 nano-adjuvant on spleen cell proliferation.

[0109] The MPLA-QS-21 nanoparticle-loaded adjuvant vaccine prepared in Example 5 was used to study its effect on mouse spleen cell proliferation. Extracted spleen cells were cultured at 5.0 × 10⁻⁶ cells / cm². 6 Cells / well (40 μL) were seeded into 96-well plates. For in vitro stimulation, 160 μL of gE diluted with RPMI 1640 complete medium (final concentration 10 μg / mL) was added to each well. Positive (Con A stimulation), negative (cells only, no vaccine), and blank control wells (medium only) were also included. After incubation at 37°C for 48 h, 20 μL of CCK-8 solution was added to each well, and incubation continued for another 4 h, with absorbance read at 450 nm.

[0110] The results showed that, among all experimental groups, the vaccine loaded with MPLA-QS-21 nano-adjuvant induced the highest level of spleen cell proliferation, which was 1.12 times that of the pure antigen group. Figure 7 A).

[0111] Example 9

[0112] The following shows a study on the effects of MPLA-QS-21 nanovaccine adjuvant on immune cells.

[0113] The MPLA-QS-21 nanovaccine adjuvant prepared in Example 5 was used to study its effect on mouse immune cells. Forty-two 4-6 week old BALB / c female mice (n=6 per group) were used to evaluate the immunization effect. Immunization was performed three times via intramuscular injection into the inner thighs on days 0, 14, and 28. Each injection consisted of 100 μL of pure antigen, gE+QS-21, gE+NPs, gE+QS-21NPs, gE+MPLA-QS-21NPs, and gE+AS01b (each dose contained 10 μg of gE antigen, 10 μg of QS-21, 10 μg of MPLA, and 3 mg / mL of nanoparticles). Mice were sacrificed on day 35 post-primary immunization, and spleen cells were collected and stimulated in vitro with gE (10 μg / well). Single cells (1 × 10⁻⁶) were collected from the spleen. 6 (cells / well) were placed in a cell culture incubator and cultured for 60 h. After washing with PBS, the cells were stained with anti-CD3, CD4, CD8, CD19, CD69, CD178, CD25, and Foxp3 mouse antibodies. Flow cytometry was used to analyze the CD4+ of spleen cells stimulated with in vitro antigens. + T cells, CD8 + The activation of T cells, B cells, and Treg cells, as well as the expression of FasL, were detected.

[0114] The results showed that the MPLA-QS-21 nanoadjuvant vaccine group was able to induce the highest level of CD4 in vivo. + CD8 + T and B cell activation were 1.36, 1.47, and 1.58 times higher than those of the single-drug QS-21 nanoparticle adjuvant group, respectively, and 3.25, 3.30, and 2.55 times higher than those of the AS01b adjuvant group, respectively. The MPLA-QS-21 nanoparticle adjuvant-loaded vaccine group induced lower levels of CD4+ in vivo. + Treg cell activation was 0.56 times that of the single-load QS-21 nanovaccine adjuvant, comparable to the AS01b group, while exhibiting the highest levels of CD4. + FasL expression on the surface of T cells was 1.11 times that of the QS-21 nanoparticle adjuvant vaccine group and 2.03 times that of the AS01b group. Figure 7 BH).

[0115] Example 10

[0116] This example illustrates the level of cytokine secretion after immunization with a vaccine loaded with MPLA-QS-21 nano-adjuvants.

[0117] The MPLA-QS-21 nanoparticle-loaded adjuvant vaccine prepared in Example 5 was used to study its effect on mouse cytokine secretion levels (cytokine kits were purchased from Shenzhen Dakwei Biotechnology Co., Ltd.). Diluted spleen cell supernatant was added to each well of a plate, followed by biotin-labeled antibody working solution. The plate was incubated at 37°C for 90 min. After incubation, the liquid in the wells was removed, and the plate was washed four times. Streptavidin-HRP working solution was then added, and the plate was incubated at 37°C for 30 min. After incubation, the plate was washed four more times. TMB chromogenic solution was then added to each well, and the plate was incubated at 37°C for 5–30 min. The reaction was terminated by adding stop solution, and the absorbance was read at 450 nm using a microplate reader within 10 min. The secretion levels of IL-2, IL-4, IL-6, IFN-γ, and TNF-α were detected using the above method.

[0118] The results showed that the MPLA-QS-21 nano-adjuvant vaccine group induced high levels of IL-2, IL-4, IFN-γ, and TNF-α secretion, which were 1.77, 2.43, 1.96, and 1.77 times higher than those in the QS-21 nano-adjuvant-only group, and 3.02, 5.49, 1.88, and 3.56 times higher than those in the AS01b group, respectively. It also exhibited lower levels of IL-6 secretion during acute shingles flare-ups, which were 0.48 times higher than those in the AS01b adjuvant group. Figure 8 ).

[0119] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. MPLA-QS-21 loaded polymeric solid lipid nanoparticle adjuvant, characterized in that, The nano-adjuvant comprises MPLA, QS-21, a polylactic acid-based biodegradable polymer, cholesterol and a cationic lipid.

2. The MPLA-QS-21 loaded polymeric solid lipid nanoparticle adjuvant as claimed in claim 1, wherein, The polylactic acid-based biodegradable polymer is at least one selected from polylactic acid, polylactic acid-polyglycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, and polylactic acid-polyglycolic acid-polyethylene glycol copolymer.

3. The MPLA-QS-21 loaded polymeric solid lipid nano-adjuvant as claimed in claim 1, wherein, The cationic lipid is at least one selected from dioctadecyldimethylammonium bromide, trimethyl-2,3-dioleyloxypropylammonium bromide, trimethyl-2,3-dioleyloxypropylammonium chloride, and 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol.

4. The MPLA-QS-21 loaded polymeric solid lipid nano-adjuvant of claim 1, wherein, The nano-adjuvant has a particle size of 50-200 nm, a polydispersity index of 0.1-0.4, and a zeta potential of +25 to +60 mV.

5. A process for the preparation of MPLA-QS-21 loaded polymeric solid lipid nanoparticle adjuvant as claimed in any one of claims 1 to 4, wherein the process comprises the steps of: The method comprises the following steps: (1) dissolving MPLA, QS-21, cholesterol, a polylactic acid-based polymer and a cationic lipid in organic solvents respectively, mixing uniformly to obtain a mixed solution; (2) adding the mixed solution to an aqueous phase to prepare MPLA-QS-21-loaded nano-adjuvant, preferably by a nanoprecipitation method.

6. A process for the preparation of MPLA-QS-21 loaded polymeric solid lipid nanoparticle adjuvant as claimed in claim 5, wherein, The mass percentage of cholesterol in the total mass of the polylactic acid-based polymer is 3%-40%, preferably 5%-35%.

7. The method for preparing the polymer solid lipid composite nanoadjuvant loaded with MPLA-QS-21 according to claim 5, characterized in that, The mass ratio of the cationic lipid to the biodegradable polymer is 1:2-1:

12.

8. A process for the preparation of MPLA-QS-21 loaded polymeric solid lipid nanoparticle adjuvant as claimed in claim 5, wherein, The mass ratio of QS-21 to the biodegradable polymer is 1:10-1:100, and the mass ratio of MPLA to the biodegradable polymer is 1:10-1:

100. The organic solvent in step (1) is acetone and anhydrous ethanol, preferably, the acetone accounts for 20%-80% of the total volume of the solvent.

9. A process for the preparation of MPLA-QS-21 loaded polymeric solid lipid nanoparticle adjuvant as claimed in claim 5, wherein, The method comprises the polymer solid lipid nanoparticle adjuvant and an antigen component according to any one of claims 1-9, preferably, the nanoparticle adjuvant and the antigen component are combined by electrostatic adsorption, Preferably, the antigen comprises at least one of an infectious disease-related antigen, such as varicella-zoster virus glycoprotein E, novel coronavirus S protein receptor binding region, hepatitis B surface antigen, human papilloma virus L1 protein and model antigen Ovalbumin.

10. A vaccine composition, characterized in that, ​ ​

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