CN118615434A - A composite liposome adjuvant and preparation method thereof - Google Patents

Abstract

The present invention discloses a stable composite liposome adjuvant with outstanding immune effect, comprising neutral lipids, cationic lipids, TLR3 and TLR4 agonists. The composite liposome adjuvant formed by neutral lipids and cationic lipids in a suitable ratio is easier to enter cells, and activates different signal pathways at the same time, exerting a synergistic effect, thereby prompting the body to obtain a more stable and long-lasting immune response.

Description

A composite liposome adjuvant and preparation method thereof

Technical Field

The present invention relates to the field of biomedical engineering, and in particular to a composite liposome adjuvant and a preparation method thereof.

Background Art

Aluminum adjuvant is the only registered adjuvant and is widely used around the world. It mainly mediates humoral immunity but is not very effective for diseases that are mainly mediated by cell-mediated immunity.

"Liposomes" are closed vesicular structures composed of one or more lipid bilayers surrounding an aqueous core. Each lipid bilayer is composed of two lipid monolayers, each of which has a hydrophobic "tail" region and a hydrophilic "head" region. In the bilayer, the hydrophobic "tail" of the lipid monolayer points to the inside of the bilayer, and the hydrophilic "head" faces the outside of the bilayer. Liposomes can have a variety of physicochemical properties, such as size, lipid composition, surface charge, fluidity, and the number of bilayer membranes. Depending on the number of lipid bilayers, liposomes can be classified as unilamellar vesicles (UV) or multilamellar vesicles (MLV), where unilamellar vesicles are composed of a single lipid bilayer and multilamellar vesicles are composed of two or more concentric bilayers separated from each other by a water layer. Water-soluble compounds are trapped in the aqueous phase/core of the liposomes, while lipophilic compounds are trapped in the core of the lipid bilayer membrane.

Cationic liposomes have been used as vaccine adjuvants for more than 40 years. They not only present antigens and protect antigens from degradation in the body, but also induce dendritic cell maturation and enhance immune response. The positive charge on their surface is an important factor in prolonging the retention of vaccines at the injection site, increasing antigen presentation, and prolonging the time of stimulating cellular immunity in the body. However, if cells take in a large amount of cationic lipids, they will have a greater toxic effect on the cells, causing cell apoptosis or necrosis. Whether they will accumulate in cells is not yet clear and requires further research [Chemistry of Life, 2016, 36(1): 50-56].

PolyI:C is a synthetic double-stranded RNA that can induce the production of antigen-specific CD8+T cells and can be recognized by the TLR3 receptor in the inclusion body. The invention patent with the authorization announcement number CN102247595B discloses the combined use of DDA and PolyI:C as a tuberculosis subunit vaccine adjuvant. After strengthening BCG immunization, it can effectively induce a specific cellular immune response in mice and effectively reduce the pathological damage caused by Mycobacterium tuberculosis infection. However, subsequent experiments showed that the adjuvant has poor stability and is prone to flocculation, which is not conducive to final production and practical application. To this end, based on the research of predecessors, we studied a more stable and effective new immune adjuvant to enhance the protective effect of the vaccine, which is suitable for later research and development and production.

Summary of the invention

The present invention discloses a stable composite liposome adjuvant with outstanding immune effect, which includes not only neutral lipids but also cationic lipids. Although its toxicity is higher than that of neutral lipids, the inventors found that the composite liposome adjuvant formed under a suitable ratio is easier to enter cells due to the negative charge of the cell membrane, and activates different signal pathways at the same time, thereby exerting a synergistic effect. The high-quality immune composition not only has strong immune stimulation, but also has higher stability, and has the potential for in-depth research and promotion and application.

The first aspect of the present invention provides a composite liposome adjuvant, the main components of which include:

A) Lipid mixture composed of DOPC and DOTAP;

B) Cholesterol;

C) 3D-MPL and PolyI:C,

The weight ratio of DOPC to DOTAP is 1 to 9:1, for example 4:1, 6:1 or 9:1, and most preferably 9:1; the weight ratio of the lipid mixture to cholesterol is 4:1, the weight ratio of sterol to 3D-MPL is 5:1, and the weight ratio of MPL to PolyI:C is 1:0.2 to 1:30, specifically 1:0.2, 1:0.5, 1:1, 1:2, 1:5, 1:10, 1:15, 1:20, 1:25 or 1:30.

In a preferred embodiment, the weight ratio of 3D-MPL to PolyI:C is 1:1 to 1:10, for example, 1:1, 1:2, 1:5 or 1:10.

Preferably, in the final concentration of the composite liposome adjuvant, the concentration of PolyI:C is 0.1-3 mg/mL, more preferably 0.1-1 mg/mL, such as 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL or 1 mg/mL.

The second aspect of the present invention provides a method for preparing a composite liposome adjuvant, comprising the following steps:

1) preparing an organic phase and an aqueous phase: dissolving a lipid mixture containing DOPC/DOTAP, cholesterol and 3D-MPL in an organic solvent at a mass ratio of 90-100:20-30:4-5 to obtain an organic phase for standby use; preparing water for injection, physiological saline or buffered saline solution as an aqueous phase for standby use;

2) preparing a liposome solution: mixing the organic phase and the aqueous phase using a microfluidic device, removing the organic solvent, and filtering to obtain a liposome solution;

3) Preparing adjuvant stock solution: using the aqueous phase to dissolve PolyI:C and mixing it with the liposome solution to obtain a composite liposome adjuvant.

In some embodiments, step 3) can be replaced by the following method:

Preparation of liposome solution: decompress the organic phase in a water bath and rotary evaporate to obtain a lipid film; add an aqueous phase solution to the lipid film for hydration, and then obtain a concentrated liposome stock solution through microfluidization homogenization, which is further diluted to a liposome solution of the required concentration.

In some embodiments, the organic solvent is selected from acetonitrile, dimethylformamide (DMF), methanol, acetone, dimethyl sulfoxide (DMSO), ethanol, n-propanol, isopropanol, chloroform or a mixture thereof, such as an ethanol/isopropanol mixed solvent.

In a preferred embodiment, the liposome solution prepared in step 3) can be filtered 1-2 times to obtain the final liposome adjuvant. The solvent is removed by dialysis, and the final concentration of the liposome adjuvant is provided after dilution.

In some embodiments, the microfluidic process in step 3) is operated at a total flow rate of 4-6 ml/min and a flow rate ratio of organic phase: aqueous phase = 1:3-1:6 at a temperature of 20-25°C. For example, it is operated at a total flow rate of 4 ml/min and a flow rate ratio of organic phase: aqueous phase = 1:3 at a temperature of 20°C. For example, it is operated at a total flow rate of 5 ml/min and a flow rate ratio of organic phase: aqueous phase = 1:4 at a temperature of 23°C. For example, it is operated at a total flow rate of 6 ml/min and a flow rate ratio of organic phase: aqueous phase = 1:5 at a temperature of 25°C, etc.

The third aspect of the present invention provides a vaccine composition, comprising the composite liposome adjuvant prepared by the present invention and an antigen component.

In some embodiments, the antigenic component can be derived from at least one of human papillomavirus (HPV), enterovirus that causes hand, foot and mouth disease, Mycobacterium tuberculosis, herpes simplex virus (HSV), cytomegalovirus (CMV), varicella-zoster virus (VZV), respiratory syncytial virus (RSV), influenza virus, new coronavirus (SARS-CoV-2), hepatitis virus and rabies virus.

The fourth aspect of the present invention provides a method for preparing a vaccine composition comprising a composite liposome adjuvant, the steps comprising:

1) preparing an organic phase and an aqueous phase: dissolving a lipid mixture containing DOPC/DOTAP, cholesterol and 3D-MPL in an organic solvent at a mass ratio of 90-100:20-30:4-5 to obtain an organic phase for standby use; dissolving antigen components in water for injection, physiological saline or buffered saline solution to obtain an aqueous phase for standby use;

2) Preparing a liposome solution: mixing an organic phase and an aqueous phase using a microfluidic device, removing the organic solvent, and filtering to obtain a liposome solution.

3) Preparation of vaccine composition: Dissolve PolyI:C with water for injection, physiological saline or buffered saline solution, and mix with liposome solution to obtain vaccine composition solution.

In some embodiments, step 3) can be replaced by the following steps:

Preparation of liposomes: The organic phase is decompressed and rotary evaporated in a water bath to obtain a lipid film; an aqueous phase is added to the lipid film for hydration, and then a concentrated liposome stock solution is obtained by microfluidization homogenization, which is further diluted to a liposome solution of the required concentration.

In some embodiments, the organic solvent is selected from acetonitrile, dimethylformamide (DMF), methanol, acetone, dimethyl sulfoxide (DMSO), ethanol, n-propanol, isopropanol, or a mixture of two thereof, such as an ethanol/isopropanol mixed solvent.

In a preferred embodiment, the liposome solution prepared in step 3) can be filtered 1-2 times to obtain the final liposome adjuvant. The solvent is removed by dialysis, and the liposome adjuvant is diluted to provide the final concentration.

In some embodiments, the microfluidic process in step 3) is operated at a total flow rate of 4-6 ml/min and a flow rate ratio of organic phase: aqueous phase = 1:3-1:6 at a temperature of 20-25°C. For example, it is operated at a total flow rate of 4 ml/min and a flow rate ratio of organic phase: aqueous phase = 1:3 at a temperature of 20°C. For example, it is operated at a total flow rate of 5 ml/min and a flow rate ratio of organic phase: aqueous phase = 1:4 at a temperature of 23°C. For example, it is operated at a total flow rate of 6 ml/min and a flow rate ratio of organic phase: aqueous phase = 1:5 at a temperature of 25°C, etc.

In some embodiments, the antigen components are derived from at least one of human papillomavirus (HPV), enterovirus that causes hand, foot and mouth disease, Mycobacterium tuberculosis, herpes simplex virus (HSV), cytomegalovirus (CMV), varicella zoster virus (VZV), respiratory syncytial virus (RSV), influenza virus, new coronavirus (SARS-CoV-2), hepatitis virus and rabies virus. In some embodiments, the antigen substances are preferably recombinant protein-type antigens.

In a preferred embodiment, the cationic lipid selected in the composite liposome adjuvant of the present invention is DOTAP (1,2-dioleoyl-3-trimethylammonium-propane). The combination of DOTAP and neutral lipids (such as DOPC) is more stable than the lipid combination of DOPC and DDA. The main advantage is that the combination of DOTAP and neutral lipids such as DOPC is more stable, and the prepared liposome mixture can maintain a precipitation-free state for more than 2 months, which is 3-5 times more stable than the use of only neutral lipids. In particular, when the ratio of DOPC to DOTAP reaches 9:1, the solution is clearer and more stable. In addition, the liposome particle size obtained by the lipid combination of DOPC and DDA is greater than 200nm, which will have an adverse effect on the filtration and sterilization in the later stage of the process, and the composite liposome adjuvant of the present invention can obtain liposome particles with a particle size between 100-200nm, which is beneficial to the later process and immune enhancement.

The composite liposome adjuvant of the present invention contains two immunostimulants, namely TLR3 agonist PolyI:C and TLR4 agonist 3D-MPL. PolyI:C is adsorbed on the outer layer of lipids, and the immune effect is more prominent, and the particle size and uniformity are better. When used in combination with 3D-MPL, it can activate two TLR signaling pathways at the same time, and more efficiently activate the innate immune system. On the other hand, there is a position difference between the MPL present in the lipid layer and the PolyI:C adsorbed on the outer layer of lipids. 3D-MPL and PolyI:C combined in a specific ratio can better play a joint role in different signaling pathways, making the immune effect more lasting.

The antigen components of the present invention are dissolved in the inner water phase of the composite liposome adjuvant, and the immune effect is better than adding antigens after re-dissolving the liposome solution, or directly mixing the liposome solution with the antigen solution.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic diagram of the structure of a composite liposome adjuvant;

FIG2 is a schematic diagram of the structure of a vaccine composition.

DETAILED DESCRIPTION

The present application is further described in detail below with reference to the accompanying drawings and embodiments.

Definition of terms

In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to FIGS. 1 and 2 .

In the present invention, DOPC and DOTAP are used as the backbone structural components of lipids, while cholesterol is used as a lipid aid, embedded between phospholipid molecules, and regulates the structure and properties of the membrane. The immunostimulants 3D-MPL and PolyI:C exist in different positions in the liposomes. 3D-MPL is embedded in the lipid membrane, while PolyI:C exists in the external aqueous phase. In the specification of the present invention, when "one embodiment" is mentioned, it means that the specific parameters, steps, etc. described in the embodiment are at least included in one embodiment according to the present invention. Therefore, in the specification of the present invention, if terms such as "according to one embodiment of the present invention" and "in one embodiment" are used, they are not used to specifically refer to the same embodiment. If terms such as "in another embodiment", "different embodiments of the present invention", "according to another embodiment of the present invention" are used, they are not used to specifically refer to the mentioned features can only be included in specific different embodiments. It should be understood by those skilled in the art that the specific parameters, steps, etc. disclosed in one or more embodiments of the specification of the present invention can be combined in any suitable manner.

TLR agonists

The TLR3 agonist in the present invention is polyinosinic acid-polycytidylic acid (Poly I:C) or its derivatives. Poly I:C is a double-stranded RNA analogue, composed of a ploy (I) chain and a ploy (C) chain, which can simulate the dsRNA formed after viral infection, stimulate the body to produce antiviral immune response and inflammatory response, and has a good antiviral effect. Studies have found that when Poly I:C is directly used as a drug in clinical practice, it will produce certain toxicity to the body. In order to reduce its toxicity to the body and improve the ability of Poly I:C to stimulate the body to produce interferon, researchers have modified it and created a variety of Poly I:C derivatives.

Poly-ICLC is a complex formed by mixing poly I:C and poly-L-lysine and dissolving them in carboxymethyl cellulose. Studies have shown that compared with poly I:C, Poly-ICLC can increase the maximum concentration of induced IFN in mice by 5-8 times. However, Poly-ICLC is also toxic to the body. After Poly-ICLC is coated with liposomes, its side effects can be significantly reduced.

Poly I:C ₁₂ U is a mismatched double-stranded RNA that can up-regulate or down-regulate the 2,5-adenylate synthetase/RNaseL system and the P68 protein kinase system, and this effect is independent of interferon.

In some embodiments, the TLR3 agonist is selected from one or more of Poly I:C, Poly ICLC and Poly I:C ₁₂ U.

In some preferred embodiments, the TLR3 agonist is Poly I:C, which has a molecular weight between 66,000 and 1200,000 Daltons, for example, between 75,000 and 1100,000 Daltons, between 96,000 and 950,000 Daltons, between 150,000 and 550,000 Daltons, and in particular between 66,000 and 660,000 Daltons.

The TLR4 agonist in the present invention is MPL or 3D-MPL. Lipopolysaccharide (LPS) and its derivatives or fragments thereof from Gram-negative bacteria, including MPL or 3D-MPL, are TLR-4 (Toll-like receptor 4) ligands. Monophosphoryl lipid A (MPL) is a non-toxic derivative of lipopolysaccharide (LPS) of Gram-negative bacteria such as Salmonella minnesota R595. It maintains the adjuvant properties of LPS while showing reduced toxicity (Johnson et al. 1987 Rev. Infect. Dis. 9 Suppl: S512-S516). 3D-MPL is 3-O-deacylated monophosphoryl lipid A (or 3 de-O-acylated monophosphoryl lipid A). Chemically, it is a mixture of 3-deacylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. In one embodiment, the immunogenic composition of the present invention comprises 3-O-deacylated monophosphoryl lipid A (3D-MPL).

Vaccine composition

In some embodiments, the antigenic component is derived from at least one of human papillomavirus (HPV), enterovirus that causes hand, foot and mouth disease, Mycobacterium tuberculosis, herpes simplex virus (HSV), cytomegalovirus (CMV), varicella zoster virus (VZV), respiratory syncytial virus (RSV), influenza virus, new coronavirus (SARS-CoV-2), hepatitis virus and rabies virus.

In some embodiments, the antigen derived from human papillomavirus (HPV) is L1 protein and/or L2 protein of each type of HPV. In some embodiments, HPV can be low-risk HPV (e.g., HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, 89), medium-risk HPV (e.g., HPV26, 53, 66, 73, 82), or high-risk HPV (e.g., HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68).

In some preferred embodiments, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from L1 proteins and/or L2 proteins of one or more of HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58.

In a preferred embodiment, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from L1 protein and/or L2 protein of HPV types 6 and 11.

In a preferred embodiment, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from L1 protein and/or L2 protein of HPV types 16 and 18.

In a preferred embodiment, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from L1 proteins and/or L2 proteins of HPV types 6, 11, 16 and 18.

In a preferred embodiment, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from L1 proteins and/or L2 proteins of HPV types 6, 11, 16, 18, 31, 33, 45, 52 and 58.

In a preferred embodiment, the antigen derived from human papillomavirus (HPV) comprises HPV virus-like particles assembled from L1 proteins and/or L2 proteins of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59.

Enteroviruses that cause hand, foot and mouth disease mainly include Coxsackie A group 4, 5, 6, 7, 9, 10, 16, etc., group B type 2, 5 and 13, and enterovirus 71 (EV71), etc. In some embodiments, the antigens derived from these enteroviruses can be from one or any combination of the above types, and the antigens are preferably virus-like particles (VLPs) composed of VP1 protein, VP2 protein, VP3 protein and VP4 protein. VP1 protein, VP2 protein, VP3 protein and VP4 protein are produced by the decomposition of precursor protein P1 under the action of 3CD protease.

In some embodiments, the antigens derived from these enteroviruses include one or more of EV71, Coxsackie A group 6, 10, and 16 virus particles. In a preferred embodiment, the antigens derived from these enteroviruses include EV71, Coxsackie A group 6, 10, and 16 virus particles.

The protein family with strong immunogenicity of Mycobacterium tuberculosis mainly includes Esx family proteins, PE/PPE family proteins and DosR family proteins. Esx family proteins preferably include ESAT-6, CFP-10, TB9.8, TB10.3, TB10.4, TB11.0, TB12.9, etc. PE/PPE family proteins preferably include PPE17, PPE18, PPE34, PPE42,, PPE57PE-PGRS33, PE35-PPE68, PE-PGRS62, PE-PGRS17, PE-PGRS11, PE25-PPE41, etc. DosR family proteins preferably include Rv2029c, Rv2031c, Rv2627c, Rv3133c, etc.

In some embodiments, the antigen derived from Mycobacterium tuberculosis comprises at least one Esx family protein, at least one PE/PPE family protein and at least one DosR family protein. In a preferred embodiment, the antigen derived from Mycobacterium tuberculosis comprises CFP-10 protein, PE35 protein, PPE68 protein and Rv2627c protein. In a preferred embodiment, the antigen derived from Mycobacterium tuberculosis comprises a fusion protein formed by CFP-10 protein, PE35 protein, PPE68 protein and Rv2627c protein.

The antigen derived from herpes simplex virus (HSV) can be gB, gC, gD, gH, gL, gI, ICP0, ICP4, etc. from HSV-1 and/or HSV-2. In some embodiments, the antigen derived from herpes simplex virus (HSV) comprises HSV gB protein or its functional fragment. In a preferred embodiment, the antigen derived from herpes simplex virus (HSV) comprises the extracellular domain of gB protein or its functional fragment. In one embodiment, the functional fragment of the extracellular domain of gB protein comprises the fusion loop (fusion-loop) domain of gB protein. In one embodiment, the extracellular domain of gB protein comprises at least one amino acid mutation, preferably, the amino acid mutation is proline substitution. In one embodiment, the extracellular domain of the gB protein of HSV-1 comprises a proline substitution at position 406. In one embodiment, the extracellular domain of the gB protein of HSV-2 comprises a proline substitution at position 408. In one embodiment, the antigen derived from herpes simplex virus (HSV) comprises a fusion protein formed by the fusion loop domain of HSV-1 gB protein and the fusion loop domain of HSV-2 gB protein.

The antigen derived from varicella zoster virus (VZV) includes glycoproteins gB, gC, gE, gH, gI, gK, gL, etc. of the VZV virus. In a preferred embodiment, the antigen derived from varicella zoster virus (VZV) includes a truncated gE protein that lacks the carboxyl-terminal hydrophobic anchor region of the gE protein. In the examples below, the gE protein is prepared according to the method described in CN202110858776.7.

The antigen derived from influenza virus comprises inactivated influenza virus, hemagglutinin (HA protein) or neuraminidase (NA protein) of influenza virus. In a preferred embodiment, the antigen derived from influenza virus comprises hemagglutinin (HA protein) of influenza virus. In a preferred embodiment, the antigen derived from influenza virus comprises inactivated influenza virus.

The antigen derived from the novel coronavirus (SARS-CoV-2) comprises the SARS-CoV-2 spike protein (S protein), the receptor binding domain (RBD) of the spike protein, or its functionally active fragment. In some embodiments, the antigen derived from the novel coronavirus (SARS-CoV-2) is a fusion protein formed by the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or its functionally active fragment and the N-terminal domain (NTD) or its functionally active fragment. In a preferred embodiment, the fusion protein further comprises a foldon domain, an Fc domain of a human immunoglobulin, or its functionally active fragment. In a more preferred embodiment, the antigen derived from the novel coronavirus (SARS-CoV-2) comprises fusion proteins from different strains, each fusion protein being a fusion protein formed by a receptor binding domain (RBD) or its functionally active fragment, an N-terminal domain (NTD) and a foldon domain or its functionally active fragment. In some embodiments, the antigen derived from the new coronavirus (SARS-CoV-2) comprises a fusion protein derived from an immunodominant strain and a fusion protein derived from a prevalent dominant strain, wherein the immunodominant strain comprises at least one of a prototype strain and a Beta strain, and the prevalent dominant strain comprises at least one of a Delta strain and an Omicron strain.

In some embodiments, the antigen derived from the novel coronavirus (SARS-CoV-2) comprises a fusion protein formed by a S protein receptor binding region or a functionally active fragment thereof derived from an immunodominant strain and a S protein receptor binding region or a functionally active fragment thereof derived from an epidemic dominant strain, wherein the immunodominant strain comprises at least one of a prototype strain and a Beta strain, and the epidemic dominant strain comprises at least one of a Delta strain and an Omicron strain. The Omicron strain includes BA.1, BA.2, BA.3, BA.4, BA.5, BF.7, BQ.1, and XBB variants.

Human hepatitis viruses include hepatitis A, B, C, D, E, and G. In some embodiments, the antigen derived from a hepatitis virus includes hepatitis B surface antigen (HBsAg) derived from hepatitis B.

The antigen derived from rabies virus includes inactivated rabies virus or recombinant protein derived from rabies virus. The recombinant protein is derived from at least one of rabies virus G protein, N protein, M protein, P protein and L protein.

Example 1 Study on the Effect of Lipid Composition on Physical Properties of Adjuvants

To study the effect of different lipid compositions on liposome production, liposome solutions were prepared with different ratios of cationic lipids to neutral lipids.

Preparation of the organic phase: 40 g of lipid mixture prepared with different ratios of DOPC/DOTAP, 10 g of cholesterol and 2 g of MPL were dissolved in 200 mL of isopropanol and dissolved in a 50°C water bath.

Prepare the aqueous phase: Prepare phosphate buffered saline as the aqueous phase.

Preparation of liposome solution: The organic phase is dried by gradient decompression and rotary evaporation in a 50°C water bath for 2-5 hours to obtain a lipid film. Aqueous phase is added to the lipid film for hydration, and then the concentrated liposome stock solution is obtained by microfluidization homogenization, and further diluted to a liposome solution of the required concentration.

Preparation of adjuvant stock solution: 4 g PolyI:C (brand: Sigma; product number: P1530; CAS number: 42424-50-0) was dissolved in phosphate buffer (50 mM PB, containing 100 mM NaCl), and mixed with the liposome solution to obtain a composite liposome adjuvant preparation at a final concentration.

Table 1 Liposome adjuvant formulations with different lipid components and ratios

The effects of different lipid components and ratios on the liposome particle size are shown in Table 2.

Table 2 Particle size of liposome adjuvants with different formulations

The results show that when DOPC and DDA are combined, no matter how the ratio of the two is adjusted, the particle size of the obtained liposomes cannot be reduced to below 200nm; when DOPC and DOTAP are combined, when the ratio of the two reaches 4:1 or higher (6:1, 9:1), smaller liposome particles can be obtained, which can effectively improve the effects of filtering and sterilization in the later stage of the process, and the stability is good, and it can be stored in a precipitation-free state for more than 2 months, significantly exceeding Gr6 (only about 2 weeks). In addition, when the ratio of DOPC to DOTAP is 9:1, the obtained liposome solution is more transparent than that of other groups, indicating that the lipid suspension formed at this time has a more uniform texture and a smaller particle size. It can be seen that, considering the properties of the preparation alone, the optimal ratio of DOPC to DOTAP is 9:1, followed by 4:1 and 6:1.

Example 2 Study on the effect of formulation on the immune effect of liposome adjuvant

In order to study the immune effects of liposome adjuvants with different formulations, composite liposome adjuvants containing different lipid ratios and ratios of PolyI: C were prepared. Two adjuvant forms were compared at the same time, one was a liposome form and the other was an adsorbed form of aluminum adjuvant.

Preparation of liposome adjuvant:

Preparation of organic phase: 40g of lipid mixture prepared with different ratios of DOPC/DOTAP, 10g of cholesterol and 2g of MPL were dissolved in 200mL of isopropanol. Preparation of aqueous phase: Phosphate buffer was used as the aqueous phase for standby. Preparation of liposome solution: The microfluidic device was operated at a total flow rate of 4ml/min and a flow rate ratio of organic phase: aqueous phase = 1:3 at 20°C to obtain a liposome solution. Preparation of adjuvant stock solution: PolyI:C (brand: Sigma; item number: P1530; CAS number: 42424-50-0) was dissolved in phosphate buffer (50mM PB, containing 100mM NaCl), and mixed with the liposome solution to obtain a final concentration of composite liposome adjuvant stock solution.

Adding antigen: After the VZV gE antigen containing buffer is freeze-dried, it is directly added to the adjuvant stock solution, mixed and reconstituted to obtain the vaccine preparation of the final concentration.

Aluminum adjuvant preparation:

MPL was dissolved in 200 mL of isopropanol solution, polyI:C (brand: Sigma; item number: P1530; CAS number: 42424-50-0) was dissolved in phosphate buffer (50 mM PB, containing 100 mM NaCl), and polyI:C and MPL were adsorbed with aluminum adjuvant (ALHYDROGEL, CAS: 21645-51-2, manufacturer: CRODA, item number: AJV3012, batch number: 0001678865) 4 mg/ml, respectively, and then mixed to a suitable concentration for use. VZV gE antigen containing buffer was mixed with adjuvant stock solution to obtain a vaccine preparation of final concentration.

2.1 Effects of different lipid ratios on the immune effect of adjuvants

Immunogenicity studies were conducted using C57BL/6 mice as an animal model and varicella zoster virus gE protein as an antigen. Immunization experiments were conducted in groups according to the design shown in Table 3, with 5 mice in each group. The mice were first immunized with varicella vaccine on day 0 (immunization dose was 1/10 of the human dose), and then vaccinated with recombinant herpes zoster vaccine on days 35 and 49, respectively. The spleens of mice were taken 15 days after the last immunization (day 63) to isolate mouse splenic lymphocytes, and the VZV gE (1-546aa) peptide library was used as a stimulus, and the expression levels of intracellular cytokines IFN-γ and IL-2 were detected by flow cytometry.

Table 3 Adjuvants with different lipid compositions and ratios and experimental design

The test results are shown in Table 4. When isopropanol is used to dissolve different proportions of DOPC/DOTAP to prepare composite liposome adjuvants, the composite liposome adjuvants prepared by different DOPC/DOTAP ratios can achieve a good cellular immune protection effect that is not inferior to the aluminum-containing composite adjuvant. Among them, when the weight ratio of MPL to PolyI:C is 1:20, although the overall level of cytokines in Gr5 is slightly lower than that of the other groups (Gr1, Gr2, Gr4), there is no significant difference. Therefore, considering the immunogenicity, process, cost, stability and other aspects, the optimal ratio of DOPC/DOTAP is 9:1.

Table 4 Cytokine levels induced by different adjuvants

Group IL-2 ⁺ IL-2 ⁺ IFN-γ ⁺ IFN-γ ⁺ total Gr1 0.251 0.251 0.421 0.923 Gr2 0.287 0.425 0.474 1.186 Gr3 0.369 0.139 0.240 0.748 Gr4 0.341 0.203 0.462 1.006 Gr5 0.353 0.211 0.237 0.801 Gr6 0.314 0.178 0.585 1.077 Gr7 0.406 0.231 0.469 1.106 Gr8 0.312 0.120 0.545 0.977 Gr9 0.535 1.320 1.480 3.335

2.2 Effect of different polyI:C dosages on adjuvant immune effects

Immunogenicity studies were conducted using C57BL/6 mice as an animal model and varicella zoster virus gE protein as an antigen. Immunization experiments were conducted in groups according to the design shown in Table 5, with 5 mice in each group. The mice were first immunized with varicella vaccine on day 0 (immunization dose was 1/10 of the human dose), and then vaccinated with recombinant herpes zoster vaccine on days 35 and 53, respectively. The spleens of mice were taken 15 days after the last immunization (day 67) to isolate mouse splenic lymphocytes, and the VZV gE (1-546aa) peptide library was used as a stimulus, and the expression levels of intracellular cytokines IFN-γ and IL-2 were detected by flow cytometry.

Table 5 Adjuvants with different PolyI:C dosages and experimental design

The test results are shown in Table 6. Overall, the cytokine level of the composite liposome adjuvant group is better than that of the aluminum adjuvant group. When the ratio of DOPC/DOTAP is 4:1, the cytokine level of Gr1 (the weight ratio of MPL to PolyI:C is 1:2) is the highest and significantly higher than that of the other groups. The cytokine level of Gr2 is slightly higher than that of Gr3 and Gr4, but no significant difference is shown. It shows that when the content of polyI:C is between 0.2 and 2 mg/mL and the weight ratio of MPL to PolyI:C is between 1:2 and 1:20, a relatively ideal cellular immune effect can be achieved.

Table 6 Cytokine levels induced by adjuvants with different amounts of PolyI:C

Group IL-2 ⁺ IL-2 ⁺ IFN-γ ⁺ IFN-γ ⁺ total Gr1 0.475 0.468 0.594 1.537 Gr2 0.421 0.258 0.527 1.206 Gr3 0.413 0.215 0.512 1.140 Gr4 0.453 0.205 0.499 1.157 Gr5 0.406 0.231 0.469 1.106 Gr6 0.314 0.178 0.585 1.077

In addition, the ratio of DOPC to DOTAP was adjusted to 9:1, and the groups were designed again according to the contents of Table 7, and the immune experiment was performed according to the same method.

Table 7 Adjuvants and experimental design with different amounts of PolyI:C when the liposome ratio is 9:1

Group Dosage form Element Mouse dosage (1/10HD) usage Gr7 Liposomes DOPC/DOTAP(9:1),MPL,polyIC(0.2mg/ml) MPL 5μg, polyIC 10μg 1×, reconstituted antigen Gr8 Liposomes DOPC/DOTAP(9:1),MPL,polyIC(1mg/ml) MPL 5μg, polyIC 50μg 1×, reconstituted antigen Gr9 Liposomes DOPC/DOTAP(9:1),MPL,polyIC(3mg/ml) MPL 5μg, polyIC 150μg 1×, reconstituted antigen

The test results are shown in Table 8. When the ratio of DOPC/DOTAP is 9:1, Gr9 has the highest cytokine level, Gr7-Gr8 is slightly lower, but there is no significant difference between them, and slightly higher than Gr2-Gr4. Considering the differences between different test batches, it can be considered that there is no significant difference between Gr7-9 and Gr1-4 groups. This shows that when the content range of polyI:C is expanded to 0.2-2 mg/mL and the weight ratio of MPL to PolyI:C is expanded to 1:2-1:30, a relatively ideal cellular immune effect can still be achieved.

Table 8 Cytokine levels induced by adjuvants with different amounts of PolyI:C when the liposome ratio was 9:1

Group IL-2 ⁺ IL-2 ⁺ IFN-γ ⁺ IFN-γ ⁺ total Gr5 0.446 0.392 0.364 1.202 Gr6 0.559 0.366 0.415 1.340 Gr7 0.553 0.479 0.607 1.639

Based on the above test results, it can be seen that in the composite liposome adjuvant system provided in the present application, the content of polyI:C is set more flexibly, and can bring ideal results within a larger range; it can even be expected that when the content of polyI:C is further reduced (for example, 0.1 mg/mL), a certain immune effect can be obtained and the use requirements can be basically met. From the perspective of saving costs and reducing the risk of adverse reactions, the amount of PolyI:C can be reduced as much as possible, for example, 0.2-0.5 mg/mL, or even a lower 0.1 mg/mL. At this time, the preferred ratio of MPL to PolyI:C is 1:2-1:10, or even 1:1.

Example 3 Study on the influence of preparation method on the immune effect of vaccine composition

Two different methods were used to prepare vaccine compositions containing liposome adjuvants, and the immune effects were compared.

Method 1: Adjuvant as a reconstitution agent to reconstitute the antigen

Preparation of organic phase: As described in Table 7, 40 g of lipid mixture prepared with different ratios of DOPC/DOTAP, 10 g of cholesterol and 2 g of MPL were dissolved in 200 mL of isopropanol for use as the organic phase.

Prepare the aqueous phase: Phosphate buffered saline is used as the aqueous phase.

Preparation of liposome solution: Run the microfluidic device at 20°C with a total flow rate of 4 ml/min and a flow rate ratio of organic phase:aqueous phase = 1:3 to obtain a liposome solution.

Preparation of adjuvant stock solution: 4 g of PolyI:C (brand: Sigma; product number: P1530; CAS number: 42424-50-0) was dissolved in phosphate buffer (50 mM PB, containing 100 mM NaCl), and mixed with the liposome solution to obtain the final concentration of adjuvant stock solution.

Addition of antigen: After the VZV gE antigen containing buffer is freeze-dried, it is directly added to the adjuvant stock solution, mixed and reconstituted to obtain a vaccine preparation of the final concentration.

Method 2: Preparation of adjuvant and antigen together

Preparation of organic phase: As described in Table 9, 40 g of lipid mixture prepared with different ratios of DOPC/DOTAP, 10 g of cholesterol and 2 g of MPL were dissolved in 200 mL of isopropanol for use as the organic phase.

Prepare the aqueous phase: dissolve the VZV gE protein antigen in phosphate buffer and use it as the aqueous phase.

Preparation of liposome solution: Run the microfluidic device at 20°C with a total flow rate of 4 ml/min and a flow rate ratio of organic phase:aqueous phase = 1:3 to obtain a liposome solution.

Preparation of vaccine composition: 4 g of PolyI:C (brand: Sigma; product number: P1530; CAS number: 42424-50-0) was dissolved in phosphate buffer (50 mM PB, containing 100 mM NaCl), and mixed with the liposome solution to obtain a vaccine composition at a final concentration.

Table 9 Experimental design of vaccine composition prepared by different methods

Immunogenicity studies were conducted using C57BL/6 mice as an animal model and varicella zoster virus gE protein as an antigen. Immunization experiments were conducted in groups according to the design shown in Table 7, with 5 mice in each group. The mice were first immunized with varicella vaccine on day 0 (immunization dose was 1/10 of the human dose), and then vaccinated with recombinant herpes zoster vaccine on days 35 and 53, respectively. The spleens of mice were taken 15 days after the last immunization (day 67) to isolate mouse splenic lymphocytes, and the VZV gE (1-546aa) peptide library was used as a stimulus, and the expression levels of intracellular cytokines IFN-γ and IL-2 were detected by flow cytometry.

The test results are shown in Table 10. The levels of various cytokines in Gr3 are significantly higher than those in Gr1 and Gr2. Although Gr4 did not reach the level of Gr3, its various indicators also exceeded those of Gr1 and Gr2 to a certain extent. It can be seen that the vaccine composition prepared by method 2 (preparation of antigen and liposome adjuvant in one body) can produce a higher level of cellular immune response than method 1 (adjuvant as a reconstructing agent to re-dissolve antigen). It can be considered that the preparation method of the vaccine composition provided in this application is more advantageous than the traditional direct mixing method.

Table 10 Cytokine levels induced by vaccine compositions prepared by different methods

Group IL-2 ⁺ IL-2 ⁺ IFN-γ ⁺ IFN-γ ⁺ total Gr1 0.369 0.139 0.240 0.748 Gr2 0.353 0.211 0.237 0.801 Gr3 0.492 0.240 0.485 1.217 Gr4 0.373 0.224 0.292 0.889

Example 4 Preparation of HPV vaccine composition

Preparation of organic phase: 40 g of lipid mixture prepared with DOPC/DOTAP in a ratio of 9:1 (36 g of DOPC, 4 g of DOTAP), 10 g of cholesterol and 2 g of MPL were dissolved in 200 mL of isopropanol as the organic phase.

Preparation of aqueous phase: Dissolve the HPV16 L1 protein stock solution (40 μg per person) in physiological saline as the aqueous phase for later use.

Preparation of liposome solution: Run the microfluidic device at 20°C with a total flow rate of 4 ml/min and a flow rate ratio of organic phase:aqueous phase = 1:3 to obtain a liposome solution.

Preparation of vaccine composition: Dissolve PolyI:C (brand: Sigma; catalog number: P1530; CAS number: 42424-50-0) in phosphate buffer (50 mM PB, containing 100 mM NaCl), and mix with liposome solution to make the final concentration of PolyI:C the same as that of MPL, thereby obtaining a vaccine composition of the final concentration.

Example 5 Preparation of RSV vaccine composition

Preparation of organic phase: 40 g of lipid mixture prepared with DOPC/DOTAP in a ratio of 9:1 (36 g of DOPC, 4 g of DOTAP), 10 g of cholesterol and 2 g of MPL were dissolved in 200 mL of isopropanol as the organic phase.

Prepare the aqueous phase: dissolve the recombinant F protein of RSV in physiological saline (refer to Example 1 in the specification of CN202211033268.6, the dose for one person is 40 μg) as the aqueous phase for later use.

Preparation of liposome solution: Run the microfluidic device at 20°C with a total flow rate of 4 ml/min and a flow rate ratio of organic phase:aqueous phase = 1:3 to obtain a liposome solution.

Preparation of vaccine composition: Dissolve PolyI:C (brand: Sigma; catalog number: P1530; CAS number: 42424-50-0) in phosphate buffer (50 mM PB, containing 100 mM NaCl), and mix with liposome solution to make the final concentration of PolyI:C twice that of MPL, thus obtaining a vaccine composition with a final concentration.

The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims (9)

1. The composite liposome adjuvant is characterized by mainly comprising the following components:

a) A lipid mixture consisting of DOPC and DOTAP;

B) Cholesterol;

c) 3D-MPL and PolyI:C;

Wherein the weight ratio of DOPC to DOTAP is 9:1, the weight ratio of lipid to cholesterol is 4:1, the weight ratio of cholesterol to 3D-MPL is 5:1, and the weight ratio of 3D-MPL to polyI:C is 1:1-1:30.

2. The complex liposome adjuvant of claim 1, wherein the weight ratio of 3D-MPL to PolyI:c is 1:1 to 1:10.

3. The method for preparing the composite lipid adjuvant according to claim 1 or 2, comprising the steps of:

1) Preparation of organic and aqueous phases: dissolving a lipid mixture containing DOPC/DOTAP, cholesterol and 3D-MPL in an organic solvent according to a mass ratio of 90-100:20-30:4-5 to obtain an organic phase for standby; preparing water for injection, physiological saline or buffer salt solution as water phase for standby;

2) Preparation of liposome solution: mixing the organic phase and the water phase by using a microfluidic device, removing the organic solvent, and filtering to obtain a liposome solution;

3) Preparing an adjuvant stock solution: and (3) dissolving PolyI:C by using the water phase, and mixing with the liposome solution to obtain the composite liposome adjuvant.

4. A process according to claim 3, wherein step 3) is replaced by:

Preparation of liposome solution: decompressing and rotary evaporating the organic phase under a water area to obtain a lipid film; adding water phase into the lipid film for hydration, homogenizing by microjet to obtain concentrated liposome stock solution, and further diluting to obtain liposome solution with required concentration.

5. The method according to claim 3 or 4, wherein the organic solvent is selected from one or two of acetonitrile, dimethylformamide, methanol, acetone, dimethyl sulfoxide, ethanol, n-propanol, isopropanol, and chloroform.

6. A vaccine composition comprising a complex liposome adjuvant according to claim 1 or 2 and an antigenic component.

7. A method of preparing a vaccine composition as claimed in claim 6, comprising the steps of:

1) Preparation of organic and aqueous phases: dissolving a lipid mixture containing DOPC/DOTAP, cholesterol and 3D-MPL in an organic solvent according to a mass ratio of 90-100:20-30:4-5 to obtain an organic phase for standby; dissolving antigen components with injectable water, physiological saline or buffer salt solution to obtain water phase;

2) Preparation of liposome solution: mixing the organic phase and the water phase by using a microfluidic device, removing the organic solvent, and filtering to obtain a liposome solution;

3) Preparing a vaccine composition: and dissolving PolyI:C with water for injection, physiological saline or buffer salt solution, and mixing with the liposome solution to obtain the vaccine composition.

8. The method of claim 7, wherein step 3) is replaced by:

Preparation of liposome solution: decompressing and rotary evaporating the organic phase under a water area to obtain a lipid film; adding water phase into the liposome to hydrate, homogenizing by microjet to obtain concentrated liposome stock solution, and further diluting to obtain liposome solution with required concentration.

9. The method according to claim 7 or 8, wherein the organic solvent is selected from one or two of acetonitrile, dimethylformamide, methanol, acetone, dimethyl sulfoxide, ethanol, n-propanol, isopropanol, and chloroform.