An immune complex, adjuvant and vaccine

Abstract

The application relates to an immune complex, an adjuvant and a vaccine and belongs to the technical field of vaccines. The immune complex comprises liposomes and quillaja saponins, the liposomes comprise cholesterols, the distribution density of the quillaja saponins on the cholesterols in the liposomes is 0.204-0.556 g / g, and the proportion of the amount of the cholesterols adsorbing the quillaja saponins in the total amount of the cholesterols is 25.7%-70%. The immune complex is applied to the preparation of vaccines, can effectively guarantee the safety of the vaccines, effectively improve the immunogenicity of the vaccines, and thus guarantees the product quality of the vaccine products.

Description

Technical Field

[0001] This application relates to the field of vaccine technology, and particularly to an immune complex, adjuvant, and vaccine. Background Technology

[0002] Saponins are commonly used in vaccines to act on antigen-presenting cells (APCs) and T cells, stimulating Th2 humoral and Th1 cell-mediated immune responses. They also activate the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, releasing caspase-1-dependent cytokines, interleukin-1β (IL-1β), and interleukin-18 (IL-18) to promote the proliferation and differentiation of immune cells, thereby enhancing the vaccine's immunogenicity. However, saponins are hemolytic. To reduce hemolytic activity and improve safety, cholesterol from liposomes is often used in vaccines to bind with saponins, thus reducing their hemolytic activity and improving vaccine safety.

[0003] However, the current simple mixed distribution of saponins and cholesterol in liposomes may have an adverse effect on the quality of vaccine products. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this application is to provide an immune complex, adjuvant, and vaccine to improve the problem that the simple mixed distribution of cholesterol in saponins and liposomes may adversely affect the quality of vaccine products.

[0005] In a first aspect, embodiments of this application provide an immune complex comprising: liposomes and saponins, wherein the liposomes contain cholesterol, and the distribution density of saponins on cholesterol in the liposomes is 0.204-0.556 g / g; the amount of cholesterol adsorbed by saponins accounts for 25.7%-70% of the total cholesterol.

[0006] This application describes an immune complex formed by controlling the distribution density of saponins on cholesterol in liposomes to be 0.204-0.556 g / g, and the proportion of cholesterol adsorbed with saponins to the total cholesterol to be 25.7%-70%. When applied to vaccine preparation, this complex can effectively ensure vaccine safety and improve vaccine immunogenicity, thereby guaranteeing the quality of the vaccine product.

[0007] Secondly, embodiments of this application provide an immune complex comprising: liposomes and saponin 21, wherein the liposomes contain cholesterol, the distribution density of saponin 21 on cholesterol in the liposomes is 0.25-0.556 g / g, and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

[0008] This application specifically uses cholesterol and saponin 21, and controls the distribution density of saponin 21 on cholesterol in liposomes to be 0.25-0.556 g / g, and the amount of cholesterol adsorbed with saponin 21 accounts for 25.7%-57.1% of the total cholesterol to form an immune complex. When applied to the preparation of vaccines, it can effectively ensure the safety of vaccines and effectively improve the immunogenicity of vaccines, thereby ensuring the quality of vaccine products.

[0009] Thirdly, embodiments of this application provide the use of an immune complex, as provided in the first or second aspect, in the preparation of adjuvants, vaccines, or pharmaceuticals.

[0010] This application, by applying the aforementioned immune complex to the preparation of adjuvants, vaccines, or drugs, can effectively ensure the safety of vaccines or drugs and effectively improve the immunogenicity of vaccines or drugs, thereby ensuring the product quality of vaccine or drug products.

[0011] Fourthly, embodiments of this application provide an adjuvant comprising an immune complex and pharmaceutical excipient composition provided in the first or second aspect.

[0012] This application uses the above-mentioned immune complex and pharmaceutical excipient composition to form an adjuvant. When this adjuvant is used in the preparation of vaccines, it can effectively ensure the safety of the vaccines and effectively improve the immunogenicity of the vaccines, thereby ensuring the quality of the vaccine products.

[0013] Fifthly, embodiments of this application provide a vaccine comprising at least one antigen and an immune complex provided in the first or second aspect or an adjuvant provided in the fourth aspect.

[0014] By using the aforementioned immune complexes or adjuvants in vaccines, this application can effectively ensure vaccine safety and improve vaccine immunogenicity, thereby guaranteeing the quality of vaccine products.

[0015] In a sixth aspect, embodiments of this application provide a recombinant protein vaccine, comprising at least one recombinant protein antigen and an immune complex provided in the first or second aspect or an adjuvant provided in the fourth aspect.

[0016] This application uses the above-mentioned immune complex or adjuvant and at least one recombinant protein antigen to form a recombinant protein vaccine, which can effectively ensure the safety of the recombinant protein vaccine and effectively improve the immunogenicity of the recombinant protein vaccine, thereby ensuring the product quality of the recombinant protein vaccine product.

[0017] In a seventh aspect, embodiments of this application provide a recombinant protein vaccine, comprising at least one recombinant F protein antigen and an immune complex provided in the first or second aspect or an adjuvant provided in the fourth aspect.

[0018] This application utilizes the aforementioned immune complex or adjuvant and at least one recombinant F protein antigen to form a recombinant protein vaccine, which can effectively ensure the safety of the recombinant protein vaccine and effectively improve its immunogenicity, thereby ensuring the product quality of the recombinant protein vaccine.

[0019] Eighthly, embodiments of this application provide a recombinant respiratory syncytial virus vaccine, comprising: recombinant respiratory syncytial virus F protein antigen and adjuvant; the distribution density of saponin 21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

[0020] This application specifically uses a recombinant respiratory syncytial virus (RSV) vaccine formed from recombinant respiratory syncytial virus (RSV) F protein antigen and adjuvants. The adjuvants include cholesterol and saponin 21, and the distribution density of saponin 21 on cholesterol is controlled to be 0.25-0.556 g / g. The amount of cholesterol adsorbed with saponin 21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant RSV vaccine has good safety and high immunogenicity.

[0021] Ninthly, embodiments of this application provide a recombinant human metapneumovirus vaccine, comprising: recombinant human metapneumovirus F protein antigen and adjuvant; the adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed with saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

[0022] This application specifically uses a recombinant human metapneumovirus vaccine formed from recombinant human metapneumovirus F protein antigen and adjuvants. The adjuvants include cholesterol and saponin 21, and the distribution density of saponin 21 on cholesterol is controlled to be 0.25-0.556 g / g. The amount of cholesterol adsorbed with saponin 21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant human metapneumovirus vaccine has good safety and high immunogenicity.

[0023] In a tenth aspect, embodiments of this application provide a recombinant parainfluenza vaccine, comprising: recombinant parainfluenza virus F protein antigen and an adjuvant; the adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

[0024] This application specifically uses a recombinant parainfluenza vaccine formed from recombinant parainfluenza virus F protein antigen and adjuvants. The adjuvants include cholesterol and saponin 21, and the distribution density of saponin 21 on cholesterol is controlled to be 0.25-0.556 g / g. The amount of cholesterol adsorbed with saponin 21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant parainfluenza vaccine has good safety and high immunogenicity.

[0025] Eleventhly, embodiments of this application provide a recombinant combined vaccine, comprising: recombinant respiratory syncytial virus F protein, recombinant human metapneumovirus F protein, and adjuvant; the adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

[0026] This application specifically employs a recombinant combined vaccine formed from recombinant respiratory syncytial virus F protein antigen, recombinant human metapneumovirus F protein antigen, and adjuvants. The adjuvants include cholesterol and saponin 21, and the distribution density of saponin 21 on cholesterol is controlled to be 0.25-0.556 g / g. The amount of cholesterol adsorbed with saponin 21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant combined vaccine has good safety and high immunogenicity.

[0027] In a twelfth aspect, embodiments of this application provide a recombinant combined vaccine, comprising: recombinant respiratory syncytial virus F protein, recombinant parainfluenza virus F protein, and an adjuvant; the adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

[0028] This application specifically employs a recombinant combined vaccine formed from recombinant respiratory syncytial virus F protein antigen, recombinant parainfluenza virus F protein antigen, and adjuvants. The adjuvants include cholesterol and saponin 21, and the distribution density of saponin 21 on cholesterol is controlled to be 0.25-0.556 g / g. The amount of cholesterol adsorbed with saponin 21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant combined vaccine has good safety and high immunogenicity.

[0029] In a thirteenth aspect, embodiments of this application provide a recombinant combined vaccine, comprising: recombinant human metapneumovirus F protein, recombinant parainfluenza virus F protein, and an adjuvant; the adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed with saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

[0030] This application specifically employs a recombinant combined vaccine formed from recombinant human metapneumovirus F protein antigen, recombinant parainfluenza virus F protein antigen, and adjuvants. The adjuvants include cholesterol and saponin 21, and the distribution density of saponin 21 on cholesterol is controlled to be 0.25-0.556 g / g. The amount of cholesterol adsorbed with saponin 21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant combined vaccine has good safety and high immunogenicity.

[0031] In a fourteenth aspect, embodiments of this application provide a recombinant combined vaccine, comprising: recombinant respiratory syncytial virus F protein, recombinant human metapneumovirus F protein, recombinant parainfluenza virus F protein, and an adjuvant; the adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

[0032] This application specifically employs a respiratory syncytial virus (RSV) vaccine formed from recombinant respiratory syncytial virus (RSV) F protein antigen, recombinant human metapneumovirus (HMV) F protein antigen, recombinant parainfluenza virus (PIV) F protein antigen, and an adjuvant. The adjuvant includes cholesterol and saponin 21, and the distribution density of saponin 21 on cholesterol is controlled to be 0.25-0.556 g / g. The amount of cholesterol adsorbed with saponin 21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant combined vaccine has good safety and high immunogenicity. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 The result diagram shows the immunogenicity of the vaccine provided in Experiment Example 1 of this application.

[0035] Figure 2 This is a graph showing the change in body weight of mice immunized with the vaccine provided in Experiment Example 1 of this application.

[0036] Figure 3 The result diagram shows the immunogenicity of the vaccine provided in Experiment Example 2 of this application.

[0037] Figure 4 This is a graph showing the change in body weight of mice immunized with the vaccine provided in Experiment Example 2 of this application. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] The study found that when saponins are mixed with liposomes, the adsorption rate of saponins on cholesterol in the liposomes is very fast, often only a few seconds. This may cause uneven distribution of saponins on cholesterol, which may have an adverse effect on the quality of vaccine products.

[0040] To address the potential issue of uneven distribution of saponins on cholesterol when mixing saponins and cholesterol in liposomes, which could adversely affect vaccine product quality, this application provides an immune complex comprising: liposomes and saponins. The liposomes contain cholesterol, and the distribution density of saponins on cholesterol in the liposomes is 0.204-0.556 g / g. The amount of cholesterol adsorbed with saponins accounts for 25.7%-70% of the total cholesterol.

[0041] This application describes an immune complex formed by controlling the distribution density of saponins on cholesterol in liposomes to be 0.204-0.556 g / g, and the proportion of cholesterol adsorbed with saponins to the total cholesterol to be 25.7%-70%. When applied to vaccine preparation, this complex can effectively ensure vaccine safety and improve vaccine immunogenicity, thereby guaranteeing the quality of the vaccine product.

[0042] In some embodiments of this application, the distribution density of saponins on cholesterol in liposomes is 0.25-0.556 g / g; the amount of cholesterol adsorbed with saponins accounts for 25.7%-57.1% of the total cholesterol. Exemplarily, the distribution density of saponins on cholesterol in liposomes may be, but is not limited to, 0.25 g / g, 0.28 g / g, 0.3 g / g, 0.323 g / g, 0.35 g / g, 0.4 g / g, 0.45 g / g, 0.5 g / g, and 0.556 g / g; the amount of cholesterol adsorbed with saponins accounts for 25.7%, 28%, 30%, 35%, 40%, 44.3%, 50%, 55%, and 57.1% of the total cholesterol. Further controlling the distribution density of saponins on cholesterol in liposomes to 0.25-0.556 g / g, and ensuring that the amount of cholesterol adsorbed by saponins accounts for 25.7%-57.1% of the total cholesterol, forms an immune complex. When applied to vaccine preparation, this complex can effectively improve the immunogenicity of the vaccine while ensuring its safety.

[0043] In some embodiments of this application, the liposomes further include dioleoylphosphatidylcholine (DOPC); the cholesterol concentration is 240-700 μg / mL; the saponin concentration is 35-100 μg / mL; and the DOPC concentration is 960-2800 μg / mL. Exemplarily, the cholesterol concentration may be, but is not limited to, 240 μg / mL, 280 μg / mL, 300 μg / mL, 340 μg / mL, 380 μg / mL, 400 μg / mL, 440 μg / mL, 480 μg / mL, 500 μg / mL, 540 μg / mL, 580 μg / mL, 600 μg / mL, 640 μg / mL, 680 μg / mL, or 700 μg / mL; and the saponin concentration may be, but is not limited to, 35 μg / mL, 40 μg / mL, 45 μg / mL, or 50 μg / mL. The concentrations of cholesterol, saponins, and DOPC can be, but are not limited to, 960 μg / mL, 1200 μg / mL, 1400 μg / mL, 1500 μg / mL, 1800 μg / mL, 2000 μg / mL, 2200 μg / mL, 2400 μg / mL, 2600 μg / mL, and 2800 μg / mL. Using appropriate concentrations of cholesterol, saponins, and DOPC facilitates the formation of immune complexes where the saponins have a suitable distribution density on cholesterol in liposomes, and the amount of cholesterol adsorbed with saponins accounts for a suitable proportion of the total cholesterol. This also ensures the safety of the vaccine when used in vaccine preparation.

[0044] In some embodiments of this application, the concentration of cholesterol is 350-550 μg / mL; the concentration of saponins is 50-100 μg / mL; and the concentration of DOPC is 1400-2200 μg / mL. Further using more suitable concentrations of cholesterol, saponins, and DOPC further facilitates the formation of immune complexes where the saponins have a suitable distribution density on cholesterol in liposomes, and the amount of cholesterol adsorbed with saponins accounts for a suitable proportion of the total cholesterol, while also ensuring vaccine safety when used in vaccine preparation.

[0045] In some embodiments of this application, the saponins are selected from saponins 21 (QS21), 7 (QS7), 17 (QS17), or 18 (QS18). By selecting saponins as QS21, QS7, QS17, or QS18, each can be adsorbed onto cholesterol to form an immune complex with a suitable distribution density of the saponins on cholesterol in the liposomes, and the amount of cholesterol adsorbed with saponins having a suitable proportion of the total cholesterol.

[0046] This application provides an immune complex comprising: liposomes and QS21, wherein the liposomes contain cholesterol, and the distribution density of QS21 on the cholesterol in the liposomes is 0.25-0.556 g / g; the amount of cholesterol adsorbed with QS21 accounts for 25.7%-57.1% of the total cholesterol.

[0047] This application specifically uses cholesterol and QS21, and controls the distribution density of QS21 on cholesterol in liposomes to be 0.25-0.556 g / g, and the amount of cholesterol adsorbed with QS21 accounts for 25.7%-57.1% of the total cholesterol to form an immune complex. When applied to the preparation of vaccines, it can effectively ensure the safety of vaccines and effectively improve the immunogenicity of vaccines, thereby ensuring the quality of vaccine products.

[0048] As an example, the distribution density of QS21 on cholesterol in liposomes may be, but is not limited to, 0.25 g / g, 0.28 g / g, 0.3 g / g, 0.323 g / g, 0.35 g / g, 0.4 g / g, 0.45 g / g, 0.5 g / g, and 0.556 g / g; the percentage of cholesterol adsorbed with QS21 in the total cholesterol is 25.7%, 28%, 30%, 35%, 40%, 44.3%, 50%, 55%, and 57.1%, respectively.

[0049] In some embodiments of this application, the immune complex further includes DOPC; the concentration of cholesterol is 240-700 μg / mL; the concentration of QS21 is 35-100 μg / mL; and the concentration of DOPC is 960-2800 μg / mL.

[0050] In some embodiments of this application, the concentration of cholesterol is 350-550 μg / mL; the concentration of QS21 is 50-100 μg / mL; and the concentration of DOPC is 1400-2200 μg / mL.

[0051] This application provides a method for preparing the above-mentioned immune complex, comprising:

[0052] (1) The above concentration of DOPC and cholesterol are mixed and dispersed to obtain lipid components. The lipid components are prepared into liposomes by any one of the following methods: ethanol injection method, thin film dispersion method, ultrasonic dispersion method, and reverse evaporation method.

[0053] (2) Add the saponins of the above concentration to the liposomes containing the above concentration of cholesterol at a speed of 1-50 mL / min, and stir at 50-300 rpm for 5-60 min to obtain immune complexes.

[0054] In some embodiments of this application, saponins can be added to liposomes in ways that are not limited to, but include, direct drop-in addition to a single pore, addition to multiple pores, addition to the liquid surface, or addition to the liquid subsurface.

[0055] In preparing immune complexes, this application controls the distribution density of saponins on cholesterol in liposomes to be 0.204-0.556 g / g by controlling the rate and method of adding appropriate concentrations of saponins to liposomes containing appropriate concentrations of cholesterol, as well as the mixing speed. The amount of cholesterol adsorbed with saponins accounts for 25.7%-70% of the total cholesterol.

[0056] This application provides a method for detecting the distribution density of saponins in the above-mentioned immune complex on cholesterol, including:

[0057] (1) Add an excess of positively charged substance to the solution containing the above immune complex and mix and adsorb it, then add an excess of negatively charged substance relative to the positively charged substance and mix and adsorb it.

[0058] (2) After mixing and adsorption, centrifuge and detect the cholesterol content in the supernatant.

[0059] The cholesterol in the supernatant is cholesterol that has not adsorbed saponins. Therefore, the amount of cholesterol that has adsorbed saponins = the total amount of cholesterol - the amount of cholesterol that has not adsorbed saponins.

[0060] It should be noted that when the ratio of saponins to cholesterol in the immune complex is not greater than 1, all saponins can be adsorbed by cholesterol.

[0061] Distribution density of saponins on cholesterol = Amount of saponins / Amount of cholesterol adsorbed by saponins.

[0062] This application involves adding an excess of a positively charged substance to a solution containing the aforementioned immune complex. Since the cholesterol adsorbed with saponins in the immune complex carries a negative charge, the excess positively charged substance can adsorb all the adsorbed cholesterol, forming an adsorption complex with a positive charge. Then, by adding an excess of a negatively charged substance relative to the positively charged substance, the positively charged adsorption complex can be adsorbed. Centrifugation and precipitation result in the complete precipitation of the cholesterol adsorbed with saponins in the immune complex, leaving only cholesterol without adsorbed saponins in the supernatant. This allows for the detection of the amount of cholesterol without adsorbed saponins in the supernatant, and the calculation of the amount of cholesterol adsorbed with saponins in the immune complex, the distribution density of saponins on cholesterol, and the proportion of cholesterol with adsorbed saponins in the total cholesterol.

[0063] This application provides an example of the use of the immune complex described above in the preparation of adjuvants, vaccines, or drugs. By applying the above-described immune complex to the preparation of adjuvants, vaccines, or drugs, the safety of the vaccines or drugs can be effectively guaranteed, and the immunogenicity of the vaccines or drugs can be effectively improved, thereby ensuring the product quality of the vaccine or drug products.

[0064] This application provides an adjuvant comprising the aforementioned immune complex and pharmaceutical excipient composition. By using the aforementioned immune complex and pharmaceutical excipient composition to form an adjuvant, its application in vaccine preparation can effectively ensure vaccine safety and improve vaccine immunogenicity, thereby guaranteeing the quality of the vaccine product.

[0065] In some embodiments of this application, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution.

[0066] In some embodiments of this application, the amount of protective agent added is 20-100 mg / mL, the amount of surfactant added is 100-500 μg / mL, the amount of buffer added is 5-10 mM, and the amount of salt added is 1.0-9.0 mg / mL. As an example, the protective agent may be, but is not limited to, at least one of sucrose, trehalose, dextran, and mannitol; the amount of protective agent added may be, but is not limited to, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL. The surfactant may be, but is not limited to, at least one of polyoxyethylene ether, polysorbate 20, and polysorbate 80; the amount of surfactant added may be, but is not limited to, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, and 500 μg / mL. The buffer may be, but is not limited to, at least one of phosphate buffer, citrate buffer, and HEPES buffer; the phosphate buffer includes sodium dihydrogen phosphate and disodium hydrogen phosphate; the amount of phosphate added may be, but is not limited to, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM. The salt may be, but is not limited to, at least one of sodium chloride and potassium chloride; the amount of salt added may be, but is not limited to, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL, 6.0 mg / mL, 7.0 mg / mL, 8.0 mg / mL, or 9.0 mg / mL. A pharmaceutical excipient composition with suitable components and concentrations, which forms an adjuvant with the aforementioned immune complex, when used in vaccine preparation, can help ensure the stability of the antigen in the vaccine and facilitate a good immunizing effect.

[0067] This application provides a vaccine comprising at least one antigen and the aforementioned immune complex or adjuvant. Using the aforementioned immune complex or adjuvant in a vaccine can effectively ensure vaccine safety and improve its immunogenicity, thereby guaranteeing the quality of the vaccine product.

[0068] In some embodiments of this application, the antigen is selected from bacteria, viruses, parasites, or genetically engineered antigens. Selecting antigens derived from bacteria, viruses, parasites, or genetically engineered antigens can, respectively, form vaccines with good safety and high immunogenicity with the aforementioned immune complexes or adjuvants.

[0069] This application provides a recombinant protein vaccine, comprising at least one recombinant protein antigen and the aforementioned immune complex or adjuvant. Using the aforementioned immune complex or adjuvant and at least one recombinant protein antigen to form a recombinant protein vaccine can effectively ensure the safety of the recombinant protein vaccine and effectively improve its immunogenicity, thereby ensuring the product quality of the recombinant protein vaccine.

[0070] This application provides a recombinant protein vaccine, comprising at least one recombinant F protein antigen and the aforementioned immune complex or adjuvant. Using the aforementioned immune complex or adjuvant and at least one recombinant F protein antigen to form a recombinant protein vaccine can effectively ensure the safety of the recombinant protein vaccine and effectively improve its immunogenicity, thereby ensuring the product quality of the recombinant protein vaccine.

[0071] In some embodiments of this application, the content of a recombinant F protein antigen is 60-480 μg / mL, calculated per milliliter of vaccine. Exemplarily, the content of a recombinant F protein antigen may be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, and 480 μg / mL. Using an appropriate concentration of recombinant F protein antigen facilitates the formation of a recombinant protein vaccine with good safety and effective immunogenicity.

[0072] In some embodiments of this application, the recombinant F protein antigen includes at least one of recombinant respiratory syncytial virus F protein antigen (RSV), recombinant human metapneumovirus F protein antigen (hMPV), and recombinant parainfluenza virus F protein antigen (PIV). Specifically, by using at least one of the recombinant F protein antigens, recombinant human metapneumovirus F protein antigen, and recombinant parainfluenza virus F protein antigen, a recombinant protein vaccine containing at least one of the above antigens can be obtained. This recombinant protein vaccine has good safety and high immunogenicity.

[0073] This application provides a recombinant respiratory syncytial virus (RSV) vaccine, comprising: recombinant RSV F protein antigen and adjuvant; the adjuvant comprises cholesterol and QS21, wherein the distribution density of QS21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbing QS21 accounts for 25.7%-57.1% of the total cholesterol.

[0074] This application specifically uses a recombinant respiratory syncytial virus (RSV) vaccine formed from recombinant respiratory syncytial virus (RSV) F protein antigen and adjuvants. The adjuvants include cholesterol and QS21, and the distribution density of QS21 on cholesterol is controlled to be 0.25-0.556 g / g. The amount of cholesterol adsorbed with QS21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant RSV vaccine has good safety and high immunogenicity.

[0075] In some embodiments of this application, the content of recombinant respiratory syncytial virus (RSV) F protein antigen, calculated per milliliter of vaccine, is 60-480 μg / mL. Exemplarily, the content of recombinant RSV F protein antigen may be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, and 480 μg / mL. Using an appropriate concentration of recombinant respiratory syncytial virus F protein antigen is beneficial for forming a recombinant respiratory syncytial virus vaccine with good safety and good immunogenicity.

[0076] In some embodiments of this application, the adjuvant further includes DOPC and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of QS21 is 35-100 μg / mL; and the concentration of DOPC is 960-2800 μg / mL. As an example, the concentrations of cholesterol, QS21, and DOPC can all be selected according to the aforementioned values. Using appropriate concentrations of cholesterol, QS21, and DOPC is beneficial for forming an adjuvant in which QS21 has a suitable distribution density on cholesterol in the recombinant respiratory syncytial virus vaccine, and the amount of cholesterol adsorbing QS21 has a suitable proportion in the total cholesterol, while also ensuring the safety of the recombinant respiratory syncytial virus vaccine.

[0077] In some embodiments of this application, the concentration of cholesterol is 350-550 μg / mL; the concentration of QS21 is 50-100 μg / mL; and the concentration of DOPC is 1400-2200 μg / mL. Further using more suitable concentrations of cholesterol, QS21, and DOPC further facilitates the formation of an adjuvant that ensures a suitable distribution density of QS21 on cholesterol in the recombinant respiratory syncytial virus vaccine, and that the amount of cholesterol adsorbing QS21 has an appropriate proportion in the total cholesterol, while also ensuring the safety of the recombinant respiratory syncytial virus vaccine.

[0078] In some embodiments of this application, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the protectant is added at an amount of 20-100 mg / mL, the surfactant at an amount of 100-500 μg / mL, the buffer solution at an amount of 5-10 mM, and the salt at an amount of 1.0-9.0 mg / mL. As an example, the components and concentrations of the protectant, surfactant, salt, and buffer solution can be selected as described above. The use of a pharmaceutical excipient composition with suitable components and concentrations, which forms an adjuvant in a recombinant respiratory syncytial virus (RSV) vaccine, can help ensure the stability of the recombinant RSV F protein antigen in the vaccine and facilitate a good immunization effect.

[0079] This application provides a recombinant human metapneumovirus vaccine, comprising: recombinant human metapneumovirus F protein antigen and an adjuvant; the adjuvant includes cholesterol and QS21, wherein the distribution density of QS21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbing QS21 accounts for 25.7%-57.1% of the total cholesterol. Specifically, the recombinant human metapneumovirus vaccine formed using recombinant human metapneumovirus F protein antigen and adjuvant, wherein the adjuvant includes cholesterol and QS21, and the distribution density of QS21 on cholesterol is controlled to be 0.25-0.556 g / g, and the amount of cholesterol adsorbing QS21 accounts for 25.7%-57.1% of the total cholesterol, exhibits good safety and high immunogenicity.

[0080] In some embodiments of this application, the content of recombinant human metapneumovirus F protein antigen, calculated per milliliter of vaccine, is 60-480 μg / mL. Exemplarily, the content of recombinant human metapneumovirus F protein antigen may be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, and 480 μg / mL. Using an appropriate concentration of recombinant human metapneumovirus F protein antigen is beneficial for forming a recombinant human metapneumovirus vaccine with good safety and good immunogenicity.

[0081] In some embodiments of this application, the adjuvant further includes DOPC and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of QS21 is 35-100 μg / mL; and the concentration of DOPC is 960-2800 μg / mL. As an example, the concentrations of cholesterol, QS21, and DOPC can all be selected according to the aforementioned values. Using appropriate concentrations of cholesterol, QS21, and DOPC is beneficial for forming an adjuvant in which QS21 has a suitable distribution density on cholesterol in the recombinant human metapneumovirus vaccine, and the amount of cholesterol adsorbing QS21 has a suitable proportion in the total cholesterol, while also ensuring the safety of the recombinant human metapneumovirus vaccine.

[0082] In some embodiments of this application, the concentration of cholesterol is 350-550 μg / mL; the concentration of QS21 is 50-100 μg / mL; and the concentration of DOPC is 1400-2200 μg / mL. Further using more suitable concentrations of cholesterol, QS21, and DOPC further facilitates the formation of an adjuvant in which QS21 has a suitable distribution density on cholesterol and the amount of cholesterol adsorbed with QS21 has an appropriate proportion in the total cholesterol, while also ensuring the safety of the recombinant human metapneumovirus vaccine.

[0083] In some embodiments of this application, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of protectant added is 20-100 mg / mL, the amount of surfactant added is 100-500 μg / mL, the amount of buffer solution added is 5-10 mM, and the amount of salt added is 1.0-9.0 mg / mL. As an example, the components and concentrations of the protectant, surfactant, salt, and buffer solution can be selected as described above. Using a pharmaceutical excipient composition with suitable components and concentrations, which is used to form an adjuvant in a recombinant human metapneumovirus vaccine, can help ensure the stability of the recombinant human metapneumovirus F protein antigen in the vaccine and facilitate a good immunization effect.

[0084] This application provides a recombinant parainfluenza vaccine, comprising: recombinant parainfluenza virus F protein antigen and adjuvant; the adjuvant comprises cholesterol and QS21, wherein the distribution density of QS21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbing QS21 accounts for 25.7%-57.1% of the total cholesterol.

[0085] This application specifically uses a recombinant parainfluenza vaccine formed from recombinant parainfluenza virus F protein antigen and adjuvants. The adjuvants include cholesterol and QS21, and the distribution density of QS21 on cholesterol is controlled to be 0.25-0.556 g / g. The amount of cholesterol adsorbed with QS21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant parainfluenza vaccine has good safety and high immunogenicity.

[0086] In some embodiments of this application, the content of recombinant parainfluenza virus F protein antigen, calculated per milliliter of vaccine, is 60-480 μg / mL. Exemplarily, the content of recombinant parainfluenza virus F protein antigen may be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, and 480 μg / mL. Using an appropriate concentration of recombinant parainfluenza virus F protein antigen facilitates the formation of a recombinant parainfluenza vaccine with good safety and effective immunogenicity.

[0087] In some embodiments of this application, the adjuvant further includes DOPC and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of QS21 is 35-100 μg / mL; and the concentration of DOPC is 960-2800 μg / mL. Exemplarily, the concentrations of cholesterol, QS21, and DOPC can all be selected according to the aforementioned values. Using appropriate concentrations of cholesterol, QS21, and DOPC facilitates the formation of an adjuvant in which QS21 has a suitable distribution density on cholesterol and the amount of cholesterol adsorbing QS21 has a suitable proportion in the total cholesterol, while also ensuring the safety of the recombinant parainfluenza vaccine.

[0088] In some embodiments of this application, the concentration of cholesterol is 350-550 μg / mL; the concentration of QS21 is 50-100 μg / mL; and the concentration of DOPC is 1400-2200 μg / mL. Further using more suitable concentrations of cholesterol, QS21, and DOPC further facilitates the formation of an adjuvant that ensures a suitable distribution density of QS21 on cholesterol in the recombinant parainfluenza vaccine, and that the amount of cholesterol adsorbed with QS21 accounts for a suitable proportion of the total cholesterol, while also ensuring the safety of the recombinant parainfluenza vaccine.

[0089] In some embodiments of this application, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of protectant added is 20-100 mg / mL, the amount of surfactant added is 100-500 μg / mL, the amount of buffer solution added is 5-10 mM, and the amount of salt added is 1.0-9.0 mg / mL. Exemplarily, the components and concentrations of the protectant, surfactant, salt, and buffer solution can be selected as described above. Using a pharmaceutical excipient composition with suitable components and concentrations, which is used to form an adjuvant in a recombinant parainfluenza vaccine, can help ensure the stability of the recombinant parainfluenza virus F protein antigen in the recombinant parainfluenza vaccine and facilitate a good immunization effect of the recombinant parainfluenza vaccine.

[0090] This application provides a recombinant combined vaccine, comprising: recombinant respiratory syncytial virus F protein, recombinant human metapneumovirus F protein, and an adjuvant; the adjuvant comprises cholesterol and QS21, wherein the distribution density of QS21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbing QS21 accounts for 25.7%-57.1% of the total cholesterol.

[0091] This application specifically employs a recombinant combined vaccine formed from recombinant respiratory syncytial virus F protein antigen, recombinant human metapneumovirus F protein antigen, and adjuvants. The adjuvants include cholesterol and QS21, and the distribution density of QS21 on cholesterol is controlled at 0.25-0.556 g / g, and the amount of cholesterol adsorbed with QS21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant combined vaccine has good safety and high immunogenicity.

[0092] In some embodiments of this application, the content of recombinant respiratory syncytial virus (RSV) F protein antigen per milliliter of vaccine is 60-480 μg / mL; the content of recombinant human metapneumovirus (HMV) F protein antigen is 60-480 μg / mL. Exemplarily, the content of recombinant RSV F protein antigen may be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, or 480 μg / mL. The concentration of recombinant human metapneumovirus F protein antigen can be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, and 480 μg / mL. Using appropriate concentrations of recombinant respiratory syncytial virus F protein antigen and recombinant human metapneumovirus F protein antigen in combination is beneficial for forming a recombinant combined vaccine with good safety and effective immunogenicity.

[0093] In some embodiments of this application, the adjuvant further includes DOPC and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of QS21 is 35-100 μg / mL; and the concentration of DOPC is 960-2800 μg / mL. As an example, the concentrations of cholesterol, QS21, and DOPC can all be selected according to the aforementioned values. Using appropriate concentrations of cholesterol, QS21, and DOPC facilitates the formation of an adjuvant in which QS21 has a suitable distribution density on cholesterol and the amount of cholesterol adsorbed with QS21 has a suitable proportion in the total cholesterol content, thus ensuring the safety of the recombinant combined vaccine containing recombinant respiratory syncytial virus F protein antigen and recombinant human metapneumovirus F protein antigen.

[0094] In some embodiments of this application, the concentration of cholesterol is 350-550 μg / mL; the concentration of QS21 is 50-100 μg / mL; and the concentration of DOPC is 1400-2200 μg / mL. Further using more suitable concentrations of cholesterol, QS21, and DOPC further facilitates the formation of an adjuvant in which QS21 has a suitable distribution density on cholesterol and the amount of cholesterol adsorbed with QS21 has a suitable proportion in the total cholesterol content, thus ensuring the safety of the recombinant combined vaccine containing recombinant respiratory syncytial virus F protein antigen and recombinant human metapneumovirus F protein antigen.

[0095] In some embodiments of this application, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of protectant added is 20-100 mg / mL, the amount of surfactant added is 100-500 μg / mL, the amount of buffer solution added is 5-10 mM, and the amount of salt added is 1.0-9.0 mg / mL. As an example, the components and concentrations of the protectant, surfactant, salt, and buffer solution can be selected as described above. Using a pharmaceutical excipient composition with suitable components and concentrations, which is used as an adjuvant in the formation of a recombinant combined vaccine containing recombinant respiratory syncytial virus F protein antigen and recombinant human metapneumovirus F protein antigen, can help ensure the stability of the antigens in the recombinant combined vaccine containing recombinant respiratory syncytial virus F protein antigen and recombinant human metapneumovirus F protein antigen, and is beneficial for the recombinant combined vaccine to obtain a good immunizing effect.

[0096] This application provides a recombinant combined vaccine, comprising: recombinant respiratory syncytial virus F protein, recombinant parainfluenza virus F protein, and an adjuvant; the adjuvant comprises cholesterol and QS21, wherein the distribution density of QS21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbing QS21 accounts for 25.7%-57.1% of the total cholesterol.

[0097] This application specifically employs a recombinant combined vaccine formed from recombinant respiratory syncytial virus F protein antigen, recombinant parainfluenza virus F protein antigen, and adjuvants. The adjuvants include cholesterol and QS21, and the distribution density of QS21 on cholesterol is controlled at 0.25-0.556 g / g, and the amount of cholesterol adsorbed with QS21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant combined vaccine has good safety and high immunogenicity.

[0098] In some embodiments of this application, the content of recombinant respiratory syncytial virus F protein antigen is 60-480 μg / mL per milliliter of vaccine; the content of recombinant parainfluenza virus F protein antigen is 60-480 μg / mL. Exemplarily, the content of recombinant respiratory syncytial virus F protein antigen may be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, or 480 μg / mL. The concentration of recombinant parainfluenza virus F protein antigen can be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, and 480 μg / mL. Using appropriate concentrations of recombinant respiratory syncytial virus F protein antigen and recombinant parainfluenza virus F protein antigen is beneficial for developing a recombinant combined vaccine with good safety and efficacy.

[0099] In some embodiments of this application, the adjuvant further includes DOPC and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of QS21 is 35-100 μg / mL; and the concentration of DOPC is 960-2800 μg / mL. Exemplarily, the concentrations of cholesterol, QS21, and DOPC can all be selected according to the aforementioned values. Using appropriate concentrations of cholesterol, QS21, and DOPC facilitates the formation of an adjuvant in which QS21 has a suitable distribution density on cholesterol and the amount of cholesterol adsorbed with QS21 has a suitable proportion in the total cholesterol content, thus ensuring the safety of the recombinant combined vaccine containing recombinant respiratory syncytial virus F protein antigen and recombinant parainfluenza virus F protein antigen.

[0100] In some embodiments of this application, the concentration of cholesterol is 350-550 μg / mL; the concentration of QS21 is 50-100 μg / mL; and the concentration of DOPC is 1400-2200 μg / mL. Further using more suitable concentrations of cholesterol, QS21, and DOPC further facilitates the formation of an adjuvant in which QS21 has a suitable distribution density on cholesterol and the amount of cholesterol adsorbed with QS21 has a suitable proportion in the total cholesterol content, thus ensuring the safety of the recombinant combined vaccine containing recombinant respiratory syncytial virus F protein antigen and recombinant parainfluenza virus F protein antigen.

[0101] In some embodiments of this application, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of protectant added is 20-100 mg / mL, the amount of surfactant added is 100-500 μg / mL, the amount of buffer solution added is 5-10 mM, and the amount of salt added is 1.0-9.0 mg / mL. Exemplarily, the components and concentrations of the protectant, surfactant, salt, and buffer solution can be selected as described above. Using a pharmaceutical excipient composition with suitable components and concentrations, which is used as an adjuvant in the formation of a recombinant combined vaccine containing recombinant respiratory syncytial virus F protein antigen and recombinant parainfluenza virus F protein antigen, can help ensure the stability of the antigens in the recombinant combined vaccine containing recombinant respiratory syncytial virus F protein antigen and recombinant parainfluenza virus F protein antigen, and is beneficial for the recombinant combined vaccine to obtain good immunization effects.

[0102] This application provides a recombinant combined vaccine, comprising: recombinant human metapneumovirus F protein, recombinant parainfluenza virus F protein, and an adjuvant; the adjuvant comprises cholesterol and QS21, wherein the distribution density of QS21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbing QS21 accounts for 25.7%-57.1% of the total cholesterol.

[0103] This application specifically uses a recombinant combined vaccine formed by recombinant human metapneumovirus F protein antigen, recombinant parainfluenza virus F protein antigen, and adjuvant. The adjuvant includes cholesterol and QS21, and the distribution density of QS21 on cholesterol is controlled to be 0.25-0.556 g / g. The amount of cholesterol adsorbed with QS21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant combined vaccine has good safety and high immunogenicity.

[0104] In some embodiments of this application, the content of recombinant human metapneumovirus F protein antigen is 60-480 μg / mL per milliliter of vaccine; the content of recombinant parainfluenza virus F protein antigen is 60-480 μg / mL. Exemplarily, the content of recombinant human metapneumovirus F protein antigen may be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, or 480 μg / mL. The concentration of recombinant parainfluenza virus F protein antigen can be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, and 480 μg / mL. Using appropriate concentrations of recombinant human metapneumovirus F protein antigen and recombinant parainfluenza virus F protein antigen is beneficial for developing a recombinant combined vaccine with good safety and efficacy.

[0105] In some embodiments of this application, the adjuvant further includes DOPC and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of QS21 is 35-100 μg / mL; and the concentration of DOPC is 960-2800 μg / mL. Exemplarily, the concentrations of cholesterol, QS21, and DOPC can all be selected according to the aforementioned values. Using appropriate concentrations of cholesterol, QS21, and DOPC is beneficial for forming the adjuvant in the recombinant combined vaccine containing recombinant human metapneumovirus F protein antigen and recombinant parainfluenza virus F protein antigen, which helps ensure the stability of the antigen in the recombinant combined vaccine and facilitates a good immunization effect of the recombinant combined vaccine.

[0106] In some embodiments of this application, the concentration of cholesterol is 350-550 μg / mL; the concentration of QS21 is 50-100 μg / mL; and the concentration of DOPC is 1400-2200 μg / mL. Further using more suitable concentrations of cholesterol, QS21, and DOPC is beneficial for forming adjuvants in recombinant combined vaccines containing recombinant human metapneumovirus F protein antigen and recombinant parainfluenza virus F protein antigen. This helps ensure the stability of the antigens in the recombinant combined vaccine and facilitates a good immunization effect.

[0107] In some embodiments of this application, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of protectant added is 20-100 mg / mL, the amount of surfactant added is 100-500 μg / mL, the amount of buffer solution added is 5-10 mM, and the amount of salt added is 1.0-9.0 mg / mL. As an example, the components and concentrations of the protectant, surfactant, salt, and buffer solution can be selected as described above. Using a pharmaceutical excipient composition with suitable components and concentrations, which is used as an adjuvant in the formation of a recombinant combined vaccine containing recombinant human metapneumovirus F protein antigen and recombinant parainfluenza virus F protein antigen, can help ensure the stability of the antigen in the recombinant combined vaccine containing recombinant human metapneumovirus F protein antigen and recombinant parainfluenza virus F protein antigen, and is beneficial for the recombinant combined vaccine to obtain a good immunizing effect.

[0108] This application provides a recombinant combined vaccine, comprising: recombinant respiratory syncytial virus F protein, recombinant human metapneumovirus F protein, recombinant parainfluenza virus F protein, and an adjuvant; the adjuvant comprises cholesterol and QS21, wherein the distribution density of QS21 on cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbing QS21 accounts for 25.7%-57.1% of the total cholesterol.

[0109] This application specifically employs a respiratory syncytial virus (RSV) vaccine formed from recombinant respiratory syncytial virus (RSV) F protein antigen, recombinant human metapneumovirus (HMV) F protein antigen, recombinant parainfluenza virus (PIV) F protein antigen, and an adjuvant. The adjuvant includes cholesterol and QS21, and the distribution density of QS21 on cholesterol is controlled at 0.25-0.556 g / g. The amount of cholesterol adsorbed with QS21 accounts for 25.7%-57.1% of the total cholesterol. This recombinant combined vaccine has good safety and high immunogenicity.

[0110] In some embodiments of this application, the content of respiratory syncytial virus F protein antigen per milliliter of vaccine is 60-480 μg / mL; the content of recombinant human metapneumovirus F protein antigen is 60-480 μg / mL; and the content of recombinant parainfluenza virus F protein antigen is 60-480 μg / mL. As an example, the content of respiratory syncytial virus F protein antigen may be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, or 480 μg / mL. The content of recombinant human metapneumovirus F protein antigen may be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, and 480 μg / mL. The concentration of recombinant parainfluenza virus F protein antigen can be, but is not limited to, 60 μg / mL, 80 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, 260 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 340 μg / mL, 360 μg / mL, 400 μg / mL, 420 μg / mL, 460 μg / mL, and 480 μg / mL. Using appropriate concentrations of recombinant respiratory syncytial virus F protein antigen, recombinant human metapneumovirus F protein antigen, and recombinant parainfluenza virus F protein antigen is beneficial for developing a recombinant combined vaccine with good safety and efficacy.

[0111] In some embodiments of this application, the adjuvant further includes DOPC and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of QS21 is 35-100 μg / mL; and the concentration of DOPC is 960-2800 μg / mL. Exemplarily, the concentrations of cholesterol, QS21, and DOPC can all be selected according to the aforementioned values. Using appropriate concentrations of cholesterol, QS21, and DOPC is beneficial for forming the adjuvant in a recombinant combined vaccine containing recombinant respiratory syncytial virus F protein antigen, recombinant human metapneumovirus F protein antigen, and recombinant parainfluenza virus F protein antigen. This helps ensure the stability of the antigens in the recombinant combined vaccine and facilitates a good immunization effect.

[0112] In some embodiments of this application, the concentration of cholesterol is 350-550 μg / mL; the concentration of QS21 is 50-100 μg / mL; and the concentration of DOPC is 1400-2200 μg / mL. Further using more suitable concentrations of cholesterol, QS21, and DOPC is beneficial for forming adjuvants in recombinant combined vaccines containing recombinant respiratory syncytial virus F protein antigen, recombinant human metapneumovirus F protein antigen, and recombinant parainfluenza virus F protein antigen. This helps ensure the stability of the antigens in the recombinant combined vaccine and facilitates a good immunization effect.

[0113] In some embodiments of this application, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of the protectant added is 20-100 mg / mL, the amount of the surfactant added is 100-500 μg / mL, the amount of the buffer solution added is 5-10 mM, and the amount of the salt added is 1.0-9.0 mg / mL. Exemplarily, the components and concentrations of the protectant, surfactant, salt, and buffer solution can be selected as described above. Using a pharmaceutical excipient composition with suitable components and concentrations as an adjuvant in the formation of a recombinant combined vaccine containing recombinant respiratory syncytial virus F protein antigen, recombinant human metapneumovirus F protein antigen, and recombinant parainfluenza virus F protein antigen can help ensure the stability of the antigens in the recombinant combined vaccine containing these antigens and facilitates a good immunization effect from the recombinant combined vaccine.

[0114] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0115] The amino acid sequence of the recombinant human metapneumovirus F protein antigen used in this application embodiment is shown in SE Q ID NO.1: MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVL RTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVCTAAAVTAGIAIAKTIRLESEVNAIKGALKTTNEAVSTLGNGVRVLAFAVRELKEFVSKNLTSALNKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWII KAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNKKDCETRGDHVFCDTACGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLPKGCSY ITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAESAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGGSSSGGFLGFLLGV.

[0116] The amino acid sequence of the recombinant parainfluenza virus F protein antigen used in this application embodiment is shown in SE Q ID NO.2: MYSMQLASCVTLTLVLLVNSQIDITKLQHVGVLVNSP KGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLKLQKDVIVTNQESNENTDPRTERFFGGVIGTIALGVATSAQITAAVALVEAKQAKSDIEKLKEAIRDTNKpVQSVCSSVGNCIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNcFGDNIGSLQEKGIKLQcIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLT RLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTTCTCNGIGNRINQPPDQGVKII THKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNSVALDPIDISIELNKvKSDLEESKEWyRRSNQKLSAIEDKIEEILSKIYHIENEIARIKKLIGEAPGGLVPRGSHHHHHHSAWSHPQFEK.

[0117] The amino acid sequence of the recombinant respiratory syncytial virus F protein antigen used in this application embodiment is shown in SEQ ID NO.3: MELLILKANAITTILTAVTFCFASGQNITEEFYQS TCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVI DTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNK NRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0118] Example 1

[0119] This embodiment provides a recombinant human metapneumovirus vaccine, comprising: recombinant human metapneumovirus F protein antigen and adjuvant; wherein, the amino acid sequence of the recombinant human metapneumovirus F protein is shown in SEQ ID NO.1; the adjuvant comprises cholesterol and QS21, the distribution density of QS21 on cholesterol is 0.25 g / g; the amount of cholesterol adsorbed with QS21 accounts for 57.1% of the total cholesterol;

[0120] The recombinant human metapneumovirus F protein antigen content per milliliter of vaccine is 240 μg / mL; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 700 μg / mL; the QS21 concentration is 100 μg / mL; the DOPC concentration is 2800 μg / mL; the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer; the protectant is sucrose, the surfactant is polysorbate 80, the salt is sodium chloride, and the buffer is a phosphate buffer containing sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate; the amount of sucrose added is 50 mg / mL, the amount of polysorbate 80 added is 500 μg / mL, the amount of phosphate added is 5 mM, and the amount of sodium chloride added is 4.0 mg / mL.

[0121] This embodiment provides a method for preparing a recombinant human metapneumovirus vaccine, including:

[0122] (1) The above concentration of DOPC and cholesterol were mixed and dispersed to obtain lipid components, and the lipid components were prepared into liposomes by ethanol injection method;

[0123] (2) Add the above concentration of QS21 to the liposome containing the above concentration of cholesterol at a speed of 4 mL / min by adding it to the liquid surface, and stir at 200 rpm for 30 min; then add the components of each concentration in the above pharmaceutical excipient composition and mix to prepare an adjuvant.

[0124] (3) The adjuvant was mixed with the above-mentioned concentration of recombinant human metapneumovirus F protein antigen to prepare a recombinant human metapneumovirus vaccine with a distribution density of QS21 on cholesterol of 0.25 g / g and a cholesterol content of adsorbed QS21 of 57.1% of the total cholesterol.

[0125] Example 2

[0126] This embodiment provides a recombinant human metapneumovirus vaccine, which differs from Embodiment 1 in that: the distribution density of QS21 on cholesterol is 0.323 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 44.3% of the total cholesterol.

[0127] This embodiment provides a method for preparing a recombinant human metapneumovirus vaccine. The difference from Embodiment 1 is that in step (2), QS21 of the corresponding concentration is added to liposomes containing cholesterol of the corresponding concentration at a speed of 8 mL / min by adding it to the liquid surface, and the mixture is stirred at 100 rpm for 30 min.

[0128] Example 3

[0129] This embodiment provides a recombinant human metapneumovirus vaccine, which differs from Embodiment 1 in that: the distribution density of QS21 on cholesterol is 0.556 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 25.7% of the total cholesterol.

[0130] This embodiment provides a method for preparing a recombinant human metapneumovirus vaccine. The difference from Embodiment 1 is that in step (2), QS21 of the corresponding concentration is added to liposomes containing cholesterol of the corresponding concentration at a speed of 30 mL / min by adding it on the liquid surface, and the mixture is stirred at 50 rpm for 30 min.

[0131] Example 4

[0132] This embodiment provides a recombinant human metapneumovirus vaccine, which differs from Example 3 in that: the distribution density of QS21 on cholesterol in liposomes is 0.538 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.0% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 500 μg / mL; the QS21 concentration is 70 μg / mL; the DOPC concentration is 2000 μg / mL; and the amount of polysorbate 80 added is 300 μg / mL.

[0133] This embodiment provides a method for preparing a recombinant human metapneumovirus vaccine, which is the same as the preparation method in Example 3.

[0134] Example 5

[0135] This embodiment provides a recombinant human metapneumovirus vaccine, which differs from Example 3 in that: the distribution density of QS21 on cholesterol in liposomes is 0.547 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.7% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 240 μg / mL; the QS21 concentration is 35 μg / mL; the DOPC concentration is 960 μg / mL; and the amount of polysorbate 80 added is 100 μg / mL.

[0136] This embodiment provides a method for preparing a recombinant human metapneumovirus vaccine. The difference from embodiment 3 is that in step (2), QS21 of the corresponding concentration is added to liposomes containing cholesterol of the corresponding concentration at a speed of 40 mL / min by adding it on the liquid surface, and the mixture is stirred at 50 rpm for 30 min.

[0137] Example 6

[0138] This embodiment provides a recombinant parainfluenza vaccine, comprising: recombinant parainfluenza virus F protein antigen and adjuvant; wherein, the amino acid sequence of the recombinant parainfluenza virus F protein is shown in SEQ ID NO.2; the adjuvant comprises cholesterol and QS21, the distribution density of QS21 on cholesterol is 0.25 g / g; the amount of cholesterol adsorbed with QS21 accounts for 57.1% of the total cholesterol;

[0139] The recombinant parainfluenza virus F protein antigen content per milliliter of vaccine is 240 μg / mL; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 700 μg / mL; the QS21 concentration is 100 μg / mL; the DOPC concentration is 2800 μg / mL; the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer; the protectant is sucrose, the surfactant is polysorbate 80, the salt is sodium chloride, and the buffer is a phosphate buffer containing sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate; the amount of sucrose added is 50 mg / mL, the amount of polysorbate 80 added is 500 μg / mL, the amount of phosphate added is 5 mM, and the amount of sodium chloride added is 4.0 mg / mL.

[0140] This embodiment provides a method for preparing a recombinant parainfluenza vaccine, which is the same as the preparation method in Example 1.

[0141] Example 7

[0142] This embodiment provides a recombinant parainfluenza vaccine, which differs from Embodiment 6 in that: the distribution density of QS21 on cholesterol is 0.323 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 44.3% of the total cholesterol.

[0143] This embodiment provides a method for preparing a recombinant parainfluenza vaccine, which is the same as the preparation method in Example 2.

[0144] Example 8

[0145] This embodiment provides a recombinant parainfluenza vaccine, which differs from Embodiment 6 in that: the distribution density of QS21 on cholesterol is 0.556 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 25.7% of the total cholesterol.

[0146] This embodiment provides a method for preparing a recombinant parainfluenza vaccine, which is the same as the preparation method in Example 3.

[0147] Example 9

[0148] This embodiment provides a recombinant parainfluenza vaccine, which differs from Example 8 in that: the distribution density of QS21 on cholesterol in liposomes is 0.538 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.0% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 500 μg / mL; the QS21 concentration is 70 μg / mL; the DOPC concentration is 2000 μg / mL; and the amount of polysorbate 80 added is 300 μg / mL.

[0149] This embodiment provides a method for preparing a recombinant parainfluenza vaccine, which is the same as the preparation method in Example 3.

[0150] Example 10

[0151] This embodiment provides a recombinant parainfluenza vaccine, which differs from Example 8 in that: the distribution density of QS21 on cholesterol in liposomes is 0.547 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.7% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 240 μg / mL; the QS21 concentration is 35 μg / mL; the DOPC concentration is 960 μg / mL; and the amount of polysorbate 80 added is 100 μg / mL.

[0152] This embodiment provides a method for preparing a recombinant parainfluenza vaccine, which is the same as the preparation method in Example 5.

[0153] Example 11

[0154] This embodiment provides a recombinant respiratory syncytial virus (RSV) vaccine, comprising: recombinant RSV F protein antigen and adjuvant; wherein, the amino acid sequence of the recombinant RSV F protein is shown in SEQ ID NO.3; the adjuvant comprises cholesterol and QS21, the distribution density of QS21 on cholesterol is 0.25 g / g; the amount of cholesterol adsorbed with QS21 accounts for 57.1% of the total cholesterol;

[0155] The recombinant respiratory syncytial virus F protein antigen content per milliliter of vaccine is 240 μg / mL; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 700 μg / mL; the QS21 concentration is 100 μg / mL; the DOPC concentration is 2800 μg / mL; the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer; the protectant is sucrose, the surfactant is polysorbate 80, the salt is sodium chloride, and the buffer is a phosphate buffer containing sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate; the amount of sucrose added is 50 mg / mL, the amount of polysorbate 80 added is 500 μg / mL, the amount of phosphate added is 5 mM, and the amount of sodium chloride added is 4.0 mg / mL.

[0156] This embodiment provides a method for preparing a recombinant respiratory syncytial virus vaccine, which is the same as the preparation method in Example 1.

[0157] Example 12

[0158] This embodiment provides a recombinant respiratory syncytial virus vaccine, which differs from Embodiment 11 in that: the distribution density of QS21 on cholesterol is 0.323 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 44.3% of the total cholesterol.

[0159] This embodiment provides a method for preparing a recombinant respiratory syncytial virus vaccine, which is the same as the preparation method in Example 2.

[0160] Example 13

[0161] This embodiment provides a recombinant respiratory syncytial virus vaccine, which differs from Embodiment 11 in that: the distribution density of QS21 on cholesterol is 0.556 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 25.7% of the total cholesterol.

[0162] This embodiment provides a method for preparing a recombinant respiratory syncytial virus vaccine, which is the same as the preparation method in Example 3.

[0163] Example 14

[0164] This embodiment provides a recombinant respiratory syncytial virus vaccine, which differs from Example 13 in that: the distribution density of QS21 on cholesterol in liposomes is 0.538 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.0% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 500 μg / mL; the QS21 concentration is 70 μg / mL; the DOPC concentration is 2000 μg / mL; and the amount of polysorbate 80 added is 300 μg / mL.

[0165] This embodiment provides a method for preparing a recombinant respiratory syncytial virus vaccine, which is the same as the preparation method in Example 3.

[0166] Example 15

[0167] This embodiment provides a recombinant respiratory syncytial virus vaccine, which differs from Example 13 in that: the distribution density of QS21 on cholesterol in liposomes is 0.547 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.7% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 240 μg / mL; the QS21 concentration is 35 μg / mL; the DOPC concentration is 960 μg / mL; and the amount of polysorbate 80 added is 100 μg / mL.

[0168] This embodiment provides a method for preparing a recombinant respiratory syncytial virus vaccine, which is the same as the preparation method in Example 5.

[0169] Example 16

[0170] This embodiment provides a recombinant combined vaccine, comprising: recombinant respiratory syncytial virus (RSV) F protein antigen, recombinant human metapneumovirus (HMV) F protein antigen, and an adjuvant; wherein, the amino acid sequence of the recombinant RSV F protein is shown in SEQ ID NO.3, and the amino acid sequence of the recombinant HMV F protein is shown in SEQ ID NO.1; the adjuvant comprises cholesterol and QS21, wherein the distribution density of QS21 on cholesterol is 0.25 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 57.1% of the total cholesterol.

[0171] The recombinant respiratory syncytial virus (RSV) F protein antigen content per milliliter of vaccine is 240 μg / mL, and the recombinant human metapneumovirus (HMV) F protein antigen content is 240 μg / mL. The adjuvant also includes DOPC and a pharmaceutical excipient composition. The cholesterol concentration is 700 μg / mL; the QS21 concentration is 100 μg / mL; the DOPC concentration is 2800 μg / mL. The pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution. The protectant is sucrose, the surfactant is polysorbate 80, the salt is sodium chloride, and the buffer solution is a phosphate buffer containing sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate. The amount of sucrose added is 50 mg / mL, the amount of polysorbate 80 added is 500 μg / mL, the amount of phosphate added is 5 mM, and the amount of sodium chloride added is 4.0 mg / mL.

[0172] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 1.

[0173] Example 17

[0174] This embodiment provides a recombinant combined vaccine, which differs from Embodiment 16 in that: the distribution density of QS21 on cholesterol is 0.323 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 44.3% of the total cholesterol.

[0175] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 2.

[0176] Example 18

[0177] This embodiment provides a recombinant combined vaccine, which differs from Embodiment 16 in that: the distribution density of QS21 on cholesterol is 0.556 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 25.7% of the total cholesterol.

[0178] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 3.

[0179] Example 19

[0180] This embodiment provides a recombinant combined vaccine, which differs from Example 18 in that: the distribution density of QS21 on cholesterol in liposomes is 0.538 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.0% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 500 μg / mL; the QS21 concentration is 70 μg / mL; the DOPC concentration is 2000 μg / mL; and the amount of polysorbate 80 added is 300 μg / mL.

[0181] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 3.

[0182] Example 20

[0183] This embodiment provides a recombinant combined vaccine, which differs from Example 18 in that: the distribution density of QS21 on cholesterol in liposomes is 0.547 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.7% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 240 μg / mL; the QS21 concentration is 35 μg / mL; the DOPC concentration is 960 μg / mL; and the amount of polysorbate 80 added is 100 μg / mL.

[0184] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 5.

[0185] Example 21

[0186] This embodiment provides a recombinant combined vaccine, comprising: recombinant respiratory syncytial virus (RSV) F protein antigen, recombinant parainfluenza virus (PINS) F protein antigen, and an adjuvant; wherein, the amino acid sequence of the recombinant RSV F protein is shown in SEQ ID NO.3, and the amino acid sequence of the recombinant PINS F protein is shown in SEQ ID NO.2; the adjuvant comprises cholesterol and QS21, wherein the distribution density of QS21 on cholesterol is 0.25 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 57.1% of the total cholesterol.

[0187] The recombinant respiratory syncytial virus F protein antigen content per milliliter of vaccine is 240 μg / mL, and the recombinant parainfluenza virus F protein antigen content is 240 μg / mL. The adjuvant also includes DOPC and a pharmaceutical excipient composition. The cholesterol concentration is 700 μg / mL; the QS21 concentration is 100 μg / mL; the DOPC concentration is 2800 μg / mL. The pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer. The protectant is sucrose, the surfactant is polysorbate 80, the salt is sodium chloride, and the buffer is a phosphate buffer containing sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate. The amount of sucrose added is 50 mg / mL, the amount of polysorbate 80 added is 500 μg / mL, the amount of phosphate added is 5 mM, and the amount of sodium chloride added is 4.0 mg / mL.

[0188] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 1.

[0189] Example 22

[0190] This embodiment provides a recombinant combined vaccine, which differs from Embodiment 13 in that: the distribution density of QS21 on cholesterol is 0.323 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 44.3% of the total cholesterol.

[0191] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 2.

[0192] Example 23

[0193] This embodiment provides a recombinant combined vaccine, which differs from Embodiment 13 in that: the distribution density of QS21 on cholesterol is 0.556 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 25.7% of the total cholesterol.

[0194] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 3.

[0195] Example 24

[0196] This embodiment provides a recombinant combined vaccine, which differs from Example 23 in that: the distribution density of QS21 on cholesterol in liposomes is 0.538 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.0% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 500 μg / mL; the QS21 concentration is 70 μg / mL; the DOPC concentration is 2000 μg / mL; and the amount of polysorbate 80 added is 300 μg / mL.

[0197] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 3.

[0198] Example 25

[0199] This embodiment provides a recombinant combined vaccine, which differs from Example 23 in that: the distribution density of QS21 on cholesterol in liposomes is 0.547 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.7% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 240 μg / mL; the QS21 concentration is 35 μg / mL; the DOPC concentration is 960 μg / mL; and the amount of polysorbate 80 added is 100 μg / mL.

[0200] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 5.

[0201] Example 26

[0202] This embodiment provides a recombinant combined vaccine, comprising: recombinant human metapneumovirus F protein antigen, recombinant parainfluenza virus F protein antigen, and adjuvant; wherein, the amino acid sequence of the recombinant human metapneumovirus F protein is shown in SEQ ID NO.1, and the amino acid sequence of the recombinant parainfluenza virus F protein is shown in SEQ ID NO.2; the adjuvant comprises cholesterol and QS21, the distribution density of QS21 on cholesterol is 0.25 g / g; the amount of cholesterol adsorbed with QS21 accounts for 57.1% of the total cholesterol;

[0203] The recombinant human metapneumovirus F protein antigen content per milliliter of vaccine is 240 μg / mL, and the recombinant parainfluenza virus F protein antigen content is 240 μg / mL. The adjuvant also includes DOPC and a pharmaceutical excipient composition. The cholesterol concentration is 700 μg / mL; the QS21 concentration is 100 μg / mL; the DOPC concentration is 2800 μg / mL. The pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer. The protectant is sucrose, the surfactant is polysorbate 80, the salt is sodium chloride, and the buffer is a phosphate buffer containing sodium dihydrogen phosphate and disodium hydrogen phosphate. The amount of sucrose added is 50 mg / mL, the amount of polysorbate 80 added is 500 μg / mL, the amount of phosphate added is 5 mM, and the amount of sodium chloride added is 4.0 mg / mL.

[0204] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 1.

[0205] Example 27

[0206] This embodiment provides a recombinant combined vaccine, which differs from Embodiment 26 in that: the distribution density of QS21 on cholesterol is 0.323 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 44.3% of the total cholesterol.

[0207] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 2.

[0208] Example 28

[0209] This embodiment provides a recombinant combined vaccine, which differs from Embodiment 26 in that: the distribution density of QS21 on cholesterol is 0.556 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 25.7% of the total cholesterol.

[0210] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 3.

[0211] Example 29

[0212] This embodiment provides a recombinant combined vaccine, which differs from Example 28 in that: the distribution density of QS21 on cholesterol in liposomes is 0.538 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.0% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 500 μg / mL; the QS21 concentration is 70 μg / mL; the DOPC concentration is 2000 μg / mL; and the amount of polysorbate 80 added is 300 μg / mL.

[0213] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 3.

[0214] Example 30

[0215] This embodiment provides a recombinant combined vaccine, which differs from Example 28 in that: the distribution density of QS21 on cholesterol in liposomes is 0.547 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.7% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 240 μg / mL; the QS21 concentration is 35 μg / mL; the DOPC concentration is 960 μg / mL; and the amount of polysorbate 80 added is 100 μg / mL.

[0216] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 5.

[0217] Example 31

[0218] This embodiment provides a recombinant combined vaccine, comprising: recombinant respiratory syncytial virus (RSV) F protein antigen, recombinant human metapneumovirus (HMV) F protein antigen, recombinant parainfluenza virus (PNV) F protein antigen, and an adjuvant; wherein, the amino acid sequence of the recombinant RSV F protein is shown in SEQ ID NO.3, the amino acid sequence of the recombinant HMV F protein is shown in SEQ ID NO.1, and the amino acid sequence of the recombinant PNV F protein is shown in SEQ ID NO.2; the adjuvant comprises cholesterol and QS21, the distribution density of QS21 on cholesterol is 0.25 g / g; the amount of cholesterol adsorbed with QS21 accounts for 57.1% of the total cholesterol;

[0219] Calculated per milliliter of vaccine, the content of recombinant respiratory syncytial virus F protein antigen is 240 μg / mL, the content of recombinant human metapneumovirus F protein antigen is 240 μg / mL, and the content of recombinant parainfluenza virus F protein antigen is 240 μg / mL; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the concentration of cholesterol is 700 μg / mL; the concentration of QS21 is 100 μg / mL; the concentration of DOPC is 2800 μg / mL; the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer; the protectant is sucrose, the surfactant is polysorbate 80, the salt is sodium chloride, and the buffer is a phosphate buffer containing sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate; the amount of sucrose added is 50 mg / mL, the amount of polysorbate 80 added is 500 μg / mL, the amount of phosphate added is 5 mM, and the amount of sodium chloride added is 4.0 mg / mL.

[0220] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 1.

[0221] Example 32

[0222] This embodiment provides a recombinant combined vaccine, which differs from Embodiment 31 in that: the distribution density of QS21 on cholesterol is 0.323 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 44.3% of the total cholesterol.

[0223] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 2.

[0224] Example 33

[0225] This embodiment provides a recombinant combined vaccine, which differs from Embodiment 31 in that: the distribution density of QS21 on cholesterol is 0.556 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 25.7% of the total cholesterol.

[0226] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 3.

[0227] Example 34

[0228] This embodiment provides a recombinant combined vaccine, which differs from Example 33 in that: the distribution density of QS21 on cholesterol in liposomes is 0.538 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.0% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 500 μg / mL; the QS21 concentration is 70 μg / mL; the DOPC concentration is 2000 μg / mL; and the amount of polysorbate 80 added is 300 μg / mL.

[0229] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 3.

[0230] Example 35

[0231] This embodiment provides a recombinant combined vaccine, which differs from Example 33 in that: the distribution density of QS21 on cholesterol in liposomes is 0.547 g / g; the amount of cholesterol adsorbed with QS21 accounts for 26.7% of the total cholesterol; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 240 μg / mL; the QS21 concentration is 35 μg / mL; the DOPC concentration is 960 μg / mL; and the amount of polysorbate 80 added is 100 μg / mL.

[0232] This embodiment provides a method for preparing a recombinant combined vaccine, which is the same as the preparation method in Example 5.

[0233] Comparative Example 1

[0234] This comparative example provides a recombinant human metapneumovirus vaccine, comprising: recombinant human metapneumovirus F protein antigen and adjuvant; wherein, the amino acid sequence of the recombinant human metapneumovirus F protein is shown in SEQ ID NO.1; the adjuvant comprises cholesterol and QS21, the distribution density of QS21 on cholesterol is 0.172 g / g; the amount of cholesterol adsorbed with QS21 accounts for 82.9% of the total cholesterol;

[0235] The recombinant human metapneumovirus F protein antigen content per milliliter of vaccine is 240 μg / mL; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 700 μg / mL; the QS21 concentration is 100 μg / mL; the DOPC concentration is 2800 μg / mL; the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer; the protectant is sucrose, the surfactant is polysorbate 80, the salt is sodium chloride, and the buffer is a phosphate buffer containing sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate; the amount of sucrose added is 50 mg / mL, the amount of polysorbate 80 added is 500 μg / mL, the amount of phosphate added is 5 mM, and the amount of sodium chloride added is 4.0 mg / mL.

[0236] This comparative example provides a method for preparing a recombinant human metapneumovirus vaccine. The difference from Example 1 is that in step (2), QS21 of the corresponding concentration is added to liposomes containing cholesterol of the corresponding concentration at a speed of 1 mL / min by adding it to the liquid surface, and the mixture is stirred at 300 rpm for 30 min.

[0237] Comparative Example 2

[0238] This comparative example provides a recombinant human metapneumovirus vaccine, which differs from Comparative Example 1 in that: the distribution density of QS21 on cholesterol is 0.204 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 70% of the total cholesterol.

[0239] This comparative example provides a method for preparing a recombinant human metapneumovirus vaccine. The difference from Example 1 is that in step (2), QS21 of the corresponding concentration is added to liposomes containing the corresponding concentration of cholesterol at a speed of 2 mL / min by adding it on the liquid surface, and the mixture is stirred at 200 rpm for 30 min.

[0240] Comparative Example 3

[0241] This comparative example provides a recombinant human metapneumovirus vaccine, which differs from Comparative Example 1 in that: the distribution density of QS21 on cholesterol is 0.909 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 15.7% of the total cholesterol.

[0242] This comparative example provides a method for preparing a recombinant human metapneumovirus vaccine. The difference from Example 1 is that in step (2), QS21 of the corresponding concentration is added to liposomes containing cholesterol of the corresponding concentration at a speed of 50 mL / min by adding it on the liquid surface, and the mixture is stirred at 50 rpm for 30 min.

[0243] Comparative Example 4

[0244] This comparative example provides a recombinant parainfluenza vaccine, comprising: recombinant parainfluenza virus F protein antigen and adjuvant; wherein, the amino acid sequence of the recombinant parainfluenza virus F protein is shown in SEQ ID NO.2; the adjuvant comprises cholesterol and QS21, the distribution density of QS21 on cholesterol is 0.172 g / g; the amount of cholesterol adsorbed with QS21 accounts for 82.9% of the total cholesterol;

[0245] The recombinant parainfluenza virus F protein antigen content per milliliter of vaccine is 240 μg / mL; the adjuvant also includes DOPC and a pharmaceutical excipient composition; the cholesterol concentration is 700 μg / mL; the QS21 concentration is 100 μg / mL; the DOPC concentration is 2800 μg / mL; the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer; the protectant is sucrose, the surfactant is polysorbate 80, the salt is sodium chloride, and the buffer is a phosphate buffer containing sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate; the amount of sucrose added is 50 mg / mL, the amount of polysorbate 80 added is 500 μg / mL, the amount of phosphate added is 5 mM, and the amount of sodium chloride added is 4.0 mg / mL.

[0246] This comparative example provides a method for preparing a recombinant parainfluenza vaccine, which is the same as the preparation method of Comparative Example 1.

[0247] Comparative Example 5

[0248] This comparative example provides a recombinant parainfluenza vaccine, which differs from Comparative Example 4 in that: the distribution density of QS21 on cholesterol is 0.204 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 70% of the total cholesterol.

[0249] This comparative example provides a method for preparing a recombinant parainfluenza vaccine, which is the same as the preparation method of Comparative Example 2.

[0250] Comparative Example 6

[0251] This comparative example provides a recombinant parainfluenza vaccine, which differs from Comparative Example 4 in that: the distribution density of QS21 on cholesterol is 0.909 g / g; and the amount of cholesterol adsorbed with QS21 accounts for 15.7% of the total cholesterol.

[0252] This comparative example provides a method for preparing a recombinant parainfluenza vaccine, which is the same as the preparation method of Comparative Example 3.

[0253] The formulations of the vaccines provided in Examples 1-35 and Comparative Examples 1-6 are shown in Table 1-2.

[0254] Table 1. Vaccine formulations provided in Examples 1-15 and Comparative Examples 1-6 (per 1000 mL).

[0255]

[0256]

[0257] Table 2 shows the vaccine formulations provided in Examples 16-35 (per 1000 mL).

[0258]

[0259]

[0260] Note: The “ / ” in Table 2 does not include this component.

[0261] Experimental Example 1

[0262] This experiment tested the immunogenicity and toxicity of the vaccines provided in Examples 1-5 and Comparative Examples 1-3.

[0263] Experimental grouping: SPF-grade BALB / c mice were randomly divided into 9 groups, with 10 mice in each vaccine group and 5 mice in each negative control group, for a total of 85 animals. The administration regimens for each group are shown in Table 3.

[0264] Table 3. Mouse grouping and immunization regimen

[0265] experimental group quantity Injection method Number of injections Injection dosage Example 1 10 Intramuscular injection 2 0.1 dose Example 2 10 Intramuscular injection 2 0.1 dose Example 3 10 Intramuscular injection 2 0.1 dose Example 4 10 Intramuscular injection 2 0.1 dose Example 5 10 Intramuscular injection 2 0.1 dose Comparative Example 1 10 Intramuscular injection 2 0.1 dose Comparative Example 2 10 Intramuscular injection 2 0.1 dose Comparative Example 3 10 Intramuscular injection 2 0.1 dose Negative control (physiological saline) 5 Intramuscular injection 2 /

[0266] Note: Each dose of vaccine is 0.5 mL.

[0267] Immunization method: Before use, the test sample was gently shaken and 50 μL of the test sample solution was drawn using a 300 μL disposable sterile syringe and needle and injected into the bilateral lateral thigh muscles of mice.

[0268] Immunization time: Dosage is administered twice, with an interval of 21 days.

[0269] Serum and spleen collection: Spleens were isolated from mice 14 days after the second immunization (D35) in each experimental group. Intracellular cytokine staining (ICS) was used to detect the specific CD4 count of the corresponding antigen. + T-cell immune response. The specific method is as follows:

[0270] After collecting mouse spleens, they were stored in EP tubes containing 4 mL of maintenance culture medium. Splenic cells were collected by grinding in a biosafety cabinet and centrifuged at room temperature (531 g, 5 min). Cells were then lysed with 5 mL of ACK lysis buffer for 3 min, followed by cell washing buffer and centrifugation at room temperature (531 g, 5 min). Cells were collected again by centrifugation at room temperature (531 g, 5 min), the supernatant was discarded, and the cells were resuspended and counted. After counting, cells were collected by centrifugation at room temperature (531 g, 5 min) and the cell density was adjusted to 2 × 10⁶ cells / mL. 7 Cells / mL; After adding cell stimulant, 100 μL / well was added to a 96-well U-shaped cell culture plate and incubated at 37°C and 5% CO2 for 1 h; After stimulation, 10 μL / well of Golgi plug working solution was added, and the final concentration was adjusted to 1 μL / mL, and the cells were incubated at 37°C and 5% CO2 for 5 h; After centrifugation at room temperature (604 g, 5 min), the cells were resuspended in 200 μL / well of D-PBS, centrifuged at room temperature (604 g, 5 min) to stain dead and live cells, and then resuspended in 150 μL / well of D-PBS; After centrifugation at room temperature (604 g, 5 min) to collect cells, 50 μL / well of FACSblock was added for blocking, and the cells were incubated at 4°C in the dark for 5 min; After blocking, 50 μL / well of surface antibody mixture was added for cell surface staining, and the cells were incubated at 4°C in the dark for 30 min; 100 μL / well of FACSblock was added. Cells were collected by centrifugation at room temperature (604g, 5min) after adding 100μL / well Cytofix / cytoperm and incubating at 4℃ in the dark for 20min. Cells were then collected by centrifugation at room temperature (604g, 5min) after adding 100μL / well Perm / wash and adding 50μL / well cytokine antibody mixture for staining and incubating at 4℃ in the dark for 30min. Cells were then collected by centrifugation at room temperature (604g, 5min) after adding 150μL / well Perm / wash and adding 200μL / well Perm / wash and adding 750g, 3min. Finally, cells were fixed with 150μL / well fixative and analyzed by flow cytometry. The results were analyzed using FlowJo software and are shown in Table 5. Figure 1 As shown.

[0271] Meanwhile, 45 SPF-grade KM mice were randomly divided into 9 groups, with 5 mice in each group, and the drug administration regimens are shown in Table 4.

[0272] Table 4. Mouse grouping and immunization regimen

[0273] experimental group quantity Injection method Number of injections Injection dosage Example 1 5 Intraperitoneal injection 1 0.5 doses Example 2 5 Intraperitoneal injection 1 0.5 doses Example 3 5 Intraperitoneal injection 1 0.5 doses Example 4 5 Intraperitoneal injection 1 0.5 doses Example 5 5 Intraperitoneal injection 1 0.5 doses Comparative Example 1 5 Intraperitoneal injection 1 0.5 doses Comparative Example 2 5 Intraperitoneal injection 1 0.5 doses Comparative Example 3 5 Intraperitoneal injection 1 0.5 doses Negative control (physiological saline) 5 Intraperitoneal injection 1 /

[0274] Note: Each dose of vaccine is 0.5 mL.

[0275] Immunization method: Before use, the test sample was gently shaken and 250 μL of the test sample solution was drawn using a 1000 μL disposable sterile syringe and needle and injected into the peritoneal cavity of mice.

[0276] Mice were weighed daily from day 0 to day 7, and their body weight was recorded. The results are as follows: Figure 2 As shown.

[0277] Table 5. Antibody levels after secondary immunization with the vaccine

[0278] Group GMT (Geosensitive Antibody) Example 1 3880234 Example 2 4158732 Example 3 3620387 Example 4 3377940 Example 5 3151730 Comparative Example 1 1940117 Comparative Example 2 3377940 Comparative Example 3 2228609 physiological saline 57

[0279] From Table 5 and Figure 1 The results showed that after the second immunization, the vaccines provided in Examples 1-5 had higher immunogenicity, while the vaccines provided in Comparative Examples 1 and 3 had lower immunogenicity. The immunogenicity of the vaccine provided in Comparative Example 2 was not significantly different from that of the vaccines provided in Examples 1-5. However, due to... Figure 2 The results showed that, after intraperitoneal injection of the vaccine provided in Comparative Example 2 into mice, the mice exhibited poor weight gain and high toxicity. This indicates that the adjuvant in the vaccine provided in this application includes cholesterol and QS21. By controlling the distribution density of QS21 on cholesterol to be 0.25-0.556 g / g, and the ratio of the amount of cholesterol adsorbed with QS21 to the total cholesterol to be 25.7%-57.1%, the vaccine achieves good safety and high immunogenicity.

[0280] Experimental Example 2

[0281] This experiment tested the immunogenicity and toxicity of the vaccines provided in Examples 6-10 and Comparative Examples 4-6. The specific methods were the same as those in Example 1. The results are shown in Table 6 and... Figure 3-4 As shown.

[0282] Table 6. Antibody levels after secondary immunization with the vaccine

[0283] Group GMT (Geosensitive Antibody) Example 6 3880234 Example 7 4160354 Example 8 3377940 Example 9 3151730 Example 10 3151730 Comparative Example 4 1940117 Comparative Example 5 3620387 Comparative Example 6 2388564 physiological saline 71

[0284] From Table 6 and Figure 3 The results showed that after the second immunization, the vaccines provided in Examples 6-10 had higher immunogenicity, while the vaccines provided in Comparative Examples 4 and 6 had lower immunogenicity. The immunogenicity of the vaccine provided in Comparative Example 5 was not significantly different from that of the vaccines provided in Examples 6-10. However, due to... Figure 4The results showed that after intraperitoneal injection of the vaccine provided in Comparative Example 5 into mice, the mice exhibited poor weight gain and high toxicity. This indicates that the adjuvant in the vaccine provided in this application includes cholesterol and QS21. By controlling the distribution density of QS21 on cholesterol to be 0.25-0.556 g / g, and the ratio of cholesterol adsorbed with QS21 to the total cholesterol to be 25.7%-57.1%, the vaccine achieves good safety and high immunogenicity.

[0285] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. An immune complex, characterized in that, include: Liposomes and saponins, wherein the liposomes contain cholesterol, and the distribution density of the saponins on the cholesterol in the liposomes is 0.204-0.556 g / g; the amount of cholesterol adsorbed by the saponins accounts for 25.7%-70% of the total cholesterol.

2. The immune complex according to claim 1, characterized in that, The distribution density of the saponins on the cholesterol in the liposomes is 0.25-0.556 g / g; the amount of cholesterol adsorbed by the saponins accounts for 25.7%-57.1% of the total cholesterol.

3. The immune complex according to claim 1 or 2, characterized in that, The liposomes also contain dioleoylphosphatidylcholine; the concentration of cholesterol is 240-700 μg / mL; the concentration of saponins is 35-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 960-2800 μg / mL. Preferably, the concentration of cholesterol is 350-550 μg / mL; the concentration of saponin is 50-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 1400-2200 μg / mL.

4. The immune complex according to claim 1 or 2, characterized in that, The saponins are selected from saponins 21, 7, 17 or 18.

5. An immune complex, characterized in that, include: The liposomes and saponin 21, wherein the liposomes contain cholesterol, and the distribution density of saponin 21 on the cholesterol in the liposomes is 0.25-0.556 g / g; the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

6. The immune complex according to claim 5, characterized in that, The immune complex also includes dioleoylphosphatidylcholine; the concentration of cholesterol is 240-700 μg / mL; the concentration of saponin 21 is 35-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 960-2800 μg / mL. Preferably, the concentration of cholesterol is 350-550 μg / mL; the concentration of saponin 21 is 50-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 1400-2200 μg / mL.

7. The use of an immune complex as described in any one of claims 1-6 in the preparation of adjuvants, vaccines or pharmaceuticals.

8. An adjuvant, characterized in that, The immune complex and pharmaceutical excipient composition included in any one of claims 1-6.

9. The adjuvant according to claim 8, characterized in that, The pharmaceutical excipient composition includes a protective agent, a surfactant, a salt, and a buffer solution; Preferably, the amount of the protective agent added is 20-100 mg / mL, the amount of the surfactant added is 100-500 μg / mL, the amount of the buffer solution added is 5-10 mM, and the amount of the salt added is 1.0-9.0 mg / mL.

10. A vaccine, characterized in that, It includes at least one antigen and the immune complex as described in any one of claims 1-6 or the adjuvant as described in claim 8 or 9.

11. The vaccine according to claim 10, characterized in that, The antigen is selected from bacteria, viruses, parasites, or genetically engineered antigens.

12. A recombinant protein vaccine, characterized in that, It includes at least one recombinant protein antigen and the immune complex according to any one of claims 1-6 or the adjuvant according to claim 8 or 9.

13. A recombinant protein vaccine, characterized in that, It includes at least one recombinant F protein antigen and the immune complex according to any one of claims 1-6 or the adjuvant according to claim 8 or 9.

14. The recombinant protein vaccine according to claim 13, characterized in that, The content of one of the recombinant F protein antigens is 60-480 μg / mL, calculated per milliliter of vaccine.

15. The recombinant protein vaccine according to claim 13 or 14, characterized in that, The recombinant F protein antigen includes at least one of recombinant respiratory syncytial virus F protein antigen, recombinant human metapneumovirus F protein antigen, and recombinant parainfluenza virus F protein antigen.

16. A recombinant respiratory syncytial virus vaccine, characterized in that, include: Recombinant respiratory syncytial virus F protein antigen and adjuvant; The adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on the cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

17. The recombinant respiratory syncytial virus vaccine according to claim 16, characterized in that, The content of the recombinant respiratory syncytial virus F protein antigen is 60-480 μg / mL, calculated per milliliter of vaccine. And / or, the adjuvant further includes dioleoylphosphatidylcholine and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of saponin 21 is 35-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 960-2800 μg / mL. Preferably, the concentration of cholesterol is 350-550 μg / mL; the concentration of saponin 21 is 50-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 1400-2200 μg / mL. Preferably, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of the protectant added is 20-100 mg / mL, the amount of the surfactant added is 100-500 μg / mL, the amount of the buffer solution added is 5-10 mM, and the amount of the salt added is 1.0-9.0 mg / mL.

18. A recombinant human metapneumovirus vaccine, characterized in that, include: Recombinant human metapneumovirus F protein antigen and adjuvant; The adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on the cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

19. The recombinant human metapneumovirus vaccine according to claim 18, characterized in that, The content of the recombinant human metapneumovirus F protein antigen per milliliter of vaccine is 60-480 μg / mL; And / or, the adjuvant further includes dioleoylphosphatidylcholine and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of saponin 21 is 35-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 960-2800 μg / mL. Preferably, the concentration of cholesterol is 350-550 μg / mL; the concentration of saponin 21 is 50-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 1400-2200 μg / mL. Preferably, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of the protectant added is 20-100 mg / mL, the amount of the surfactant added is 100-500 μg / mL, the amount of the buffer solution added is 5-10 mM, and the amount of the salt added is 1.0-9.0 mg / mL.

20. A recombinant parainfluenza vaccine, characterized in that, include: Recombinant parainfluenza virus F protein antigen and adjuvant; The adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on the cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

21. The recombinant parainfluenza vaccine according to claim 20, characterized in that, The content of the recombinant parainfluenza virus F protein antigen is 60-480 μg / mL, calculated per milliliter of vaccine. And / or, the adjuvant further includes dioleoylphosphatidylcholine and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of saponin 21 is 35-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 960-2800 μg / mL. Preferably, the concentration of cholesterol is 350-550 μg / mL; the concentration of saponin 21 is 50-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 1400-2200 μg / mL. Preferably, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of the protectant added is 20-100 mg / mL, the amount of the surfactant added is 100-500 μg / mL, the amount of the buffer solution added is 5-10 mM, and the amount of the salt added is 1.0-9.0 mg / mL.

22. A recombinant combination vaccine, characterized in that, include: Recombinant respiratory syncytial virus F protein, recombinant human metapneumovirus F protein and adjuvant; The adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on the cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

23. The recombinant combined vaccine according to claim 22, characterized in that, The content of the recombinant respiratory syncytial virus F protein antigen is 60-480 μg / mL per milliliter of vaccine; the content of the recombinant human metapneumovirus F protein antigen is 60-480 μg / mL. And / or, the adjuvant further includes dioleoylphosphatidylcholine and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of saponin 21 is 35-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 960-2800 μg / mL. Preferably, the concentration of cholesterol is 350-550 μg / mL; the concentration of saponin 21 is 50-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 1400-2200 μg / mL. Preferably, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of the protectant added is 20-100 mg / mL, the amount of the surfactant added is 100-500 μg / mL, the amount of the buffer solution added is 5-10 mM, and the amount of the salt added is 1.0-9.0 mg / mL.

24. A recombinant combination vaccine, characterized in that, include: Recombinant respiratory syncytial virus F protein, recombinant parainfluenza virus F protein, and adjuvant; The adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on the cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

25. The recombinant combined vaccine according to claim 24, characterized in that, The content of the recombinant respiratory syncytial virus F protein antigen is 60-480 μg / mL per milliliter of vaccine; the content of the recombinant parainfluenza virus F protein antigen is 60-480 μg / mL. And / or, the adjuvant further includes dioleoylphosphatidylcholine and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of saponin 21 is 35-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 960-2800 μg / mL. Preferably, the concentration of cholesterol is 350-550 μg / mL; the concentration of saponin 21 is 50-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 1400-2200 μg / mL. Preferably, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of the protectant added is 20-100 mg / mL, the amount of the surfactant added is 100-500 μg / mL, the amount of the buffer solution added is 5-10 mM, and the amount of the salt added is 1.0-9.0 mg / mL.

26. A recombinant combination vaccine, characterized in that, include: Recombinant human metapneumovirus F protein, recombinant parainfluenza virus F protein, and adjuvant; The adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on the cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

27. The recombinant combined vaccine according to claim 26, characterized in that, The content of the recombinant human metapneumovirus F protein antigen is 60-480 μg / mL per milliliter of vaccine; the content of the recombinant parainfluenza virus F protein antigen is 60-480 μg / mL. And / or, the adjuvant further includes dioleoylphosphatidylcholine and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of saponin 21 is 35-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 960-2800 μg / mL. Preferably, the concentration of cholesterol is 350-550 μg / mL; the concentration of saponin 21 is 50-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 1400-2200 μg / mL. Preferably, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of the protectant added is 20-100 mg / mL, the amount of the surfactant added is 100-500 μg / mL, the amount of the buffer solution added is 5-10 mM, and the amount of the salt added is 1.0-9.0 mg / mL.

28. A recombinant combination vaccine, characterized in that, include: Recombinant respiratory syncytial virus F protein, recombinant human metapneumovirus F protein, recombinant parainfluenza virus F protein, and adjuvant; The adjuvant comprises cholesterol and saponin 21, wherein the distribution density of saponin 21 on the cholesterol is 0.25-0.556 g / g; and the amount of cholesterol adsorbed by saponin 21 accounts for 25.7%-57.1% of the total cholesterol.

29. The recombinant combined vaccine according to claim 28, characterized in that, The content of the respiratory syncytial virus F protein antigen per milliliter of vaccine is 60-480 μg / mL; the content of the recombinant human metapneumovirus F protein antigen is 60-480 μg / mL; and the content of the recombinant parainfluenza virus F protein antigen is 60-480 μg / mL. And / or, the adjuvant further includes dioleoylphosphatidylcholine and a pharmaceutical excipient composition; the concentration of cholesterol is 240-700 μg / mL; the concentration of saponin 21 is 35-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 960-2800 μg / mL. Preferably, the concentration of cholesterol is 350-550 μg / mL; the concentration of saponin 21 is 50-100 μg / mL; and the concentration of dioleoylphosphatidylcholine is 1400-2200 μg / mL. Preferably, the pharmaceutical excipient composition includes a protectant, a surfactant, a salt, and a buffer solution; the amount of the protectant added is 20-100 mg / mL, the amount of the surfactant added is 100-500 μg / mL, the amount of the buffer solution added is 5-10 mM, and the amount of the salt added is 1.0-9.0 mg / mL.

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