Recombinant respiratory syncytial virus vaccine as well as preparation method and application thereof

Abstract

The invention provides a recombinant respiratory syncytial virus vaccine as well as a preparation method and application thereof, and belongs to the technical field of viral vaccine preparations. Wherein the recombinant respiratory syncytial virus vaccine is a vaccine preparation in a liquid dosage form comprising pre-F protein antigen; wherein the amino acid sequence of the pre-F protein antigen is any one of the amino acid sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 3. The recombinant respiratory syncytial virus vaccine provided by the invention is a liquid vaccine preparation comprising a recombinant respiratory syncytial virus (RSV) protein stock solution, the vaccine preparation has better stability, and the self-made recombinant respiratory syncytial virus vaccine is proved to have good immunogenicity through immune experiment research on a BALB / c mouse by intramuscular injection.

Description

Technical Field

[0001] The invention belongs to the technical field of virus vaccine preparations, and in particular relates to a recombinant respiratory syncytial virus vaccine and a preparation method and application thereof. Background Art

[0002] Human Respiratory Syncytial Virus (hRSV) belongs to the genus Orthopneumovirus of the family Pneumoviridae. It is an enveloped, non-segmented, single-stranded negative-strand RNA virus. hRSV is one of the main pathogens causing acute lower respiratory tract diseases in infants, immunocompromised people and the elderly worldwide. It is highly contagious and is mainly transmitted through droplets and direct contact (body fluid secretions).

[0003] In 2015, approximately 1.5 million elderly people in industrialized countries suffered from RSV-associated acute respiratory infections (RSV-ARI). It is estimated that 336,000 elderly people were hospitalized due to RSV-ARI, and there were approximately 14,000 RSV-ARI-related hospitalization deaths. In 2019, there were more than 3.6 million hospitalizations and more than 100,000 deaths due to RSV infection in children under 5 years of age worldwide, and 36% of deaths in infants aged 1-6 months were related to RSV infection. RSV has brought a heavy disease burden to infants, children and the elderly. Due to its high infection rate and high incidence of severe illness, it has become a serious public health problem. Currently, there is still a lack of specific treatments in clinical practice.

[0004] Active vaccination or passive immunity using antibody preparations is an important measure to avoid severe infection and reduce death. WHO has listed the development of RSV vaccine as one of its priority projects, and the development of a safe and effective RSV vaccine is also an urgent clinical need in my country.

[0005] RSV surface fusion protein (F) and adhesion glycoprotein (G) are the only viral components that can induce RSV to produce neutralizing antibodies, and therefore are important targets for vaccinology research. Among them, the F protein is highly conserved and mediates the fusion of the viral envelope and the cell membrane through conformational changes. The F protein is expressed on the surface of the viral envelope using a metastable prefusion F protein (Prefusion Fusion Protein, PreF, Pre-F). When in contact with the host cell membrane, the structure is rearranged into a highly stable postfusion F protein (Postfusion Fusion Protein, PostF). Compared with PostF, PreF has a stronger ability to induce neutralizing antibodies. Studies on high- and medium-neutralizing antibodies have found that they mainly recognize the conformational epitopes of PreF, and some high- and medium-neutralizing antibodies only recognize PreF, not PostF. Therefore, PreF has become the preferred choice for RSV vaccines as a target antigen. However, Pre-F is a metastable structure, which is extremely unstable and easily converted into PostF. Solving the conformational stability of PreF has been an important difficulty in the development of RSV vaccines for decades.

[0006] Among them, Novavax modified the F protein and designed a "Pre-fusogenic" conformation, believing that this is an intermediate conformation between pre-fusion and post-fusion. However, the Phase III clinical trials of the vaccine for the elderly and pregnant women all ended in failure, mainly due to the design of the vaccine antigen structure. GSK and Pfizer used modified stable PreF to develop RSV vaccines. Phase III clinical trials showed that both were safe and effective; this further proves that a stable PreF protein conformation is crucial to the effectiveness of RSV vaccines.

[0007] At present, there is no RSV vaccine on the market in China. In the GSK patent (CN112566654A), recombinant RSV soluble F protein is used as the antigen to prepare a vaccine composition. The results of its stability study showed that when the liquid dosage form is used and stored at 5°C for 1 month, the PreF protein site Loss occurs (i.e. the conformation of the PreF protein changes). However, the use of a lyophilized formulation can maintain the long-term stability of the PreF protein. AREXVY and ABRYSVO, which have been launched overseas, are also lyophilized preparations. However, the production process of lyophilized vaccines is complex, costly, and the medication process is cumbersome.

[0008] Compared with freeze-dried preparations, RSV vaccines in liquid form can better meet the needs of clinical use. Therefore, how to improve the stability of RSV vaccine liquid preparations is an urgent problem to be solved.

[0009] In addition, my country currently uses traditional aluminum adjuvants to prepare vaccines, but aluminum adjuvants cannot stimulate cellular immunity. Therefore, there is an urgent need to develop vaccines based on new adjuvants that can induce high-titer neutralizing antibodies and cellular immune responses. and The comparison results of clinical data also verified the key role of the new adjuvant system in RSV vaccine development.

[0010] In summary, the development of a new adjuvant and a RSV all-liquid vaccine based on the new adjuvant is of great significance for the prevention and treatment of RSV. Summary of the invention

[0011] In order to solve the above problems, the present invention provides a recombinant respiratory syncytial virus vaccine, wherein the recombinant respiratory syncytial virus vaccine is a liquid dosage form vaccine preparation including pre-F protein antigen;

[0012] Wherein, the amino acid sequence of the pre-F protein antigen is any one of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3;

[0013] Preferably, the content of the pre-F protein antigen in a unit dose of the recombinant respiratory syncytial virus vaccine is 120 μg / mL to 480 μg / mL;

[0014] Preferably, the osmotic pressure of the recombinant respiratory syncytial virus vaccine is 210mOsmol / kg to 420mOsmol / kg;

[0015] Preferably, the average particle size of the recombinant respiratory syncytial virus vaccine is 60nm to 220nm;

[0016] Preferably, the average particle size of the recombinant respiratory syncytial virus vaccine is 80nm to 160nm;

[0017] Preferably, the pH value of the recombinant respiratory syncytial virus vaccine is 6.0 to 7.0;

[0018] Preferably, the recombinant respiratory syncytial virus vaccine further comprises: pharmaceutical excipients and adjuvant compositions.

[0019] Preferably, in the unit dose of the recombinant respiratory syncytial virus vaccine, the pharmaceutical excipients include components in the following concentrations:

[0020] Polysorbate 80, 100 μg / mL to 500 μg / mL;

[0021] Sucrose or trehalose, 20mg / mL~100mg / mL;

[0022] Sodium chloride, 1.3mg / mL~9mg / mL.

[0023] Preferably, in a unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition comprises components at the following concentrations:

[0024] DOPC, 700μg / mL~3000μg / mL;

[0025] Cholesterol, 175 μg / mL to 750 μg / mL;

[0026] QS-21, 35μg / mL~120μg / mL.

[0027] Preferably, in a unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition comprises components at the following concentrations:

[0028] DOPC, 1200μg / mL~2600μg / mL;

[0029] Cholesterol, 300 μg / mL to 650 μg / mL;

[0030] QS-21, 50μg / mL~100μg / mL.

[0031] Preferably, in a unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition comprises components at the following concentrations:

[0032] DOTAP, 70μg / mL~240μg / mL;

[0033] DOPC, 500μg / mL~2000μg / mL;

[0034] Cholesterol, 175 μg / mL to 600 μg / mL;

[0035] QS-21, 50μg / mL~120μg / mL;

[0036] Polysorbate 80, 100 μg / mL to 500 μg / mL;

[0037] Sucrose, 20 mg / mL to 100 mg / mL;

[0038] Sodium chloride, 1 mg / mL to 9 mg / mL;

[0039] Poly I:C, 400μg / mL~900μg / mL.

[0040] Preferably, in a unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition comprises components at the following concentrations:

[0041] DOTAP, 100μg / mL~175μg / mL;

[0042] DOPC, 1200μg / mL~2000μg / mL;

[0043] Cholesterol, 350 μg / mL to 600 μg / mL;

[0044] QS-21, 60μg / mL~100μg / mL;

[0045] Polysorbate 80, 300 μg / mL to 500 μg / mL;

[0046] Sucrose, 30 mg / mL to 75 mg / mL;

[0047] Sodium chloride, 3 mg / mL to 7 mg / mL;

[0048] Poly I:C, 550μg / mL~900μg / mL.

[0049] In addition, to solve the above problems, the present invention also provides a method for preparing the recombinant respiratory syncytial virus vaccine as described above, comprising:

[0050] S1, taking the purified pre-F protein antigen, adding polysorbate 80, sucrose or trehalose, and sodium chloride among the pharmaceutical excipients, and adjusting the pH to obtain an antigen stock solution;

[0051] S2. According to DOPC and cholesterol in the adjuvant composition, neutral liposomes are prepared; or, according to DOTAP, DOPC and cholesterol in the adjuvant composition, neutral liposomes and cationic liposomes are prepared respectively;

[0052] S3, taking the product prepared in step S1 and step S2, adding the remaining raw materials in the adjuvant composition, mixing and packaging, and obtaining the recombinant respiratory syncytial virus vaccine;

[0053] Wherein, in the step S2, when neutral liposomes are prepared according to DOPC and cholesterol in the adjuvant composition, the remaining raw material in the adjuvant composition is QS-21;

[0054] In step S2, when neutral liposomes and cationic liposomes are prepared respectively according to DOTAP, DOPC and cholesterol in the adjuvant composition, the remaining raw materials in the adjuvant composition are QS-21, polysorbate 80, sucrose, sodium chloride and Poly I:C.

[0055] Preferably, in step S2, neutral liposomes are prepared according to DOPC and cholesterol in the adjuvant composition, comprising:

[0056] S21, taking DOPC and cholesterol, mixing them according to a mass ratio of 4:1, and dispersing them to obtain a first lipid component;

[0057] S22, preparing the neutral liposome by using any one of an ethanol injection method, a thin film dispersion method, an ultrasonic dispersion method, and a reverse evaporation method to the first lipid component;

[0058] In the step S2, neutral liposomes and cationic liposomes are prepared respectively according to DOTAP, DOPC and cholesterol in the adjuvant composition, comprising:

[0059] The neutral liposomes are prepared according to the method of step S21 and step S22; and the cationic liposomes are prepared by the following method:

[0060] S23, mixing DOTAP, DOPC and cholesterol in a mass ratio of 2:2:1 to obtain a second lipid component;

[0061] S24, preparing the cationic liposomes by using any one of an ethanol injection method, a thin film dispersion method, an ultrasonic dispersion method and a reverse evaporation method on the second lipid component.

[0062] Preferably, the ethanol injection method is used in both step S22 and step S24 to prepare the neutral liposome and the cationic liposome respectively;

[0063] The ethanol injection method comprises:

[0064] The neutral liposome or the cationic liposome is obtained by extruding the first lipid component or the second lipid component using extrusion membranes with pore sizes of 200 nm and 100 nm, respectively, and then filtering and sterilizing.

[0065] Preferably, the first lipid component or the second lipid component is extruded using an extrusion membrane with a pore size of 200 nm and 100 nm in sequence, and then filtered and sterilized to obtain the neutral liposome or the cationic liposome, comprising:

[0066] Dissolving the first lipid component or the second lipid component in ethanol, and performing online shearing with the aqueous phase for 1 to 3 times to obtain a sheared raw material;

[0067] The sheared raw material is subjected to 200 nm liposome extrusion treatment, and filtered using a filter membrane with a pore size of 200 nm to obtain a 200 nm extrudate;

[0068] The 200 nm extrudate is subjected to 100 nm liposome extrusion treatment, and filtered using a filter membrane with a pore size of 100 nm to obtain a 100 nm extrudate;

[0069] The 100 nm extrudate is subjected to ultrafiltration replacement with a phosphate buffer solution, and is filtered and sterilized using a sterile filter to obtain the neutral liposome; and the 100 nm extrudate is subjected to ultrafiltration replacement with an acetate buffer solution, and is filtered and sterilized using a sterile filter to obtain the cationic liposome;

[0070] Preferably, the concentration multiple of the ultrafiltration substitution is 3 to 6 times, and the diafiltration multiple is 6 to 8 times;

[0071] Preferably, the average particle size D50 of the neutral liposome is 80nm to 140nm;

[0072] Preferably, the pH value of the neutral liposome solution is 5.5 to 7.0;

[0073] Preferably, the pH value of the neutral liposome solution is 6.4 to 6.6;

[0074] Preferably, the average particle size D50 of the cationic liposome is 60nm to 120nm;

[0075] Preferably, the pH value of the solution of the cationic liposome is 4.5 to 6.5;

[0076] Preferably, the Zeta potential of the cationic liposome is 50 mV to 90 mV.

[0077] In addition, the present invention also provides a use of the recombinant respiratory syncytial virus vaccine as described above in the preparation of products for treating and / or preventing diseases caused by respiratory syncytial virus, wherein the products include vaccine products, drugs, diagnostic reagents and detection kits.

[0078] The present invention provides a recombinant respiratory syncytial virus vaccine and a preparation method and application thereof, wherein the recombinant respiratory syncytial virus vaccine is a liquid dosage vaccine preparation comprising a pre-F protein antigen; wherein the amino acid sequence of the pre-F protein antigen is any one of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.7. The recombinant respiratory syncytial virus vaccine provided by the present invention is a liquid vaccine preparation comprising a recombinant respiratory syncytial virus (RSV) protein stock solution, and the vaccine preparation has better stability. The immune experimental study of BALB / c mice administered by intramuscular injection proves that the homemade recombinant respiratory syncytial virus vaccine has good immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1This is a graph showing the effect of different amounts of cholesterol on the hemolytic activity of QS-21 in Example 1 of the present invention (the OD570 value below the dotted line in the figure is judged as non-hemolytic, and the OD570 value above the dotted line is judged as hemolytic);

[0080] Figure 2 This is a graph showing the results of DOPC and cholesterol content at 2°C to 8°C and 37°C for one week in Example 1 of the present invention;

[0081] Figure 3 This is a graph showing the results of QS-21 content in Example 1 of the present invention when placed at 2°C to 8°C and at 37°C for one week;

[0082] Figure 4 This is a bar graph of IgG and its subtype antibody titers after the second immunization in each experimental group in Example 9 of the present invention;

[0083] Figure 5 The bar graph of the number of immune cells (CD4 + T cell response);

[0084] Figure 6 The bar graph of the number of immune cells (CD8 + T cell response);

[0085] Figure 7 This is a bar graph of the neutralizing antibody titer of each experimental group after the second immunization in Example 11 of the present invention.

[0086] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0087] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0088] A recombinant respiratory syncytial virus vaccine is provided in an embodiment of the present application. The recombinant respiratory syncytial virus vaccine is a liquid dosage form vaccine preparation including a pre-F protein antigen;

[0089] Wherein, the amino acid sequence of the pre-F protein antigen is any one of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3.

[0090] In the embodiment of the present application, the pre-F protein antigen is numbered 901, and the corresponding amino acid sequence is SEQ ID NO.1; the pre-F protein antigen is numbered 902, and the corresponding amino acid sequence is SEQ ID NO.2; the pre-F protein antigen is numbered 903, and the corresponding amino acid sequence is SEQ ID NO.3.

[0091] The above-mentioned recombinant respiratory syncytial virus vaccine is a RSV vaccine preparation. Among them, the vaccine preparation provided in the present invention is a liquid dosage form, and can achieve the same technical effect of long-term stability as the freeze-dried dosage form.

[0092] Furthermore, the content of the pre-F protein antigen in the unit dose of the recombinant respiratory syncytial virus vaccine is 120 μg / mL to 480 μg / mL; for example, it can be 120 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 480 μg / mL and the like.

[0093] It should be noted that the unit dose of the recombinant respiratory syncytial virus vaccine in the present invention can be 0.5 mL, that is, 0.5 mL per dose of vaccine.

[0094] Further, the osmotic pressure of the recombinant respiratory syncytial virus vaccine is 210mOsmol / kg to 420mOsmol / kg. For example, it can be 210mOsmol / kg, 220mOsmol / kg, 250mOsmol / kg, 280mOsmol / kg, 300mOsmol / kg, 320mOsmol / kg, 350mOsmol / kg, 380mOsmol / kg, 400mOsmol / kg, 410mOsmol / kg, 420mOsmol / kg, and the like.

[0095] Furthermore, the average particle size of the recombinant respiratory syncytial virus vaccine is 60nm to 220nm; for example, it can be 60nm, 80nm, 100nm, 120nm, 140nm, 160nm, 180nm, 200nm, 220nm, etc.

[0096] Furthermore, the average particle size of the recombinant respiratory syncytial virus vaccine is 80nm to 160nm; for example, it can be 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, etc.

[0097] Furthermore, the pH value of the recombinant respiratory syncytial virus vaccine is 6.0 to 7.0, for example, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, etc.

[0098] Furthermore, the recombinant respiratory syncytial virus vaccine also includes: pharmaceutical excipients and adjuvant compositions.

[0099] The recombinant respiratory syncytial virus vaccine provided in the present invention may include the following three main components: (1) pre-F protein antigen; (2) pharmaceutical excipients; and (3) adjuvant composition.

[0100] Furthermore, the amino acid sequence of protein antigen 1 is any one of the following amino acid sequences: SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0101] The recombinant respiratory syncytial virus vaccine provided by the present invention is obtained by adding an adjuvant composition to the recombinant respiratory syncytial virus (RSV) protein stock solution to obtain a liquid dosage form of the recombinant respiratory syncytial virus vaccine, which has better stability than the freeze-dried vaccine preparation. The immune experimental study of BALB / c mice injected intramuscularly proved that the homemade recombinant respiratory syncytial virus vaccine has good immunogenicity.

[0102] Furthermore, in the unit dose of the recombinant respiratory syncytial virus vaccine, the pharmaceutical excipients include components in the following concentrations:

[0103] (1) Polysorbate 80, 100 μg / mL to 500 μg / mL; for example, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, etc.

[0104] (2) Sucrose or trehalose, 20 mg / mL to 100 mg / mL; for example, 20 mg / mL, 40 mg / mL, 60 mg / mL, 80 mg / mL, 100 mg / mL, etc.

[0105] (3) Sodium chloride, 1.3 mg / mL to 9 mg / mL. For example, 1.3 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, etc.

[0106] In the present invention, the pharmaceutical excipients mainly include polysorbate 80, sucrose or trehalose, and sodium chloride, which can be used to maintain the stability of the vaccine. Among them, polysorbate 80, that is, Tween 80, is an organic substance with a chemical formula of C 64 H 124 O26 It is a non-ionic surfactant with a peculiar smell, warm and slightly bitter. It is one of the polyoxyethylene sorbitan fatty acid ester series products.

[0107] As mentioned above, the two auxiliary materials, sucrose and trehalose, can be used interchangeably, and can also be used in combination.

[0108] Further, in the vaccine preparation, for the adjuvant composition, the following two combinations are provided, which can be a first adjuvant composition (adjuvant 1) and a second adjuvant composition (adjuvant 2), respectively as follows:

[0109] 1. The first adjuvant composition:

[0110] In a unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition comprises components at the following concentrations:

[0111] (1) DOPC, 700 μg / mL to 3000 μg / mL; for example, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1000 μg / mL, 1500 μg / mL, 2000 μg / mL, 2500 μg / mL, 3000 μg / mL, etc.;

[0112] (2) Cholesterol, 175 μg / mL to 750 μg / mL; for example, 175 μg / mL, 180 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, 550 μg / mL, 600 μg / mL, 700 μg / mL, 750 μg / mL, etc.;

[0113] (3) QS-21, 35 μg / mL to 120 μg / mL. For example, it can be 35 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, etc.

[0114] The above-mentioned adjuvant composition can be used to improve the antibody level and cellular immunity level induced by antigen, and includes DOPC, cholesterol and QS-21.

[0115] As mentioned above, DOPC is the abbreviation of dioleoylphosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine). It is a phospholipid substance used in the biomedical field and drug research. DOPC is a phospholipid whose main component comes from natural sources and has important physiological functions in cell membranes. It is one of the main components in the phospholipid bilayer and can regulate the stability, fluidity and permeability of the cell membrane. As a phospholipid substance, DOPC consists of two oleic acid groups and one phosphate group with choline. The choline part is hydrophilic and can interact with water molecules, while the oleic acid group is hydrophobic. This allows DOPC molecules to self-assemble in the form of a bilayer phospholipid structure in an aqueous environment to form the basic structural unit of the cell membrane.

[0116] As mentioned above, QS-21 is a saponin compound with multiple biological activities and pharmacological effects. It is mainly used as an immune adjuvant, which can enhance the immunogenicity of vaccines and promote immune response. In the present invention, QS-21 is used together with antigens to improve the immunogenicity of antigens, activate the immune system, and increase the immune response to vaccines in vivo.

[0117] Further, in the unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition includes components at the following concentrations:

[0118] (1) DOPC, 1200 μg / mL~2600 μg / mL; (2) Cholesterol, 300 μg / mL~650 μg / mL; (3) QS-21, 50 μg / mL~100 μg / mL.

[0119] The above is a preferred embodiment of the first adjuvant composition for use in vaccine preparations for improving the antibody level and cellular immunity level induced by antigens.

[0120] 2. The second adjuvant composition:

[0121] In a unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition comprises components at the following concentrations:

[0122] (1) DOTAP, 70 μg / mL to 240 μg / mL; for example, 70 μg / mL, 90 μg / mL, 100 μg / mL, 120 μg / mL, 140 μg / mL, 180 μg / mL, 200 μg / mL, 220 μg / mL, 240 μg / mL, etc.;

[0123] (2) DOPC, 500 μg / mL to 2000 μg / mL; for example, 500 μg / mL, 800 μg / mL, 1000 μg / mL, 1200 μg / mL, 1500 μg / mL, 1700 μg / mL, 2000 μg / mL, etc.

[0124] (3) Cholesterol, 175 μg / mL to 600 μg / mL; for example, 175 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, etc.;

[0125] (4) QS-21, 50 μg / mL to 120 μg / mL; for example, 50 μg / mL, 60 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, etc.;

[0126] (5) Polysorbate 80, 100 μg / mL to 500 μg / mL; for example, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, etc.;

[0127] (6) Sucrose, 20 mg / mL to 100 mg / mL; for example, 20 mg / mL, 30 mg / mL, 50 mg / mL, 60 mg / mL, 80 mg / mL, 100 mg / mL, etc.;

[0128] (7) Sodium chloride, 1 mg / mL to 9 mg / mL; for example, 1 mg / mL, 3 mg / mL, 5 mg / mL, 7 mg / mL, 9 mg / mL, etc.;

[0129] (8) Poly I: C, 400 μg / mL to 900 μg / mL. For example, it can be 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, etc.

[0130] The above provides an adjuvant composition, which can be used to improve the antibody level and cellular immunity level induced by antigen, and includes DOTAP, DOPC, cholesterol and QS-21, polysorbate 80, sucrose, sodium chloride and Poly I:C.

[0131] As mentioned above, the full name of DOTAP is 1,2-dioleoyl-3-trimethylammonium-propane. It is a synthetic cationic surfactant.

[0132] As mentioned above, poly I:C is a synthetic double-stranded RNA, an interferon inducer with antiviral and immunomodulatory functions. It is used for chronic hepatitis B, hemorrhagic fever, epidemic encephalitis B, viral keratitis, herpes zoster, various warts and respiratory tract infections, etc.

[0133] Further, in the unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition includes components at the following concentrations:

[0134] (1) DOTAP, 100 μg / mL~175 μg / mL; (2) DOPC, 1200 μg / mL~2000 μg / mL; (3) Cholesterol, 350 μg / mL~600 μg / mL; (4) QS-21, 60 μg / mL~100 μg / mL; (5) Polysorbate 80, 300 μg / mL~500 μg / mL; (6) Sucrose, 30 mg / mL~75 mg / mL; (7) Sodium chloride, 3 mg / mL~7 mg / mL; (8) Poly I:C, 550 μg / mL~900 μg / mL.

[0135] The above is a preferred embodiment of the second adjuvant composition for use in vaccine preparations for improving the antibody level and cellular immunity level induced by antigens.

[0136] In summary, in the present invention, the first adjuvant composition (mainly composed of QS-21 and liposomes) or the second adjuvant composition (mainly composed of Poly I:C, QS-21 and liposomes) independently developed can induce high-titer neutralizing antibodies and cellular immune responses.

[0137] At the same time, by adding pharmaceutical excipients such as disaccharides and surfactants, the PreF protein conformation in the vaccine preparation can remain stable even if it is stored for a long time, which greatly enhances the stability of the protein in the liquid preparation and avoids the problem that the existing vaccine uses liquid dosage forms and is stored at 5°C for a long time (1 month or more). Loss occurs, i.e., a defect occurs in which the conformation of the PreF protein changes.

[0138] Compared with the currently available RSV products, the vaccine preparation prepared by the present invention adopts the first adjuvant composition or the second adjuvant composition as an adjuvant, and together with pharmaceutical excipients and pre-F protein antigens, constitutes a vaccine preparation, so that the preparation is in the form of a liquid single preparation, which is easy to use and has better stability.

[0139] In addition, the present invention also provides a method for preparing the recombinant respiratory syncytial virus vaccine as described above, comprising:

[0140] S1, taking the purified pre-F protein antigen, adding polysorbate 80, sucrose or trehalose, and sodium chloride among the pharmaceutical excipients, and adjusting the pH to obtain an antigen stock solution;

[0141] S2. According to DOPC and cholesterol in the adjuvant composition, neutral liposomes are prepared; or, according to DOTAP, DOPC and cholesterol in the adjuvant composition, neutral liposomes and cationic liposomes are prepared respectively;

[0142] S3, taking the product prepared in step S1 and step S2, adding the remaining raw materials in the adjuvant composition, mixing and packaging, and obtaining the recombinant respiratory syncytial virus vaccine;

[0143] Wherein, in step S2, there are the following two situations according to the different adjuvant compositions used:

[0144] (1) When the step S2 is: preparing neutral liposomes according to DOPC and cholesterol in the adjuvant composition, the remaining raw material in the adjuvant composition in the step S3 is QS-21;

[0145] (2) When the step S2 is: preparing neutral liposomes and cationic liposomes respectively according to DOTAP, DOPC and cholesterol in the adjuvant composition, the remaining raw materials in the adjuvant composition in step S3 are QS-21, polysorbate 80, sucrose, sodium chloride and Poly I:C.

[0146] Further in the preparation process, based on the difference between the treatment method in step S2 and the obtained product, the following two situations are included:

[0147] (1) In step S2, neutral liposomes are prepared according to DOPC and cholesterol in the adjuvant composition, comprising:

[0148] S21, taking DOPC and cholesterol, mixing them according to a mass ratio of 4:1, and dispersing them to obtain a first lipid component;

[0149] S22, preparing the neutral liposome by using any one of an ethanol injection method, a thin film dispersion method, an ultrasonic dispersion method, and a reverse evaporation method to the first lipid component;

[0150] (2) In the step S2, neutral liposomes and cationic liposomes are prepared respectively according to DOTAP, DOPC and cholesterol in the adjuvant composition, comprising:

[0151] The neutral liposomes are prepared according to the method of step S21 and step S22; and the cationic liposomes are prepared by the following method:

[0152] S23, mixing DOTAP, DOPC and cholesterol in a mass ratio of 2:2:1 to obtain a second lipid component;

[0153] S24, preparing the cationic liposomes by using any one of an ethanol injection method, a thin film dispersion method, an ultrasonic dispersion method and a reverse evaporation method on the second lipid component.

[0154] In the second case (2), neutral liposomes and cationic liposomes need to be prepared. Either one of the two products can be prepared first, or they can be prepared simultaneously. The order of preparation does not need to be limited.

[0155] Furthermore, the ethanol injection method is used in both step S22 and step S24 to prepare the neutral liposome and the cationic liposome respectively;

[0156] The ethanol injection method comprises:

[0157] The neutral liposome or the cationic liposome is obtained by extruding the first lipid component or the second lipid component using extrusion membranes with pore sizes of 200 nm and 100 nm, respectively, and then filtering and sterilizing.

[0158] Furthermore, the first lipid component or the second lipid component is extruded using an extrusion membrane with a pore size of 200 nm and 100 nm in sequence, and then filtered and sterilized to obtain the neutral liposome or the cationic liposome, comprising:

[0159] Dissolving the first lipid component or the second lipid component in ethanol, and performing online shearing with the aqueous phase for 1 to 3 times to obtain a sheared raw material;

[0160] The sheared raw material is subjected to 200 nm liposome extrusion treatment, and filtered using a filter membrane with a pore size of 200 nm to obtain a 200 nm extrudate;

[0161] The 200 nm extrudate is subjected to 100 nm liposome extrusion treatment, and filtered using a filter membrane with a pore size of 100 nm to obtain a 100 nm extrudate;

[0162] (1) ultrafiltration replacement of the 100 nm extrudate with phosphate buffer and filtration sterilization with a sterile filter to obtain the neutral liposome;

[0163] (2) and ultrafiltration replacement of the 100 nm extrudate with acetate buffer, and sterilization by filtration using a sterile filter to obtain the cationic liposomes;

[0164] Furthermore, the concentration multiple of the ultrafiltration substitution is 3 to 6 times; for example, it can be 3 times, 4 times, 5 times, 6 times, etc.

[0165] The diafiltration multiple is 6 to 8 times; for example, it can be 6 times, 7 times, 8 times, etc.

[0166] Furthermore, the average particle size of the neutral liposome is 80 nm to 140 nm; for example, it can be 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, etc.

[0167] Furthermore, the pH value of the neutral liposome solution is 5.5-7.0; for example, it can be 5.5, 5.8, 6.0, 6.2, 6.4, 6.8, 7.0, etc.

[0168] Furthermore, the pH value of the neutral liposome solution is 6.4-6.6; for example, it can be 6.4, 6.5, 6.6, etc.

[0169] Furthermore, the average particle size of the cationic liposome is 40 nm to 100 nm; for example, it can be 40 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, and the like.

[0170] Furthermore, the pH value of the solution of the cationic liposome is 4.5 to 6.5; for example, it can be 4.5, 5.0, 5.5, 6.0, 6.5, etc.

[0171] Furthermore, the zeta potential of the cationic liposome at pH 6.5 is 50 mV to 90 mV, for example, 50 mV, 55 mV, 60 mV, 65 mV, 70 mV, 75 mV, 80 mV, 85 mV, 90 mV, etc.

[0172] In addition, the present invention also provides a use of the recombinant respiratory syncytial virus vaccine as described above in the preparation of products for treating and / or preventing diseases caused by respiratory syncytial virus, wherein the products include vaccine products, drugs, diagnostic reagents and detection kits.

[0173] The above-mentioned products may include, but are not limited to, vaccine preparations, drugs, diagnostic reagents and test kits for treating and / or preventing diseases caused by respiratory syncytial virus. Among them, the drug may be a suitable dosage form for treating or preventing diseases caused by respiratory syncytial virus, and may be a single-use or combined-use drug.

[0174] The present invention is further described below by means of specific examples, but it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0175] Example 1: Stability study of antigens

[0176] In this example, the physical stability of the antigens and DS-Cav1 provided in the present application was investigated, wherein the 901 protein corresponds to the amino acid sequence of the pre-F protein antigen SEQ ID NO.1; the 902 protein corresponds to the amino acid sequence of the pre-F protein antigen SEQ ID NO.2; the 903 protein corresponds to the amino acid sequence of the pre-F protein antigen SEQ ID NO.3; the DS-Cav1 protein is an antigen protein based on the GSK patent (CN112566654A), and is investigated for comparison as a conventional technique.

[0177] Experimental method: The stability of 901, 902 and 903 antigen proteins was compared with that of DS Cav 1 antigen protein. They were placed at 37°C for 3 days and 7 days, at 50°C for 1 hour, and freeze-thawed for 3, 5 and 10 times, respectively. The protein purity and antigen content were examined by SEC and ELISA, respectively, to examine the antigen stability.

[0178] Experimental results:

[0179] Table 1-1. Results of stability study of DS Cav 1 antigen protein

[0180]

[0181] Table 1-2, 901 / antigen protein stability test results

[0182]

[0183] Table 1-3, 902 / antigen protein stability test results

[0184]

[0185] Table 1-4. Results of the 903 / antigen protein stability study

[0186]

[0187] (1) From the data in the above table, it can be seen that the purity and antigen content of DS Cav1 protein, 901 protein, 902 protein and 903 protein did not change significantly after being placed at 37°C for 3 days and 7 days and at 50°C for 1 hour;

[0188] (2) When placed at 70°C for 1 h, the protein purity and antigen content decreased significantly, with the degree of decrease being DSCav 1 protein > 901 protein > 903 protein > 902 protein;

[0189] (3) After freezing and thawing for 3, 5 and 7 times, the purity and antigen content of 901 protein, 902 protein and 903 protein did not change significantly. However, the purity and antigen content of DS Cav 1 protein showed a significant downward trend with the increase of freezing and thawing times.

[0190] In summary, it is shown that proteins 901 (SEQ ID NO.1), 902 (SEQ ID NO.2) and 903 (SEQ ID NO.3) all exhibit better stability under different conditions of 37°C, 50°C and freeze-thaw, and in horizontal comparison, protein 902 is slightly better than protein 903 and protein 901, and are significantly better than DS Cav 1 protein in conventional technology.

[0191] From the above data, it can be seen that 901, 902 and 903 proteins can maintain a certain stability under different conditions. The reasons can be analyzed as follows:

[0192] The Pre-F protein in this example, based on its amino acid sequence, and the DS Cav 1 protein, replaces one or more amino acid mutations in the internal cavity of the Pre-F protein with hydrophobic amino acids to fill the cavity, which can reduce the variability of the Pre-F conformation, thereby enhancing the stability of the Pre-F conformation; introduces one or more pairs of disulfide bond (cysteine ​​pair) mutations in the Pre-F protein monomer to reduce the variability of the Pre-F conformation, thereby enhancing the stability of the Pre-F conformation; introduces a heterologous trimerization domain at the C-terminus of the Pre-F protein extracellular domain, thereby enhancing the stability of the Pre-F conformation and the Pre-F trimer structure.

[0193] In addition, the Pre-F proteins in this embodiment, i.e., proteins 901 (SEQ ID NO.1), 902 (SEQ ID NO.2), and 903 (SEQ ID NO.3), further include one or more of the following reasons to enable them to further maintain stability: one or more hydrophobic amino acids at the contact interface of different Pre-F monomers of the Pre-F trimer are mutated and replaced with charged hydrophilic amino acids to increase the attraction between ions, or one or more charged hydrophilic amino acids are mutated and replaced with hydrophobic amino acids to reduce the repulsion between ions, thereby enhancing the stability of the Pre-F conformation and the structural stability of the Pre-F trimer; one or more pairs of disulfide bonds (cysteine ​​pairs) between Pre-F protein monomers are introduced to reduce the variability of the Pre-F conformation and increase the binding force between Pre-F monomers, thereby enhancing the stability of the Pre-F conformation and the structural stability of the Pre-F trimer; the fusion peptide and Pep27 peptide of the Pre-F protein are deleted, and the F1 subunit and F2 subunit of the Pre-F protein are genetically fused into a single chain, thereby enhancing the stability of the Pre-F conformation.

[0194] Example 2: Investigation of adjuvant prescription.

[0195] The first adjuvant composition is a liposome preparation containing a quillaja saponin (QS-21) immunopotentiating component. In this example, the formulation of the first adjuvant composition was determined by formulation screening.

[0196] (1) Changes in physical and chemical properties before and after the addition of QS-21:

[0197] According to the first adjuvant composition preparation process, three batches of neutral liposomes (DOPC and cholesterol) were used to add QS-21 respectively, and the changes in particle size distribution and Zeta potential before and after the addition of QS-21 were detected. The results are shown in the following table.

[0198] Wherein, the neutral liposome is prepared by the following method:

[0199] DOPC and cholesterol are mixed in a mass ratio of 4:1 and dispersed to obtain a first lipid component;

[0200] The neutral liposomes are prepared by using the ethanol injection method to inject the first lipid component.

[0201] According to the first adjuvant composition preparation process, three batches of neutral liposomes were used to add QS-21 respectively, and the changes in particle size distribution and Zeta potential before and after the addition of QS-21 were detected. The results are shown in the following table.

[0202] Table 2. Particle size distribution and Zeta potential before and after adding QS-21 in the preparation process of the first adjuvant composition

[0203]

[0204] As can be seen from the table above, after adding QS-21, the average particle size and Zeta potential of the first adjuvant composition did not change significantly, indicating that the addition of QS-21 had no significant effect on the particle size distribution and Zeta potential of the system.

[0205] (2) Changes in hemolytic activity after addition of QS-21:

[0206] Free QS-21 will bind to cholesterol on the cell membrane to form an irreversible complex, which will cause hemolysis when it binds to red blood cells. When QS-21 is adsorbed to cholesterol in neutral liposomes, it prevents it from binding to the cell membrane and reduces hemolytic activity.

[0207] In this experiment, the amount of QS-21 was fixed at 70 μg, mixed with different amounts of neutral liposomes (the amounts of cholesterol were 0 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg and 500 μg, respectively), and chicken red blood cells were added and incubated at room temperature for 30 minutes. The OD value of the supernatant was measured at 570 nm using a UV spectrophotometer. The higher the OD value, the stronger the hemolysis.

[0208] Table 3. Hemolytic activity test table of different samples

[0209] sample QS-21 addition amount (μg) Cholesterol content (μg) <![CDATA[OD 570 Value]]> Normal saline control 0 0 0.307 1 70 0 11.714 2 70 50 4.671 3 70 100 0.720 4 70 200 0.742 5 70 300 0.803 6 70 400 0.878 7 70 500 0.942

[0210] Note: The hemolysis standard is OD 570 ≥Blank control OD 570 value × 4.5 = 1.382

[0211] From the above table and Figure 1 It can be seen that QS-21 has strong hemolytic activity; with the addition of cholesterol, OD 570 When the amount of cholesterol added was ≥100 μg, OD 570 The values ​​were all lower than the hemolysis standard (1.382) and tended to be stable, indicating that 100 μg of cholesterol can fully adsorb 70 μg of QS-21 and make it non-hemolytic. Therefore, at least 100 μg of cholesterol is required for every 70 μg of QS-21.

[0212] (3) pH range of adjuvant system:

[0213] Samples with different pH values ​​were placed at 2°C-8°C and 37°C for 1 week, and the content of each adjuvant component was measured. The results are shown in the following table.

[0214] Table 4. Content of components in adjuvants at different pH conditions at 2℃~8℃ and 37℃ for 1 week

[0215]

[0216] From the above table and Figure 2 and Figure 3 It can be seen that in the pH range of 6.0 to 7.5, DOPC and cholesterol have no obvious changes at 2°C to 8°C. Their contents decrease to a certain extent after being placed at 37°C for one week, and there is no significant difference in the extent of decrease among samples with different pH values. Considering that the QS-21 content in the sample with pH 7.5 decreases most significantly, and QS-21 is easily hydrolyzed in an alkaline environment, the pH value of the product should not be higher than 7.0.

[0217] (4) Adjuvant dosage study:

[0218] Experimental grouping: SPF BALB / c mice were randomly divided into 8 groups, with 5 mice in each negative control group and 10 mice in each other vaccine groups, for a total of 75 animals. The dosing design for each group is as shown in the following table:

[0219] Table 5. Mouse grouping and immunization scheme

[0220] Group No. Experimental Group quantity Injection method Number of injections Injection dosage Group 1 RSV vaccine (with adjuvant)-1.5 times neutral liposome 10 Intramuscular injection 2 0.1 dose Group 2 RSV vaccine (with adjuvant) - 1x neutral liposome 10 Intramuscular injection 2 0.1 dose Group 3 RSV vaccine (with adjuvant)-0.5 times neutral liposome 10 Intramuscular injection 2 0.1 dose Group 4 RSV vaccine (with adjuvant)-0x neutral liposome 10 Intramuscular injection 2 0.1 dose Group 5 RSV vaccine (with adjuvant) - 100μg / mL QS-21 10 Intramuscular injection 2 0.1 dose Group 6 RSV vaccine (with adjuvant) - 40μg / mL QS-21 10 Intramuscular injection 2 0.1 dose Group 7 RSV vaccine (with adjuvant) - 0μg / mL QS-21 10 Intramuscular injection 2 0.1 dose Group 8 Negative control 5 Intramuscular injection 2 0.1 dose

[0221] Note: The negative control was injected with normal saline. The above vaccines all used 902 protein, corresponding to SEQ ID No. 2. In Group 2, RSV vaccine (with adjuvant) - 1 times neutral liposomes contained 70 μg / mL QS-21. The amounts of other groups were calculated accordingly based on this dose, the same below.

[0222] Immunization method: After the test sample is gently shaken before use, 50 μL of the test sample solution is drawn using a 300 μL disposable sterile syringe and needle and injected into the bilateral lateral thigh muscles of the mouse.

[0223] Immunization time: Two doses with an interval of 21 days.

[0224] Serum and spleen collection and detection: 14 days after the second immunization (D35), B2 blood was collected from each group of experimental animals, and serum was separated. The enzyme-linked immunosorbent assay (ELISA) was used to detect the titer of anti-RSV PreF protein (antigen) binding antibody IgG and its subtype antibody in the serum, and the rapid fluorescent focus inhibition test (RFFIT) was used to detect the titer of serum anti-RSV A2 virus neutralizing antibody. At the same time, the spleen of mice was separated on D35, and intracellular cytokine staining (ICS) was used to detect the CD4+ T cells that produce cytokines (IL-2 / IFN-γ / TNF-α). + T and CD8 + T cell immune response level.

[0225] The results are as follows:

[0226] Table 6. GMT results of serum specific IgG antibodies and subtype detection after the second immunization:

[0227] Group No. Test groups IgG IgG1 IgG2a IgG2b Group 1 RSV vaccine-1.5 times neutral liposome 3435669 1162846 429821 94404 Group 2 RSV vaccine-1x neutral liposome 3697919 967267 480554 89291 Group 3 RSV vaccine-0.5x neutral liposome 3984802 1231244 593520 122228 Group 4 RSV vaccine-0x neutral liposome 2516322 756188 412551 62999 Group 5 RSV Vaccine-100μg / mL QS-21 4592876 1261774 808419 132504 Group 6 RSV Vaccine - 40μg / mL QS-21 3012341 839730 311759 84279 Group 7 RSV Vaccine - 0μg / mL QS-21 5436 2021 50 204 Group 8 Negative control 300 50 50 50

[0228] The results showed that at the same QS-21 content, the serum antibody titers of various types were lowly correlated with the neutral liposome content, and there was no statistical difference in the serum antibody titers of the animals in different neutral liposome content groups. At the same neutral liposome content, the antibody titers still increased with the increase of QS-21 content, and there was no statistically significant difference between the QS-21=70μg / mL group and the QS-21=100μg / mL group, and between the QS-21=70μg / mL group and the QS-21=40μg / mL group.

[0229] Table 7. GMT results of anti-RSV A2 virus neutralizing antibody detection:

[0230] Group No. Test groups Anti-RSV A2 neutralizing antibodies Group 1 RSV vaccine-1.5 times neutral liposome 1311 Group 2 RSV vaccine-1x neutral liposome 1114 Group 3 RSV vaccine-0.5x neutral liposome 1549 Group 4 RSV vaccine-0x neutral liposome 854 Group 5 RSV Vaccine-100μg / mL QS-21 1885 Group 6 RSV Vaccine - 40μg / mL QS-21 970 Group 7 RSV Vaccine - 0μg / mL QS-21 22 Group 8 Negative control 20

[0231] The results showed that at the same QS-21 content, the difference in anti-RSV A2 virus neutralizing antibody titer between groups with different neutral liposome contents was small. At the same neutral liposome content, the anti-RSV A2 virus neutralizing antibody titer was positively correlated with the QS-21 content in the adjuvant formula. When the QS-21 content in the adjuvant ranged from 40 μg / mL to 100 μg / mL, the anti-RSV A2 virus neutralizing antibody titer showed an upward trend, but there was no statistically significant difference between the groups.

[0232] Table 8, Cellular Immunity Test Results:

[0233] Group No. Test groups <![CDATA[CD4 + T]]> <![CDATA[CD8 + T]]> Group 1 RSV vaccine-1.5 times neutral liposome 0.0278％ 0.0844％ Group 2 RSV vaccine-1x neutral liposome 0.0288％ 0.1295％ Group 3 RSV vaccine-0.5x neutral liposome 0.0346％ 0.0635％ Group 4 RSV vaccine-0x neutral liposome 0.0208％ 0.0471％ Group 5 RSV Vaccine-100μg / mL QS-21 0.0430％ 0.1044％ Group 6 RSV Vaccine - 40μg / mL QS-21 0.0207％ 0.0452％ Group 7 RSV Vaccine - 0μg / mL QS-21 0.0023％ 0.0023％ Group 8 Negative control 0.0007％ 0.0008％

[0234] The results showed that at the same QS-21 content, CD4 + / CD8 + The T cell immune response was poorly correlated with the neutral liposome content. The neutral liposome 0.5-fold group induced CD4 + The T cell immune response was higher than that of the other neutral liposome content groups. The neutral liposome 1x group induced CD8 + T cell immune response was higher than that of other neutral liposome content groups. + The T cell immune response was positively correlated with the QS-21 content. The QS-21=100μg / mL group induced CD4 + The T cell immune response was higher than that of the other QS-21 content groups. The QS-21 = 70 μg / mL group induced CD8 + The T cell immune response was higher than that of the other QS-21 content groups.

[0235] Therefore, the concentration range of QS-21 is 40μg / mL to 100μg / mL, and the concentration range of neutral liposomes is 0.5 to 1.5 times, that is, DOPC: 1000μg / mL to 3000μg / mL; cholesterol: 250μg / mL to 750μg / mL. Considering that 100μg of cholesterol in Example 2 can sufficiently adsorb 70μg of QS-21, the lowest cholesterol concentration for 100μg / mL QS-21 is 150μg / mL, and the lowest DOPC concentration is 750μg / mL.

[0236] Example 3: Antigen dosing studies.

[0237] Experimental grouping: SPF BALB / c mice were randomly divided into 7 groups, with 5 mice in the negative control group and 10 mice in the other vaccine groups, for a total of 65 animals. The dosing design for each group is as follows:

[0238] Table 9. Mouse grouping and immunization scheme

[0239] Group No. Experimental Group quantity Injection method Number of injections Injection dosage Group 1 No adjuvant - low antigen dose 10 Intramuscular injection 2 0.1 dose Group 2 No adjuvant - Medium antigen dose 10 Intramuscular injection 2 0.1 dose Group 3 No adjuvant - high antigen dose 10 Intramuscular injection 2 0.1 dose Group 4 Adjuvanted-Low Antigen Dose 10 Intramuscular injection 2 0.1 dose Group 5 Adjuvant-containing antigen dose 10 Intramuscular injection 2 0.1 dose Group 6 Adjuvanted-High Antigen Dose 10 Intramuscular injection 2 0.1 dose Group 7 Negative control 5 Intramuscular injection 2 0.1 dose

[0240] Note: The negative control was injected with saline, and the antigen was 902 protein, corresponding to SEQ ID No. 2. Adjuvant 1 (500 μg / mL cholesterol, 70 μg / mL QS-21, 2000 μg / mL DOPC) was used.

[0241] Immunization method: After the test sample is gently shaken before use, 50 μL of the test sample solution is drawn using a 300 μL disposable sterile syringe and needle and injected into the bilateral lateral thigh muscles of the mouse.

[0242] Immunization time: Two doses with an interval of 20 days.

[0243] Serum and spleen collection and detection: 14 days after the second immunization (D34), B2 blood was collected from each group of experimental animals, and serum was separated. The enzyme-linked immunosorbent assay (ELISA) was used to detect the titer of anti-RSV PreF protein (antigen) binding antibody IgG and its subtype antibody in the serum, and the rapid fluorescent focus inhibition test (RFFIT) was used to detect the titer of serum anti-RSV A2 virus neutralizing antibody. At the same time, the spleen of mice was separated on D34, and intracellular cytokine staining (ICS) was used to detect the CD4+ T cells that produce cytokines (IL-2 / IFN-γ / TNF-α). + T and CD8 + T cell immune response level. The results are as follows:

[0244] Table 10. GMT results of serum specific IgG antibodies and subtype detection after the second immunization:

[0245] Group No. Test groups IgG IgG1 IgG2a IgG2b Group 1 No adjuvant - low antigen dose 5454 987 125 206 Group 2 No adjuvant - Medium antigen dose 10740 4541 286 399 Group 3 No adjuvant - high antigen dose 58099 36059 692 1009 Group 4 Adjuvanted-Low Antigen Dose 2589975 519003 475829 78676 Group 5 Adjuvant-containing antigen dose 1864098 544035 338838 53026 Group 6 Adjuvanted-High Antigen Dose 3300048 927901 584499 80941 Group 7 Negative control 50 50 50 50

[0246] From the above, it can be seen that after the second immunization, each vaccine test group can induce the production of high levels of anti-RSV PreF protein (antigen) binding antibody IgG and its subtype antibody titers and are significantly higher than those of the negative control group.

[0247] The titers of various types of binding antibodies in the sera of animals in the non-adjuvanted vaccine groups were positively correlated with the antigen dose level, and were significantly higher in the high antigen dose group (240 μg / dose) than in the low antigen dose group (60 μg / dose).

[0248] The titers of various types of antibodies in the serum of animals in the adjuvanted vaccine groups reached saturation when the antigen dose was 60μg / dose, and there was no significant difference in the antibody titers among the high, medium and low antigen dose groups.

[0249] Table 11. GMT results of anti-RSV A2 virus neutralizing antibody detection:

[0250] Group No. Test groups Anti-RSV A2 neutralizing antibodies Group 1 No adjuvant - low antigen dose 27 Group 2 No adjuvant - Medium antigen dose 25 Group 3 No adjuvant - high antigen dose 36 Group 4 Adjuvanted-Low Antigen Dose 775 Group 5 Adjuvant-containing antigen dose 648 Group 6 Adjuvanted-High Antigen Dose 970 Group 7 Negative control 20

[0251] From the above, we can see that after the second immunization, the anti-RSV A2 virus neutralizing antibody titers induced by the test samples of the adjuvanted vaccine in mice were significantly higher than those of the negative control group, and there was no significant difference in the anti-RSV A2 virus neutralizing antibody titers between the high, medium and low antigen dose groups; the anti-RSV A2 virus neutralizing antibody titer levels of the test samples of the non-adjuvanted vaccine groups were equivalent to those of the negative control group.

[0252] Table 12, Cellular Immunity Test Results:

[0253] Group No. Test groups <![CDATA[CD4 + T]]> <![CDATA[CD8 + T]]> Group 1 No adjuvant - low antigen dose 0.0004％ 0.0004％ Group 2 No adjuvant - Medium antigen dose 0.0031％ 0.0006％ Group 3 No adjuvant - high antigen dose 0.0019％ 0.0007％ Group 4 Adjuvanted-Low Antigen Dose 0.0239％ 0.0527％ Group 5 Adjuvant-containing antigen dose 0.0212％ 0.0460％ Group 6 Adjuvanted-High Antigen Dose 0.0197％ 0.0423％ Group 7 Negative control 0.0008％ 0.0005％

[0254] From the above, we can know that each test sample containing adjuvant vaccine induces the production of CD4 + / CD8 + The level of T cell immune response was significantly higher than that of the negative control group, and the CD4 + / CD8 + There was no statistical difference in the level of T cell immune response; the non-adjuvanted vaccine induced CD4 + / CD8 + The level of T cell immune response was low and comparable to that of the negative control group.

[0255] Conclusion: After immunization of mice with RSV vaccines (containing adjuvants) at different antigen doses, they can induce high levels of binding antibodies, anti-RSV A2 virus neutralizing antibodies and cellular immune responses. The high, medium and low antigen dose vaccines containing adjuvants have comparable immunogenicity in mice. In the presence of adjuvants, the immune response induced at an antigen dose of 60 μg / dose is saturated.

[0256] After mice were immunized with RSV vaccines (without adjuvant) with different antigen doses, a lower level of binding antibodies was induced, and the binding antibody titer was positively correlated with the antigen dose level; high, medium, and low antigen dose vaccines without adjuvant failed to induce anti-RSV A2 virus neutralizing antibodies and cellular immune responses.

[0257] In summary, for this adjuvanted vaccine, the antigen dose selection range is 60 μg / dose to 240 μg / dose, that is, 120 μg / mL to 480 μg / mL.

[0258] Example 4: Sodium chloride concentration screening.

[0259] The sodium chloride concentration not only affects the osmotic pressure of the vaccine product, but may also affect the stability of the antigen protein. In this example, liquid preparations containing different sodium chloride concentrations were prepared, and the samples with different sodium chloride concentrations were placed at 2°C to 8°C and 37°C for 1 day and 1 week, and the antigen content was determined by ELISA (AM14).

[0260] Table 13. Antigen content under different sodium chloride conditions at 2℃~8℃ and 37℃ for 1 week

[0261]

[0262] Note: The antigen used is 902 protein, corresponding to SEQ ID No.2.

[0263] It can be seen from the above table that when the sodium chloride concentration ranges from 0.13% to 0.90%, the antigen content does not decrease when placed at 37°C for 1 day and 7 days respectively (which means that the PreF protein conformation is stable). Therefore, the sodium chloride concentration range can be selected as 0.13% to 0.90%.

[0264] It should be noted that since NaCl needs to be added to the antigen protein stock solution in advance to keep the stock solution stable, the overall sodium chloride content of the vaccine preparation can only be configured to a minimum of 0.13%, and cannot be reduced to within 0.13%. Therefore, 0.13% is used as the lower limit; in addition, if the total sodium chloride content in the vaccine preparation increases to more than 0.9%, the osmotic pressure of the vaccine preparation will be significantly higher than the physiological osmotic pressure. Therefore, based on the above reasons and the experimental results in this embodiment, the concentration (content) range of sodium chloride is finally determined to be 0.13% to 0.90%.

[0265] Example 5: Polysorbate 80 concentration screening.

[0266] In order to select the appropriate polysorbate 80 concentration, in this embodiment, vaccine formulations with different polysorbate 80 concentrations were designed according to the table below, and the protein purity and antigen content were determined by SEC and ELISA (AM14) methods, respectively, through a 37°C accelerated test.

[0267] Table 14 Accelerated stability (SEC) at different polysorbate 80 concentrations

[0268]

[0269] Note: The antigen used is 902 protein, corresponding to SEQ ID No.2.

[0270] As can be seen from the table above, after 14 days at 37°C, the protein purity of the group without polysorbate 80 did not change, but the antigen content decreased. In other groups containing polysorbate 80, there was no significant change in protein purity and antigen content. This shows that the concentration of Tween 80 at 0.01% to 0.05% can maintain the stability of protein antigens.

[0271] In the preparation and production of vaccine preparations, the concentration of polysorbate 80 in general vaccine preparations is not higher than 0.05%. Therefore, in this embodiment, the concentration of polysorbate 80 is limited to the range of 0.01% to 0.05%.

[0272] Example 6: Comparison of sucrose and trehalose.

[0273] Sucrose and trehalose may play a protective role on pre-F protein antigen. In order to compare the protective effects of sucrose and trehalose and select the appropriate concentration, vaccine formulations with different sucrose and trehalose concentrations were designed according to the table below. The antigen content was determined by ELISA (AM14) through a 28-day accelerated test at 37°C.

[0274] Table 15. Accelerated stability under different sucrose and trehalose concentrations

[0275]

[0276] Note: The antigen in the table uses 902 protein, corresponding to SEQ ID No.2.

[0277] As can be seen from the above table, the formula containing sucrose or trehalose, when placed at 37°C for 28 days, did not show significant changes in antigen content compared with the samples at 2°C to 8°C. However, after the control group was accelerated for 28 days, the antigen content decreased significantly compared with the samples at 2°C to 8°C, indicating that sucrose or trehalose improved the stability of the pre-F protein antigen.

[0278] In addition, sucrose in the concentration range of 2% to 10% or trehalose in the concentration range of 2% to 10% can provide sufficient protection for the pre-F protein.

[0279] Since the antibody stock solution contains disaccharides (sucrose and / or trehalose) to maintain stability, the sugar substances in the vaccine preparation cannot be prepared to less than 2%. Therefore, 2% is used as the lower limit of sugar substances in this embodiment; in addition, if the concentration of sucrose and / or trehalose exceeds 10%, the overall osmotic pressure of the vaccine preparation will be significantly higher than the physiological osmotic pressure. Therefore, the concentration (content) range of sucrose and / or trehalose is determined to be 2% to 10%.

[0280] Example 6: Preparation of recombinant respiratory syncytial virus vaccine.

[0281] The recombinant respiratory syncytial virus vaccine (containing the first adjuvant composition) was prepared according to the following table:

[0282] Table 16. Recombinant respiratory syncytial virus vaccine formula

[0283]

[0284] It should be noted that the pre-F stock solution of formula 1 is 901 protein stock solution, corresponding to SEQ ID No.1; the pre-F stock solution of formula 2 is 902 protein stock solution, corresponding to SEQ ID No.2; the pre-F stock solution of formula 3 is 903 protein stock solution, corresponding to SEQID No.3, the same below.

[0285] According to the above formula, phosphate buffer (prepared with sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and sucrose, as shown in the table above), neutral liposomes, QS-21 solution, polysorbate 80 solution, and pre-F stock solution are added in sequence, and the pH is adjusted to 6.0-7.0. After filtration and sterilization, the pre-filling filling machine is used to fill the syringes into 1mL pre-filled syringes (0.5mL each). The filled samples are labeled and outpackaged to obtain the recombinant respiratory syncytial virus vaccine.

[0286] Example 8: Preliminary stability study of recombinant respiratory syncytial virus vaccine.

[0287] In this example, an accelerated stability study was conducted on the homemade RSV vaccine (the vaccine preparation prepared in Example 7). The vaccine was placed at 37°C for 14 days and 28 days, and at 25°C for 1 month and 3 months, respectively. The changes in protein purity and antigen content were investigated using SEC and ELISA methods, respectively.

[0288] Table 17. Accelerated stability results

[0289]

[0290] It should be noted that the pre-F stock solution of formula 1 is 901 protein stock solution, corresponding to SEQ ID No.1; the pre-F stock solution of formula 2 is 902 protein stock solution, corresponding to SEQ ID No.2; the pre-F stock solution of formula 3 is 903 protein stock solution, corresponding to SEQID No.3; the antigen content unit is mg / mL. The protein purity is detected by SEC method; the antigen content is detected by Elisa (AM14) method.

[0291] As can be seen from the above table, there is no significant change in protein purity and antigen content.

[0292] It should be noted that, in this example, Elisa (AM14) did not decrease, indicating that the trimer conformation of pre-F did not change, which further showed that the homemade RSV vaccine was stable when placed at 37°C for 28 days and at 25°C for 3 months.

[0293] In the accelerated experiment of this embodiment, if the pre-F protein The loss of sites or the decomposition of trimers into monomers will cause a decrease in Elisa (AM14). Therefore, this example proves that the pre-F protein can maintain a stable pre-F trimer conformation in the current formulation. The stable pre-F trimer conformation is the key to the vaccine allowing the immune system to produce sufficient neutralizing antibodies.

[0294] Example 9: Mouse immunization test of respiratory syncytial virus vaccine.

[0295] Experimental grouping: SPF BALB / c mice were randomly divided into 10 groups, 10 mice in each group, with a total of 100 animals. The dosing design for each group is shown in the following table.

[0296] Table 18. Mouse grouping and immunization scheme

[0297] Experimental Group quantity Injection method Number of injections Injection dosage Adjuvant 2+901 protein stock solution (10μg) 10 Intramuscular injection 2 0.1 dose Adjuvant 2+903 protein stock solution (10μg) 10 Intramuscular injection 2 0.1 dose Adjuvant 2+902 protein stock solution (10μg) 10 Intramuscular injection 2 0.1 dose Adjuvant 2L + 902 protein stock solution (10μg) 10 Intramuscular injection 2 0.1 dose Adjuvant 2H+902 protein stock solution (10μg) 10 Intramuscular injection 2 0.1 dose Adjuvant 1+902 protein stock solution (10μg) 10 Intramuscular injection 2 0.1 dose MF59+902 protein stock solution (10μg) 10 Intramuscular injection 2 0.1 dose <![CDATA[Al(OH) 3 +902 protein stock solution (10μg)]]> 10 Intramuscular injection 2 0.1 dose Buffer + 902 protein stock solution (10 μg) 10 Intramuscular injection 2 0.1 dose 0.9% NaCl 10 Intramuscular injection 2 0.1 dose

[0298] Note: In the above table, it should be noted that the 901 protein stock solution corresponds to SEQ ID No. 1; the 902 protein stock solution corresponds to SEQ ID No. 2; and the 903 protein stock solution corresponds to SEQ ID No. 3. The buffer is 20 mM tris buffer.

[0299] In the table, adjuvant 1 corresponds to the first adjuvant composition (500 μg / mL cholesterol, 70 μg / mL QS-21, 2000 μg / mL DOPC), and adjuvant 2 corresponds to the second adjuvant composition; adjuvant 2L and adjuvant 2H are the low and high doses of adjuvant 2, respectively, and the corresponding doses of each component are as follows:

[0300] Table 19. Adjuvant 2L and Adjuvant 2H are the low and high doses of each component of Adjuvant 2, respectively.

[0301] Adjuvant DOTAP DOPC cholesterol QS-21 Poly I:C Adjuvant 2L 70 630 175 50 400 Adjuvant 2 140 1860 500 70 800 Adjuvant 2H 240 2000 560 120 900

[0302] Note: The dosage units of each adjuvant component in the above table are μg / mL.

[0303] Immunization method: After the test product is gently shaken before use, 100 μL of the test product solution is drawn using a 300 μL disposable sterile syringe and needle and injected into the bilateral lateral thigh muscles of the mouse.

[0304] Immunization time: Two doses with an interval of 14 days.

[0305] Serum and spleen collection and detection: Blood was collected from experimental animals in each test group 14 days after the second immunization (D28), and serum was separated and serum-specific IgG antibodies and antibody subtype titers were determined by enzyme-linked immunosorbent assay (ELISA) (see Example 9 for details). Mouse spleens were separated on D28 (the first 5 animals in each group), and antigen-specific CD4 + T and CD8 + T cell immune response (see Example 10 for details). The neutralization activity of the animal sera (first 5 animals in each group) 14 days after the second immunization against RSV virus was detected by immunoplaque assay (see Example 11 for details).

[0306] Example 10: ELISA detection of specific antibodies in mouse immunization test antiserum.

[0307] (1) Specific IgG antibody detection

[0308] The RSV PreF protein (antigen) was diluted to 4 μg / mL with coating solution, and 100 μL / well was coated on the ELISA plate at 4°C overnight. After washing 3 times with washing solution, 200 μL of blocking solution was added to each well and blocked at 25°C for 1 hour. After blocking, the blocking solution was poured out, and the mouse antiserum was diluted in multiples and added to the ELISA plate with mouse negative serum, 100 μL / well, and incubated at 25°C for 2 hours. After washing 3 times with washing solution, HRP-labeled goat anti-mouse IgG (1:2500) was added, 100 μL / well, and incubated at 25°C for 1 hour. After washing 5 times with washing solution, TMB substrate colorimetric solution was added, and the reaction was allowed to react at 25°C in the dark for 15 minutes. 100 μL of stop solution was added to stop the reaction. The OD of each well was measured using an enzyme reader. 450 The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of the sample.

[0309] (2) Specific IgG antibody subtype detection

[0310] RSV PreF protein (antigen) was diluted to 4 μg / mL with coating solution, and 100 μL / well was coated on the ELISA plate at 4°C overnight. After washing 3 times with washing solution, 200 μL of blocking solution was added to each well and blocked at 25°C for 1 hour. After blocking, the blocking solution was discarded, and the mouse antiserum was diluted in multiples and added to the ELISA plate with mouse negative serum, 100 μL / well, and incubated at 25°C for 2 hours. After washing 3 times with washing solution, biotin-labeled goat anti-mouse IgG1 (1:4000), IgG2a (1:2500), and IgG2b (1:5000) were added, 100 μL / well, and incubated at 25°C for 1 hour. After washing 3 times with washing solution, HRP-Labeled Streptavidin (1:10000) was added, and after washing 5 times with washing solution, TMB substrate colorimetric solution was added, and the reaction was incubated at 25°C in the dark for 15 minutes, and 100 μL of stop solution was added to terminate the reaction. Measure the OD of each well with an enzyme-labeled 450 The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of the sample.

[0311] Results: The specific IgG and its subtype antibody titers in serum were detected by ELISA. The bar graph of IgG and its subtype antibody titers in each experimental group after the second immunization (D28) is shown in Figure 4 After the second immunization, the serum IgG antibody and subtype titer levels of each test group were significantly higher than those of the negative control group. The serum IgG antibody and subtype titer of the test group prepared with antigen + adjuvant were significantly higher than those of the antigen group without adjuvant (buffer + 902). The serum IgG2a and IgG2b titers of the test group prepared with antigen + novel adjuvant (adjuvant 1, adjuvant 2) were significantly higher than those of Al(OH) 3 Adjuvant and MF59 adjuvant vaccine groups. The serum IgG antibody and subtype titer levels were comparable between the test groups with the same adjuvant (Adjuvant 2) and different sequence antigens (901, 902, 903). There was no significant difference in the serum IgG antibody and subtype titer between the test groups with the same antigen (902 protein stock solution) and different doses of adjuvant 2 (Adjuvant 2L, Adjuvant 2, Adjuvant 2H).

[0312] Example 11: Cytokine detection in mouse immunization assay.

[0313] After the mouse spleen was collected, it was stored in an EP tube containing 4 mL of maintenance medium. The spleen cells were collected by grinding in a biosafety cabinet and centrifuged at room temperature (531 g, 5 minutes). The collected cells were lysed by adding 5 mL of ACK lysis buffer for 3 minutes, and the cell washing solution was added and centrifuged at room temperature (531 g, 5 minutes). The collected cells were centrifuged again at room temperature (531 g, 5 minutes), the supernatant was discarded, the collected cells were resuspended and counted; after the counting was completed, the collected cells were centrifuged at room temperature (531 g, 5 minutes) to adjust the cell density to 2×10 7After adding cell stimulants, 100 μL / well was placed in a 96-well U-shaped cell culture plate at 37°C and 5% CO 2 Stimulate in the incubator for 1 hour; after stimulation, add 10 μL / well of Golgi plug working solution, adjust the final concentration to 1 μL / mL, and continue to incubate at 37℃5% CO 2 Culture in an incubator for 5 hours; centrifuge at room temperature (604g, 5 minutes), add 200μL / well D-PBS to resuspend the cells, centrifuge at room temperature (604g, 5 minutes) to stain live and dead cells, and then add 150μL / well D-PBS to resuspend the cells; centrifuge at room temperature (604g, 5 minutes) to collect the cells, add 50μL / well FACS block to block, and incubate at 4℃ in the dark for 5 minutes; after blocking, add 50μL / well surface antibody mixture to stain the cell surface, and incubate at 4℃ in the dark for 30 minutes; add 100μL / well FACS Buffer, collect cells by centrifugation at room temperature (604g, 5 minutes), add 100μL / well Cytofix / cytoperm, and incubate at 4℃ in dark for 20 minutes; add 100μL / well Perm / wash and centrifuge at room temperature (604g, 5 minutes) to collect cells, then add 50μL / well cytokine antibody mixture for staining, and incubate at 4℃ in dark for 30 minutes; add 150μL / well Perm / wash and centrifuge at room temperature (604g, 5 minutes) to collect cells, add 200μL / well Perm / wash and centrifuge at room temperature (750g, 3 minutes) to collect cells again, and finally add 150μL / well fixative to fix the cells and detect them by flow cytometer.

[0314] Results: On D28, spleens of mice were isolated and intracellular cytokine staining (ICS) was used to detect antigen-specific CD4 + T and CD8 + T cell immune response. The test results were analyzed using FlowJo software. Figure 5 and Figure 6 .

[0315] After the second immunization, except for MF59 and Al(OH) 3 In addition to adjuvant, each antigen + adjuvant test group can induce specific CD4 + / CD8 + T cell immune response, induction of CD4 + / CD8 + There was no significant difference in the level of T cell immune response (P>0.05). 3 Adjuvant vaccine group (Al(OH) 3No specific CD4 was detected in the antigen group without adjuvant (buffer + 902 protein solution) or negative control group (0.9% NaCl). + / CD8 + T cell immune response.

[0316] Example 12: Detection of serum anti-RSV virus neutralizing antibodies in mouse immunization assay.

[0317] The day before the experiment, cells were plated in a 24-well plate with 4×105 cells / well. The cells were cultured for about 16 hours. The serum samples to be tested were diluted in multiples with serum-free medium and 3×10 4 The virus amount of PFU / well was shaken and mixed, and then incubated in a 37℃ incubator for 1 hour. The cell culture medium was discarded, and 200μL / well serum-virus mixture was added to the 24-well plate, and the infection was carried out at 37℃ for 2 hours. The infection solution was discarded, and 1mL / well of 3% methylcellulose medium was added, and the culture was continued in a 37℃ incubator for 6 days. The methylcellulose medium in the well plate was discarded, and the residual methylcellulose medium was washed with PBS, and the tissue fixative was added, and fixed at room temperature overnight. The tissue fixative was discarded in the fume hood, and the residual liquid of the well plate was washed with PBS. After washing on a shaker for 5 minutes, the PBS was discarded, and the wash was washed 3 times in total. About 500uL / well blocking solution (5% milk, 0.2% Triten-100) was added for blocking, and the block was blocked on a shaker at room temperature for 1 hour. After the blocking was completed, the primary antibody diluted in the blocking solution (1:1000) was added and incubated in a 4℃ refrigerator for 2 hours. The primary antibody was discarded, and the shaker was used to wash 3 times with PBST (0.5% Tween-20), each time for 10 minutes. Add anti-goat secondary antibody (1:300) and incubate at room temperature for 1 hour, then wash 3 times on a PBST shaker, 10 minutes each time. Finally, use DBA colorimetric solution for 5 to 10 minutes, until obvious plaques can be seen, then rinse the colorimetric solution with water, drain and count the plaques. Calculate the PRNT50 of the serum (serum dilution that reduces the plaques caused by the virus by 50%).

[0318] Results: Neutralization activity of serum from animals 14 days after the second immunization against RSV virus was detected by immunoplaque assay. Figure 7 .

[0319] The serum neutralization titers of the test groups prepared with each antigen + novel adjuvant were significantly higher than those of the negative control group (0.9% NaCl), the antigen group without adjuvant (buffer + 902), the MF59 adjuvant vaccine group (MF59 + 902) and the Al(OH) 3 Adjuvant vaccine group (Al(OH) 3+902). The results of comparative analysis between the test groups of the same adjuvant (Adjuvant 2) with different sequence antigens (901, 902, 903) showed that the level of neutralizing antibodies induced by the group containing 902 antigen was higher than that of 901 and 903. The serum neutralizing antibody levels of the test groups of the same antigen (902 protein stock solution) with different doses of adjuvant 2 (Adjuvant 2L, Adjuvant 2, Adjuvant 2H) were comparable.

[0320] Conclusion: In summary, the independently developed vaccines with adjuvant 1 and adjuvant 2 can produce good specific immune responses in BALB / c mice. In short, the recombinant respiratory syncytial virus vaccine provided by the present invention is obtained by adding an adjuvant composition to the recombinant respiratory syncytial virus (RSV) protein stock solution to obtain a liquid dosage form of the recombinant respiratory syncytial virus vaccine. The immune experimental study of BALB / c mice by intramuscular injection proved that the homemade recombinant respiratory syncytial virus vaccine has good immunogenicity.

[0321] The above are preferred embodiments and corresponding examples of the present invention. It should be noted that, for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to the adjustment of the proportion, process, dosage and reaction vessel, such as the use of a continuous flow reactor, which all fall within the scope of protection of the present invention. The above are preferred embodiments and corresponding examples of the present invention. It should be noted that, for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to the adjustment of the proportion, process, dosage, which all fall within the scope of protection of the present invention.

Claims

1. A recombinant respiratory syncytial virus vaccine, characterized in that: The recombinant respiratory syncytial virus vaccine is a liquid dosage form vaccine preparation including pre-F protein antigen; Wherein, the amino acid sequence of the pre-F protein antigen is any one of the amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3; Preferably, the content of the pre-F protein antigen in a unit dose of the recombinant respiratory syncytial virus vaccine is 120 μg / mL to 480 μg / mL; Preferably, the osmotic pressure of the recombinant respiratory syncytial virus vaccine is 210mOsmol / kg to 420mOsmol / kg; Preferably, the average particle size of the recombinant respiratory syncytial virus vaccine is 60nm to 220nm; Preferably, the average particle size of the recombinant respiratory syncytial virus vaccine is 80nm to 160nm; Preferably, the pH value of the recombinant respiratory syncytial virus vaccine is 6.0 to 7.0; Preferably, the recombinant respiratory syncytial virus vaccine further comprises: pharmaceutical excipients and adjuvant compositions.

2. The recombinant respiratory syncytial virus vaccine according to claim 1, characterized in that In the unit dose of the recombinant respiratory syncytial virus vaccine, the pharmaceutical excipients include components in the following concentrations: Polysorbate 80, 100 μg / mL to 500 μg / mL; Sucrose or trehalose, 20mg / mL~100mg / mL; Sodium chloride, 1.3mg / mL~9mg / mL.

3. The recombinant respiratory syncytial virus vaccine according to claim 1, characterized in that In a unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition comprises components at the following concentrations: DOPC, 700μg / mL~3000μg / mL; Cholesterol, 175 μg / mL to 750 μg / mL; QS-21, 35μg / mL~120μg / mL.

4. The recombinant respiratory syncytial virus vaccine according to claim 1, characterized in that In a unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition comprises components at the following concentrations: DOPC, 1200μg / mL~2600μg / mL; Cholesterol, 300 μg / mL to 650 μg / mL; QS-21, 50μg / mL~100μg / mL.

5. The recombinant respiratory syncytial virus vaccine according to claim 1, characterized in that In a unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition comprises components at the following concentrations: DOTAP, 70μg / mL~240μg / mL; DOPC, 500μg / mL~2000μg / mL; Cholesterol, 175 μg / mL to 600 μg / mL; QS-21, 50μg / mL~120μg / mL; Polysorbate 80, 100 μg / mL to 500 μg / mL; Sucrose, 20 mg / mL to 100 mg / mL; Sodium chloride, 1 mg / mL to 9 mg / mL; Poly I:C, 400μg / mL~900μg / mL.

6. The recombinant respiratory syncytial virus vaccine according to claim 1, characterized in that In a unit dose of the recombinant respiratory syncytial virus vaccine, the adjuvant composition comprises components at the following concentrations: DOTAP, 100μg / mL~175μg / mL; DOPC, 1200μg / mL~2000μg / mL; Cholesterol, 350 μg / mL~600 μg / mL; QS-21, 60 μg / mL~100 μg / mL; Polysorbate 80, 300 μg / mL~500 μg / mL; Sucrose, 30 mg / mL~75 mg / mL; Sodium chloride, 3 mg / mL~7 mg / mL; Poly I:C, 550μg / mL~900μg / mL.

7. A method for preparing the recombinant respiratory syncytial virus vaccine according to any one of claims 1 to 6, characterized in that: include: S1, taking the purified pre-F protein antigen, adding polysorbate 80, sucrose or trehalose, and sodium chloride among the pharmaceutical excipients, and adjusting the pH to obtain an antigen stock solution; S2. According to DOPC and cholesterol in the adjuvant composition, neutral liposomes are prepared; or, according to DOTAP, DOPC and cholesterol in the adjuvant composition, neutral liposomes and cationic liposomes are prepared respectively; S3, taking the product prepared in step S1 and step S2, adding the remaining raw materials in the adjuvant composition, mixing and packaging, and obtaining the recombinant respiratory syncytial virus vaccine; Wherein, in the step S2, when neutral liposomes are prepared according to DOPC and cholesterol in the adjuvant composition, the remaining raw material in the adjuvant composition is QS-21; In step S2, when neutral liposomes and cationic liposomes are prepared respectively according to DOTAP, DOPC and cholesterol in the adjuvant composition, the remaining raw materials in the adjuvant composition are QS-21, polysorbate 80, sucrose, sodium chloride and Poly I:C; Preferably, in step S2, neutral liposomes are prepared according to DOPC and cholesterol in the adjuvant composition, comprising: S21, taking DOPC and cholesterol, mixing them according to a mass ratio of 4:1, and dispersing them to obtain a first lipid component; S22, preparing the neutral liposome by using any one of an ethanol injection method, a thin film dispersion method, an ultrasonic dispersion method, and a reverse evaporation method to the first lipid component; Preferably, in step S2, neutral liposomes and cationic liposomes are prepared respectively according to DOTAP, DOPC and cholesterol in the adjuvant composition, comprising: The neutral liposomes are prepared according to the method of step S21 and step S22; and the cationic liposomes are prepared by the following method: S23, mixing DOTAP, DOPC and cholesterol in a mass ratio of 2:2:1 to obtain a second lipid component; S24, preparing the cationic liposomes by using any one of an ethanol injection method, a thin film dispersion method, an ultrasonic dispersion method and a reverse evaporation method on the second lipid component.

8. The method for preparing the recombinant respiratory syncytial virus vaccine according to claim 7, characterized in that: In both step S22 and step S24, the ethanol injection method is used to prepare the neutral liposome and the cationic liposome respectively; The ethanol injection method comprises: The neutral liposome or the cationic liposome is obtained by extruding the first lipid component or the second lipid component using extrusion membranes with pore sizes of 200 nm and 100 nm, respectively, and then filtering and sterilizing.

9. The method for preparing the recombinant respiratory syncytial virus vaccine according to claim 8, characterized in that: The first lipid component or the second lipid component is extruded using an extrusion membrane with a pore size of 200 nm and an extrusion membrane with a pore size of 100 nm in sequence, and then filtered and sterilized to obtain the neutral liposome or the cationic liposome, comprising: Dissolving the first lipid component or the second lipid component in ethanol, and performing online shearing with the aqueous phase for 1 to 3 times to obtain a sheared raw material; The sheared raw material is subjected to 200 nm liposome extrusion treatment, and filtered using a filter membrane with a pore size of 200 nm to obtain a 200 nm extrudate; The 200 nm extrudate is subjected to 100 nm liposome extrusion treatment, and filtered using a filter membrane with a pore size of 100 nm to obtain a 100 nm extrudate; The 100 nm extrudate is subjected to ultrafiltration replacement with a phosphate buffer solution, and is filtered and sterilized using a sterile filter to obtain the neutral liposome; and the 100 nm extrudate is subjected to ultrafiltration replacement with an acetate buffer solution, and is filtered and sterilized using a sterile filter to obtain the cationic liposome; Preferably, the concentration multiple of the ultrafiltration substitution is 3 to 6 times, and the diafiltration multiple is 6 to 8 times; Preferably, the average particle size D50 of the neutral liposome is 80nm to 140nm; Preferably, the pH value of the neutral liposome solution is 5.5 to 7.0; Preferably, the pH value of the neutral liposome solution is 6.4 to 6.6; Preferably, the average particle size D50 of the cationic liposome is 40 nm to 100 nm; Preferably, the pH value of the solution of the cationic liposome is 4.5 to 6.5; Preferably, the Zeta potential of the cationic liposome is 50 mV to 90 mV.

10. Use of the recombinant respiratory syncytial virus vaccine according to any one of claims 1 to 6 in the preparation of a product for treating and / or preventing diseases caused by respiratory syncytial virus, characterized in that: The products include vaccine products, medicines, diagnostic reagents and test kits.

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