Water-in-oil type polypeptide vaccine composition

Abstract

The invention discloses a water-in-oil type polypeptide vaccine composition. The composition comprises an oily component and a water-based component, the water-based component comprises antigen peptide or pharmaceutically acceptable salt thereof, an excipient and acid radical ions; the oily component comprises a surfactant and an oil adjuvant. The invention also provides a polypeptide vaccine prepared from the polypeptide vaccine composition. The polypeptide vaccine is specifically obtained by shearing and emulsifying the water phase and the oily component of the water-in-oil polypeptide vaccine composition. According to the water-in-oil type polypeptide vaccine composition provided by the invention, the water-based component containing the antigen peptide library and the excipient is matched with the oil-based component, so that the water-in-oil type polypeptide vaccine composition has relatively high immunogenicity and stability. The vaccine prepared from the composition has moderate pH value and isotonic concentration, and in the subcutaneous / intramuscular injection process, the medication compliance can be improved, and the pain of a patient in the medication process can be reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccines, and in particular to a water-in-oil type polypeptide vaccine composition. Background Art

[0002] At present, the vaccine layout for the novel coronavirus (SARS-CoV-2) Omicron variant is mainly monovalent and multivalent vaccines, but the prevention method is to produce antibodies to neutralize the virus through humoral immunity. Once the virus mutates, breakthrough infection will occur. At the same time, because people with humoral immune deficiency and low immunity in the population cannot stimulate humoral immunity to produce antibodies or enough antibodies, the current vaccines cannot effectively build a complete population immune barrier.

[0003] T cells play a very important role in virus clearance, and vaccines based on T cells can effectively prevent and control the infection and spread of the new coronavirus. The antigenic peptide library of the new crown prevention polypeptide vaccine HY3001 is composed of 6 conserved T cell dominant epitopes. It not only produces antibodies by activating B cells, but also produces CD8+Tc cells by activating T cells. It is suitable for people with humoral immunity deficiency or immunodeficiency, and tolerates the mutation of the new crown strain to avoid immune escape of mutant strains. In addition, as one of the subunit vaccines, the polypeptide vaccine has a polypeptide as its antigenic substance, which is completely chemically synthesized and has the advantages of high purity, good stability, strong specificity, wide population coverage, high safety and low cost. However, the immunogenicity of polypeptides is relatively weak, and the polypeptide library is usually composed of multiple epitope peptides. At present, the physical and chemical stability of polypeptide mixtures is a major challenge facing the formulation process, and adjuvants are also needed to enhance the immune effect of polypeptide vaccines. Based on literature reports, most antigen peptide libraries do not have good hydrophilicity. The commonly used treatment methods in the literature are to use a certain concentration of DMSO for reconstitution, or to use water and acetonitrile as solvents to dissolve the peptides and then freeze-dry them for clinical trials. However, organic solvents cannot be completely removed, which not only increases the risk of incompatibility between the drug solution and the production components, but also inevitably leads to residual organic volatile compounds in the preparation. In addition, due to the large number of target antigen peptides, the characteristics of each peptide are different, which may lead to physical and chemical stability problems. Therefore, it is crucial to develop a peptide composition with good stability and ideal immunogenicity. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a water-in-oil polypeptide vaccine composition to improve the physical and chemical stability of the polypeptide mixture and enhance the immunogenicity of the vaccine.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] On the one hand, the present invention provides a water-in-oil polypeptide vaccine composition, comprising an oily component and an aqueous component; the aqueous component comprises an antigen peptide or a pharmaceutically acceptable salt thereof, an excipient and an acid ion; the oily component comprises a surfactant and an oil adjuvant.

[0007] As a preferred embodiment, the antigenic peptide is selected from at least one of the polypeptides having the following amino acid sequences:

[0008] M23: ATSRTLSYYK, as shown in SEQ ID NO. 1;

[0009] N25: KTFPPTEPK, as shown in SEQ ID NO. 2;

[0010] N37: KRTATKAYNVTQAFGRR, as shown in SEQ ID NO. 3;

[0011] LJ43: VGYLQPRTFLLKYNENGT, as shown in SEQ ID NO. 4;

[0012] LJ71:DSKVGGNYNYLYRLFRK, as shown in SEQ ID NO.5;

[0013] LJ158: ITGRLQSLQTYVTQQLIR, as shown in SEQ ID NO. 6;

[0014] Preferably, the pharmaceutically acceptable salt of the antigen peptide is selected from at least one of acetate, hydrochloride and citrate, preferably at least one of acetate and hydrochloride;

[0015] Preferably, the salt form of the polypeptide M23 salt is hydrochloride;

[0016] Preferably, the salt form of the polypeptide N25 salt is acetate;

[0017] Preferably, the salt form of the polypeptide N37 salt is acetate;

[0018] Preferably, the salt form of the polypeptide LJ43 salt is acetate;

[0019] Preferably, the salt form of the polypeptide LJ71 salt is acetate;

[0020] Preferably, the salt form of the polypeptide LJ158 salt is acetate.

[0021] As a preferred embodiment, the excipient is selected from at least one of mannitol, trehalose and sorbitol, preferably mannitol.

[0022] As a preferred embodiment, the acid ions are selected from one or both of chloride ions and acetate ions; wherein the chloride ions are derived from hydrochloric acid, and the acetate ions are derived from acetic acid;

[0023] Preferably, the acid ions are chloride ions and acetate ions used in combination.

[0024] As a preferred embodiment, in the aqueous component, the weight ratio of the total weight of the antigen peptide or its pharmaceutically acceptable salt to the excipient and the acid ion is (0.3-6): (5-75): (0.09-1.6);

[0025] Preferably, the weight ratio of the antigen peptide or its pharmaceutically acceptable salt to the excipient and the acid ion is 3:10:(0.1-1.6);

[0026] More preferably, the weight ratio of the antigen peptide or its pharmaceutically acceptable salt to the excipient and the acid ion is 3:10:(0.1-1).

[0027] As a preferred embodiment, the oily component is Montanide ISA;

[0028] Preferably, the oily component is selected from at least one of Montanide ISA 720VG and Montanide ISA51 VG, and more preferably Montanide ISA 720VG.

[0029] As a preferred embodiment, the aqueous component is a lyophilized preparation;

[0030] Preferably, the preparation process of the lyophilized preparation comprises the following steps: uniformly mixing an aqueous solution of an excipient with an aqueous solution of an antigen peptide or a pharmaceutically acceptable salt thereof, adjusting the pH to 2.5 to 4.0 by a pH regulator; sterilizing and filtering; freeze drying;

[0031] Specifically, the pH adjuster is selected from at least one of acetic acid and dilute hydrochloric acid.

[0032] Preferably, the aqueous solution of the antigenic peptide or a pharmaceutically acceptable salt thereof is an aqueous solution acidified with acetic acid;

[0033] Specifically, the freeze-drying includes pre-freezing, vacuuming, primary drying and secondary drying; preferably, the pre-freezing temperature is ≤-38°C, preferably ≤-45°C; the pre-freezing time is at least 2h; the primary drying temperature is ≤-5°C, preferably -15°C to -10°C; the primary drying time is 10 to 16h; the secondary drying temperature is 30 to 35°C; the secondary drying time is at least 4h.

[0034] In another aspect, the present invention provides a polypeptide vaccine prepared from the above-mentioned water-in-oil polypeptide vaccine composition.

[0035] As a preferred embodiment, the preparation process comprises the following steps:

[0036] The aqueous phase formed after the aqueous component is re-dissolved is shear-emulsified with the oily component to form a water-in-oil type polypeptide vaccine;

[0037] Preferably, the volume ratio of the aqueous phase formed after the aqueous component is re-dissolved to the oily component is 3:3-7;

[0038] Specifically, the amount of the aqueous component after re-dissolution and the oily component is in the following relationship:

[0039] The oily component is Montanide ISA 720VG, and the volume ratio of the aqueous component to the oily component after reconstitution is 3:7;

[0040] Alternatively, the oily component is Montanide ISA 51VG, and the volume ratio of the aqueous component to the oily component after redissolution is 1:1.

[0041] The above technical solution has the following advantages or beneficial effects:

[0042] The present invention provides a water-in-oil (W / O) polypeptide vaccine composition with good stability, comprising an aqueous component containing an antigen peptide library (used as an aqueous phase after reconstitution) and an oily component (containing a surfactant and an oil adjuvant). Before administration, the polypeptide vaccine composition provided by the present invention can be obtained by shearing and emulsifying the aqueous phase formed by reconstitution of the aqueous component and the oily component to obtain a polypeptide vaccine.

[0043] The water-in-oil polypeptide vaccine composition provided by the present invention adopts a combination of an aqueous component containing an antigen peptide library and an excipient and an oily component, and has high immunogenicity and stability.

[0044] The vaccine prepared by the polypeptide vaccine composition provided by the present invention has a moderate pH value (4-7) and isotonic concentration, and can improve medication compliance and reduce the pain of patients during medication during subcutaneous / muscular injection.

[0045] The water-in-oil polypeptide vaccine composition provided by the present invention has an aqueous component which is a lyophilized preparation. After freeze drying, 95-99% of the water can be removed, greatly reducing the risk of physical aggregation and chemical degradation. The excipient added to the aqueous component can enable the freeze-dried product to maintain a good cake-like solid appearance while also having an osmotic pressure regulating function. The excipient selected in the present invention is compatible with other ingredients, especially multiple antigen peptides, to avoid interactions and reduce drug degradation, such as oxidation reactions, reduction reactions, and the generation of compatible impurities in raw and auxiliary materials.

[0046] The water-in-oil polypeptide vaccine composition provided by the present invention does not introduce organic solvents, thereby reducing safety risks and clinical use risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a graph showing the stability test results of the N37 antigen peptide in Example 3 of the present invention.

[0048] Figure 2 This is a graph showing the stability test results of the N25 antigen peptide in Example 3 of the present invention.

[0049] Figure 3 This is a graph showing the stability test results of the M23 antigen peptide in Example 3 of the present invention.

[0050] Figure 4 This is a graph showing the stability test results of the LJ71 antigen peptide in Example 3 of the present invention.

[0051] Figure 5 This is a graph showing the stability test results of the LJ43 antigen peptide in Example 3 of the present invention.

[0052] Figure 6 This is a graph showing the stability test results of the LJ158 antigen peptide in Example 3 of the present invention.

[0053] Figure 7 This is an appearance diagram of the polypeptide vaccine emulsion prepared in Example 5 of the present invention.

[0054] Figure 8 This is a graph showing the water drop test results of the polypeptide vaccine prepared in Example 5 of the present invention.

[0055] Fig. 9 This is a graph showing the results of the ELISPOT spot counting experiment in Example 6 of the present invention. DETAILED DESCRIPTION

[0056] The following embodiments are only some embodiments of the present invention, rather than all embodiments. Therefore, the detailed description in the embodiments of the present invention provided below is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention. In the present invention, unless otherwise specified, all equipment and raw materials, etc. can be purchased from the market or are commonly used in the industry. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0057] The sequences of the antigenic peptides used in the following examples of the present invention are as follows:

[0058] M23: ATSRTLSYYK, as shown in SEQ ID NO. 1;

[0059] N25: KTFPPTEPK, as shown in SEQ ID NO. 2;

[0060] N37: KRTATKAYNVTQAFGRR, as shown in SEQ ID NO. 3;

[0061] LJ43: VGYLQPRTFLLKYNENGT, as shown in SEQ ID NO. 4;

[0062] LJ71:DSKVGGNYNYLYRLFRK, as shown in SEQ ID NO.5;

[0063] LJ158:ITGRLQSLQTYVTQQLIR, as shown in SEQ ID NO.6.

[0064] Embodiment 1:

[0065] This embodiment provides an aqueous component freeze-dried reagent containing antigen peptides, and its components and contents are shown in Table 1:

[0066] Table 1

[0067]

[0068] The preparation process includes the following steps:

[0069] (1) Take about 150 mL of water for injection, add 15 g of mannitol, and stir to dissolve;

[0070] (2) Take 7.2 g of glacial acetic acid and dilute it to 72 g with water for injection, or take 20 g of acetic acid (36%) and dilute it to 72 g to dissolve LJ158 acetate;

[0071] (3) Add water for injection to 400 g, then add the remaining peptides in the following order: N37 acetate, LJ43 acetate, LJ71 acetate, N25 acetate, M23 hydrochloride, and stir to dissolve;

[0072] (4) Using 10% acetic acid as a pH adjuster, adjust the pH to 3.0 and adjust the volume to 500 mL;

[0073] (5) After filtration and sterilization, the drug solution is dispensed into 2 mL medium borosilicate glass controlled injection bottles with a filling volume of 0.5 mL / vial, half-stoppered with bromobutyl rubber stoppers, and freeze-dried; the specific freeze-drying process includes: loading at 20°C, pre-freezing at -45°C for 2 h; drying for 12 h at a gradient temperature of -15°C; secondary drying at 30°C for 4 h; after freeze-drying, re-pressurize with nitrogen, press stoppers; unpack and cap.

[0074] Performance Testing:

[0075] In this example, the above-mentioned aqueous component freeze-dried reagent containing antigen peptides was reconstituted and its osmotic pressure, viscosity and stability were tested.

[0076] The aqueous component lyophilized reagent prepared in this example was reconstituted in water for injection at a concentration of 4 mg / mL of total antigen peptide content, and its viscosity was tested using a BROOKFIELD DV2T viscometer. The test process was as follows: a suitable rotor was selected, about 0.5 mL of the reconstituted sample was sampled and added to the sample pool, and the contact points were adjusted to be well connected, the speed was set to 30 rpm, and the operation was started. The viscosity was 1.8 mPa.s, which was a low viscosity product and was easy to dissolve and administer.

[0077] The aqueous component lyophilized reagent prepared in this example was reconstituted in water for injection at a concentration of 4 mg / mL of the total antigen peptide content, and its osmotic pressure was tested using an osmotic pressure tester. The test process was as follows: the instrument was calibrated using a standard, and about 0.05 mL of the reconstituted sample was added to a clean and dry test tube to ensure that there were no bubbles in the sample. The sample was loaded and tested, and its osmotic pressure was 299 mOsmol / kg.

[0078] The aqueous component lyophilized reagent prepared in this example was re-dissolved in water for injection at a total antigen peptide content of 4 mg / mL. A pH meter was used to test the pH value, and the pH value was measured to be in the range of 4.1 to 4.6 based on the test results of multiple samples.

[0079] The aqueous component freeze-dried reagent prepared in this embodiment was measured for moisture by coulometric titration, and its moisture content was 0.68%. The acetic acid content was measured by high performance liquid chromatography, and the test results of multiple samples showed that the acetic acid content in every 1.2g of antigen peptide preparation was 0.15-0.25g. Therefore, the weight ratio of the antigen peptide or its pharmaceutically acceptable salt to the excipient and the acid radical ion is 1.2:15:0.15-0.25.

[0080] The aqueous component freeze-dried reagent prepared in this embodiment was subjected to 40±5°C, 75%RH±5%RH stability test for 1 month, 2 months, 3 months and 25±5°C, 60%RH±5%RH stability test for 3 months, 6 months, and 12 months. After reaching the time point, samples were taken to detect the relevant substances and contents. Common quantitative methods include impurity reference method, main component self-control method with correction factor, main component self-control method without correction factor, and peak area normalization method. The response of each antigen peptide of this product is quite different, so the single peptide area normalization method is used for calculation, and the detection conditions are as follows: Take the freeze-dried powder test sample, dissolve it in water and dilute it to make a solution containing about 1.0mg per 1ml, and mix it evenly. The chromatographic column was filled with octadecylsilane bonded silica gel; trifluoroacetic acid-water (0.5:1000) solution was used as mobile phase A, and acetonitrile was used as mobile phase B; gradient elution was performed according to Table 2; the detection wavelength was 220 nm; the flow rate was 0.3 mL per minute; the column temperature was 70°C; the sample chamber temperature was 10°C; and the injection volume was 10 μL.

[0081] Table 2

[0082] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 25 85 15 40 75 25 60 50 50 65 90 10 70 95 5

[0083] The test results are shown in Table 3-4, which shows that the total impurities of each polypeptide in the relevant substances only show a slight increase trend with the extension of storage time, and the stability of the product is good.

[0084] Table 3 Stability test results - related substances

[0085]

[0086] Table 4 Stability test results - content

[0087]

[0088] Embodiment 2:

[0089] The components, preparation method and test method of the aqueous component freeze-dried reagent containing antigen peptides provided in this example are the same as those in Example 1, except that the quality of the polypeptide, the type and content of the excipient, and the amount of the acid radical ion in this example are different from those in Example 1, as shown in Table 5:

[0090] Table 5

[0091]

[0092] The appearance of the aqueous component lyophilized preparation in this example is significantly different due to the different types of excipients. The total content of the antigen peptide was reconstituted at a concentration of 4 mg / mL and then the osmotic pressure and pH were tested. Mannitol was used as an excipient and the osmotic pressure met the isotonic requirements. The results are shown in Table 6:

[0093] Table 6 Appearance of preparations prepared with different excipients and osmotic pressure and pH after reconstitution

[0094]

[0095]

[0096] The aqueous component freeze-dried reagent in this embodiment was tested for stability at 40±5°C and 75%RH±5%RH. The test comparison results are shown in Table 7. Among them, Examples 2-5 and 2-6 using mannitol clearly showed the best stability, and trehalose and sorbitol were second only to mannitol.

[0097] Table 7 Stability of formulations prepared with different excipients

[0098]

[0099]

[0100] The aqueous component freeze-dried reagents prepared in Examples 2-5 and 2-6 were measured for moisture by coulometric titration, and the moisture contents were 0.81% and 0.99%, respectively. The acetic acid content was measured by high performance liquid chromatography, and the acetic acid content of every 6 g of antigen peptide in the freeze-dried composition of Example 2-5 was 0.96 g, and the acetic acid content of every 0.3 g of antigen peptide in the freeze-dried composition of Example 2-6 was 0.09 g. Therefore, the weight ratio of the antigen peptide or its pharmaceutically acceptable salt to mannitol and acid ion is (0.3-6):(5-75):(0.09-0.96), and the product has good stability.

[0101] Embodiment 3:

[0102] The components, preparation method and test method of the aqueous component freeze-dried reagent containing antigen peptides provided in this example are the same as those in Example 1, except that the quality of the polypeptide, the type of excipient, the type of pH adjuster, the pH of the drug solution, and the amount of acid radical ions in this example are different from those in Example 1, as shown in Table 8 and Table 9:

[0103] Table 8

[0104] Example Antigenic peptide Excipient dosage Types of pH adjusters Acid ion weight 3-1 0.5g / peptide, 3g in total Mannitol 10g 10% acetic acid 0.66g 3-2 0.5g / peptide, 3g in total Mannitol 10g 10% acetic acid 0.49g 3-3 0.5g / peptide, 3g in total Mannitol 10g 10% acetic acid 0.13g 3-4 0.5g / peptide, 3g in total Mannitol 10g 10% acetic acid, 1% dilute hydrochloric acid 1.58g 3-5 0.5g / peptide, 3g in total Mannitol 10g 10% acetic acid, 1% dilute hydrochloric acid 1.14g 3-6 0.5g / peptide, 3g in total Mannitol 10g 10% acetic acid, 1% dilute hydrochloric acid 1.05g 3-7 0.5g / peptide, 3g in total Mannitol 10g 10% acetic acid, 1% dilute hydrochloric acid 0.99g 3-8 0.5g / peptide, 3g in total Mannitol 10g 1% citric acid, 1% dilute hydrochloric acid 1.47g

[0105] Table 9

[0106] Example Types of pH adjusters Adjust the pH of the solution to pH after freeze-drying and reconstitution 3-1 10% acetic acid 2.5 4.29 3-2 10% acetic acid 3.0 4.35 3-3 10% acetic acid 4.0 4.68 3-4 10% acetic acid, 1% dilute hydrochloric acid 2.0 3.14 3-5 10% acetic acid, 1% dilute hydrochloric acid 2.5 3.53 3-6 10% acetic acid, 1% dilute hydrochloric acid 3.0 3.78 3-7 10% acetic acid, 1% dilute hydrochloric acid 4.0 3.97 3-8 1% citric acid, 1% dilute hydrochloric acid 2.5 2.75

[0107] The aqueous component freeze-dried preparation in this embodiment was subjected to pH test and acid ion weight test using HPLC method. Since most of the acetic acid is removed during the freeze-drying process, different types and amounts of pH adjusters directly affect the final content of acid ions in the composition. The table summarizes the total weight of acid ions as a percentage of the weight of the antigen peptide (free base). It can be seen that the weight ratio of the antigen peptide or its pharmaceutically acceptable salt to mannitol and acid ions is 3:10:(0.1~1.6). Combined with the stability of Table 10, the preferred weight ratio of the antigen peptide or its pharmaceutically acceptable salt to mannitol and acid ions is 3:10:(0.1~1).

[0108] The stability of the aqueous component freeze-dried preparation in this example was investigated. The results showed that the products of Examples 3-8 using a combination of dilute hydrochloric acid and citric acid were significantly degraded. The products of the combination of acetic acid and dilute hydrochloric acid, except for Examples 3-4, had good stability. It can be seen that the pH of the drug solution is preferably adjusted to 2.5-4.0 by using acetic acid and / or dilute hydrochloric acid, and the quality of the products obtained is good. The specific results are shown in Tables 10 and Figure 1-6 :

[0109] Table 10 Stability results

[0110]

[0111] Example 4

[0112] The components and preparation method of the aqueous component freeze-dried reagent containing antigen peptides provided in this embodiment are the same as those in Example 1, except that the freeze-drying process in the preparation method of this embodiment is different from that in Example 1, as shown in Table 11:

[0113] Table 11 Different freeze-drying processes

[0114]

[0115] Therefore, the preparation method provided by the present invention can obtain a freeze-dried product with good properties under the conditions of primary drying at -15 to -10°C and secondary drying at 30 to 35°C.

[0116] Example 5

[0117] In this example, the aqueous component freeze-dried reagent containing antigen peptides and the oil phase component in Example 1 are prepared into a polypeptide vaccine, and the preparation process is as follows:

[0118] The aqueous component lyophilized reagent containing the antigen peptide in Example 1 was reconstituted with 0.3 mL of water for injection, and then sheared and emulsified with the oily component at a volume ratio of W / O=3 / 7 (the oily component was Montanide ISA 720VG, hereinafter referred to as 720VG) or a volume ratio of W / O=1 / 1 (the oily component was Montanide ISA 51VG, hereinafter referred to as 51VG) to form a water-in-oil polypeptide vaccine.

[0119] The appearance of the two polypeptide vaccine emulsions prepared in this example is shown in Figure 7 , Figure 7 In the figure, the left picture is a fresh image, and the right picture is an appearance picture after being placed at room temperature for 2 hours. In both pictures, the reagent on the left is 720VG, and the reagent on the right is 51VG.

[0120] The test results of the vaccine prepared with 720VG as the oily component in the water droplet experiment are shown in Figure 8 It can be seen that the water-in-oil emulsion prepared by the present invention does not disperse or break when dropped into an aqueous solution, which meets the requirements of the water drop test, indicating that the prepared emulsion has good performance.

[0121] In this example, the vaccine prepared with 720VG as the oily component was analyzed by Malvern laser diffraction particle size analyzer. The results are shown in Table 12, where D(v; 0.5) means that 50% of the droplet size of the sample is smaller than the output particle size value, and D(v; 0.9) means that 90% of the droplet size of the sample is smaller than the output particle size value. The results show that the emulsion has good particle size uniformity and stability within 2 hours at room temperature.

[0122] Table 12

[0123] Room temperature storage time Particle size-D(v;0.5) Particle size-D(v;0.9) 0h 1.0μm 1.1μm 24h 1.0μm 1.2μm

[0124] From the above test results, it can be seen that the vaccine emulsion prepared by the present invention has a stable appearance and does not show any instability phenomena such as demulsification and stratification.

[0125] Example 6

[0126] This example evaluates the efficacy of the vaccine prepared in Example 5 in animals. The test uses a mouse model with a human HLA genotype as the test object, and sets up a saline control group (Group 1: Saline), an antigen peptide group (Group 2: M23, N25, N37, LJ43, LJ71, LJ158 antigen peptide combination group), a polypeptide vaccine group 1 (a vaccine emulsion prepared with an oily component of 51VG in Example 5, Group 4), and a polypeptide vaccine group 2 (a vaccine emulsion prepared with an oily component of 720VG in Example 5, Group 5). The administration method is intramuscular injection, and the administration volume is 200μL per mouse. Each group is set up with 6 mice (3 males and 3 females), and the number of vaccinations is 2 times, with an interval of 14 days, and anatomical sampling is performed on the 24th day. After the injection of the above four groups of mice, their fur, injection site, eyes, and breathing, eating, drinking, urination and defecation behaviors are observed every day. The test results show that the indicators of Group 2 and Group 5 are no different from those of Group 1.

[0127] The spleens of the four groups of mice were dissected and cultured in vitro. The spleen cells were stimulated with antigen peptides (M23, N25, N37, LJ43, LJ71, LJ158, 10 μg / mL / strip) and the IFN-γ secreted by T cells was detected. The results of ELISPOT spot counting experiments showed that (see Fig. 9 ): The IFN-γ concentration produced by the vaccine prepared using Montanide ISA 51VG and Montanide ISA 720VG as the oily component was significantly higher than that of the negative saline control group and the group injected with antigen peptide only (Group 2), indicating that the oil emulsion vaccine composition effectively stimulated the body to produce T cell immunity after vaccination. Therefore, the polypeptide vaccine composition provided by the present invention has a good immune effect. Considering that Montanide ISA51 VG is composed of non-metabolizable mineral oil and is prone to form granulomas at the injection site, Montanide ISA720 VG is preferably used as the oily component.

[0128] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A water-in-oil polypeptide vaccine composition, characterized in that: It comprises an oily component and an aqueous component; the aqueous component comprises an antigen peptide or a pharmaceutically acceptable salt thereof, an excipient and an acid ion; and the oily component comprises a surfactant and an oil adjuvant.

2. The water-in-oil polypeptide vaccine composition according to claim 1, characterized in that: The antigenic peptide is selected from at least one of the polypeptides having the following amino acid sequences: M23: ATSRTLSYYK, as shown in SEQ ID NO. 1; N25: KTFPPTEPK, as shown in SEQ ID NO. 2; N37: KRTATKAYNVTQAFGRR, as shown in SEQ ID NO. 3; LJ43: VGYLQPRTFLLKYNENGT, as shown in SEQ ID NO. 4; LJ71:DSKVGGNYNYLYRLFRK, as shown in SEQ ID NO.5; LJ158: ITGRLQSLQTYVTQQLIR, as shown in SEQ ID NO. 6; Preferably, the pharmaceutically acceptable salt of the antigen peptide is selected from at least one of acetate, hydrochloride and citrate, preferably at least one of acetate and hydrochloride; Preferably, the salt form of the polypeptide M23 salt is hydrochloride; Preferably, the salt form of the polypeptide N25 salt is acetate; Preferably, the salt form of the polypeptide N37 salt is acetate; Preferably, the salt form of the polypeptide LJ43 salt is acetate; Preferably, the salt form of the polypeptide LJ71 salt is acetate; Preferably, the salt form of the polypeptide LJ158 salt is acetate.

3. The water-in-oil polypeptide vaccine composition according to claim 1, characterized in that: The excipient is selected from at least one of mannitol, trehalose and sorbitol, preferably mannitol.

4. The water-in-oil polypeptide vaccine composition according to claim 1, characterized in that: The acid ions are selected from one or both of chloride ions and acetate ions; wherein the chloride ions are derived from hydrochloric acid, and the acetate ions are derived from acetic acid; Preferably, the acid ions are chloride ions and acetate ions used in combination.

5. The water-in-oil polypeptide vaccine composition according to claim 1, characterized in that: In the aqueous component, the weight ratio of the total weight of the antigen peptide or its pharmaceutically acceptable salt to the excipient and the acid ion is (0.3-6): (5-75): (0.09-1.6); Preferably, the weight ratio of the antigen peptide or its pharmaceutically acceptable salt to the excipient and the acid ion is 3:10:(0.1-1.6); More preferably, the weight ratio of the antigen peptide or its pharmaceutically acceptable salt to the excipient and the acid ion is 3:10:(0.1-1).

6. The water-in-oil polypeptide vaccine composition according to claim 1, characterized in that: The oily component is Montanide ISA; Preferably, the oily component is selected from at least one of Montanide ISA720 VG and Montanide ISA 51VG, and more preferably Montanide ISA 720VG.

7. The water-in-oil polypeptide vaccine composition according to claim 1, characterized in that: The aqueous component is a lyophilized preparation; Preferably, the preparation process of the lyophilized preparation comprises the following steps: uniformly mixing an aqueous solution of an excipient with an aqueous solution of an antigen peptide or a pharmaceutically acceptable salt thereof, adjusting the pH to 2.5 to 4.0 by a pH regulator; sterilizing and filtering; freeze drying; Preferably, the aqueous solution of the antigen peptide or a pharmaceutically acceptable salt thereof is an aqueous solution acidified with acetic acid.

8. The water-in-oil polypeptide vaccine composition according to claim 7, characterized in that: The freeze drying includes pre-freezing, vacuuming, primary drying and secondary drying; Preferably, the pre-freezing temperature is ≤-38°C, preferably ≤-45°C; the pre-freezing time is at least 2 hours; the primary drying temperature is ≤-5°C, preferably -15°C to -10°C; the primary drying time is 10 to 16 hours; the secondary drying temperature is 30 to 35°C; and the secondary drying time is at least 4 hours.

9. A polypeptide vaccine, characterized in that: The vaccine is prepared from the water-in-oil polypeptide vaccine composition described in any one of claims 1 to 8.

10. The polypeptide vaccine according to claim 9, characterized in that: The preparation process comprises the following steps: The aqueous phase formed after the aqueous component is re-dissolved is shear-emulsified with the oily component to form a water-in-oil type polypeptide vaccine; Preferably, the volume ratio of the aqueous phase formed after the aqueous component is redissolved to the oily component is 3:3-7.

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