A vaccine and a method of making the same

Abstract

The application discloses a vaccine and a preparation method thereof, and the preparation method comprises the following steps: mixing an acidic protein antigen solution containing a phosphate buffer solution with an aluminum adjuvant to make the molar ratio of phosphorus and aluminum below 0.9, so as to obtain an acidic protein vaccine; or mixing the aluminum adjuvant with a solution containing phosphate ions to make the molar ratio of phosphorus and aluminum above 0.2, and then mixing the aluminum adjuvant with an alkaline protein antigen solution containing a phosphate buffer solution, so as to obtain an alkaline protein vaccine; wherein the acidic protein antigen is any kind of protein antigen with a net negative charge; and the alkaline protein antigen is any kind of protein antigen with a net positive charge. According to the preparation method, the molar ratio of phosphorus and aluminum is adjusted to a specific range according to the net charge of the protein antigen, so that the isoelectric point of the aluminum adjuvant can be effectively changed, the charge carried by the aluminum adjuvant is opposite to the charge carried by the protein antigen, and then the aluminum adjuvant and the protein antigen are attracted to each other through electrostatic interaction. The vaccine antigen prepared by the method has high antigen adsorption rate and good stability, the preparation method is simple, and the method is suitable for industrial application.

Description

Technical Field

[0001] The present invention belongs to the technical field of vaccines, and in particular relates to a vaccine and a preparation method thereof. Background Art

[0002] Protein antigen is a protein that can stimulate the immune system to produce antibodies. It is the active ingredient of the vaccine. It can be divided into acidic protein antigens and basic protein antigens according to the amount of charge it carries. Specifically, when the entire protein carries more negative charge, it is an acidic protein antigen (isoelectric point tends to be acidic), such as the Norovirus GI.1 antigen, and when it carries more positive charge, it is a basic protein antigen (isoelectric point tends to be alkaline), such as the new crown antigen OD-RBD.

[0003] Regarding the preparation methods of vaccines disclosed in the prior art, there are few targeted studies on the above different protein antigens. CN113730566A discloses a combined influenza and COVID-19 vaccine and a preparation method thereof, comprising: diluting a recombinant COVID-19 vaccine containing RBD-Fc fusion protein with PBS phosphate buffer and mixing it with an aluminum hydroxide solution to obtain vaccine A. CN114129719A discloses a preparation method for a broad-spectrum vaccine, comprising: obtaining a recombinant protein of a pathogen; diluting the recombinant protein with a low-temperature buffer, and then diluting it with a cross-linking agent diluent, then refrigerating it, adding glycine, and concentrating and collecting it at 37°C for at least 2 hours, washing it, filtering, concentrating it, collecting it, and then mixing it with an aluminum adjuvant. CN115300620A discloses a preparation method for a novel composite adjuvant bivalent COVID-19 vaccine, wherein a composite adjuvant (Nano Al-CpG) formed by 0.19 mg / mL homemade nano-aluminum and 10 μg CpG is added to the vaccine stock solution. However, the existing vaccine preparation methods, especially those used for the preparation of basic protein antigens, have generally low antigen adsorption rates, which affects the immune effect of the vaccine and becomes a technical problem that needs to be solved urgently. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides a vaccine and a preparation method thereof, which can significantly increase the adsorption rate of aluminum adjuvants on acidic protein antigens and basic protein antigens, thereby improving the quality of the vaccine.

[0005] In a first aspect of the present invention, a method for preparing a vaccine is provided, wherein the raw materials for the preparation include: a) a first antigen solution: an acidic protein antigen solution containing a phosphate buffer; or a second antigen solution: an alkaline protein antigen solution containing a phosphate buffer; b) an aluminum adjuvant; and optionally c) a solution containing phosphate ions, the preparation method comprising:

[0006] The first antigen solution is mixed with an aluminum adjuvant to obtain an acidic protein vaccine at a phosphorus-to-aluminum molar ratio of less than 0.9; alternatively, the aluminum adjuvant is mixed with a phosphate ion-containing solution to obtain a phosphorus-to-aluminum molar ratio of greater than 0.2, and then mixed with the second antigen solution to obtain a basic protein vaccine;

[0007] Among them, the acidic protein antigen is any protein antigen with a net negative charge; the basic protein antigen is any protein antigen with a net positive charge.

[0008] Proteins are composed of amino acids, which are divided into acidic and basic amino acids. When a protein contains more acidic amino acids, the entire protein has a net negative charge. At this time, more acidic amino acids release protons when dissociating in aqueous solution, thereby increasing the acidity of the protein's surrounding environment and making the isoelectric point of such proteins acidic (pI < 7), so they are also called acidic proteins. Conversely, basic amino acids have a positive charge, while acidic amino acids have a negative charge. When a protein contains more basic amino acids, the entire protein has a net positive charge. At this time, more basic amino acids absorb protons when dissociating in aqueous solution, thereby reducing the acidity of the protein's surrounding environment and making the isoelectric point of such proteins alkaline (pI > 7), so they are also called basic proteins.

[0009] According to a specific embodiment of the present invention, the acidic protein antigen is a norovirus antigen.

[0010] According to a specific embodiment of the present invention, the basic protein antigen is a new coronavirus antigen.

[0011] According to a specific embodiment of the present invention, the method for preparing the acidic protein vaccine includes: mixing the first antigen solution with an aluminum adjuvant so that the phosphorus to aluminum molar ratio is 0.2 to 0.9.

[0012] According to a specific embodiment of the present invention, the preparation method of the basic protein vaccine includes: mixing an aluminum adjuvant with a solution containing phosphate ions to make the phosphorus to aluminum molar ratio between 0.2-0.6, preferably between 0.4 and 0.6, and then mixing with a second antigen solution.

[0013] According to a specific embodiment of the present invention, the second antigen solution does not contain sodium ions and / or chloride ions.

[0014] According to a specific embodiment of the present invention, the second antigen solution further contains organic sugars and histidine.

[0015] According to a specific embodiment of the present invention, the final concentration of the organic sugar is 1.8-3.0% w / v, and the final concentration of the histidine is 24 mM.

[0016] According to a specific embodiment of the present invention, the first antigen solution further contains histidine; preferably, the final concentration of the histidine is 20 mM.

[0017] The second aspect of the present invention provides a vaccine prepared by the aforementioned preparation method.

[0018] The beneficial effects of the present invention are:

[0019] The preparation method provided by the present invention adjusts the phosphorus-aluminum molar ratio to a specific range based on the net charge of the protein antigen, effectively changing the isoelectric point of the aluminum adjuvant, so that the charge it carries is opposite to that of the protein antigen, leading to mutual attraction through electrostatic interaction. The vaccine prepared by this method has a high antigen adsorption rate, good stability, and a simple preparation method, making it suitable for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a trend diagram of the change of the isoelectric point of aluminum hydroxide with the concentration of phosphate;

[0021] Figure 2 This is a graph showing the change trend of the isoelectric point of aluminum hydroxide and the P / Al ratio with phosphate concentration;

[0022] Figure 3 This is a trend chart showing the effect of different phosphate concentrations on antigen adsorption rate;

[0023] Figure 4 The results of isoelectric point, pH value and adsorption rate of norovirus monovalent vaccine at different PB concentrations are shown;

[0024] Figure 5 This is the trend diagram of the relationship between sodium chloride and adsorption rate;

[0025] Figure 6 This is a graph showing the maximum adsorption capacity of aluminum hydroxide adjuvant to OD-RBD antigen changing with NaCl concentration;

[0026] Figure 7 This is a graph showing the adsorption rate of aluminum hydroxide adjuvant on acidic protein (GI.1 antigen) changing with NaCl concentration. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. In the following examples, OD-RBD stock solution (OD-RBD + phosphate buffer system) and Noro GI.1 stock solution (Noro GI.1 + phosphate buffer system) are both provided by Anhui Zhifei Longcom Biopharmaceutical Co., Ltd.

[0028] A basic protein vaccine, the composition ratio of each group is shown in Table 1, and the preparation method is as follows:

[0029] The OD-RBD stock solution is diluted with a histidine solution to obtain solution I; the aluminum hydroxide solution is diluted with a phosphate buffer solution to adjust the phosphorus-aluminum molar ratio to a certain range to obtain solution II; and solution I and solution II are mixed to obtain the product.

[0030] Table 1 Content of each component of the vaccine

[0031]

[0032] An acidic protein vaccine, the composition ratio of each group is shown in Table 2, and the preparation method is as follows:

[0033] The Noro GI.1 stock solution is diluted with histidine, and then mixed with aluminum hydroxide solution to make the phosphorus-aluminum molar ratio within a certain range to obtain the product.

[0034] Table 2 Content of each component of the vaccine

[0035]

[0036] 1. Study on the adsorption mechanism of protein and aluminum hydroxide adjuvant

[0037] 1. Charge detection results

[0038] By detecting the isoelectric points of basic proteins, acidic proteins and aluminum hydroxide adjuvants, the charge of each component in the system is evaluated, and the adsorption mechanism of the basic / acidic protein is predicted.

[0039] 1.1 Experimental Results

[0040] Table 3 Test results of vaccine intermediate products

[0041]

[0042] 1.2 Results Analysis

[0043] The results of sample pH, isoelectric point, and zeta potential tests indicate that the pH of the ReNov GI.1-VLP monovalent stock solution was higher than its isoelectric point, indicating that the GI.1 antigen was negatively charged (-); the pH of the OD-RBD stock solution was lower than its isoelectric point, indicating that the OD-RBD antigen was positively charged (+); and the pH of the aluminum hydroxide adjuvant was lower than its isoelectric point, indicating that the aluminum hydroxide adjuvant was positively charged (+). The charge of the protein and the aluminum adjuvant are opposite, and the positive and negative charges attract each other, indicating a strong electrostatic attraction between the two. This force is the primary factor in the adsorption of the antigen and adjuvant.

[0044] In summary, in a near-neutral buffer system, there is an electrostatic attraction between acidic proteins and aluminum hydroxide adjuvants, which is the main factor promoting the adsorption of the two. The electrostatic attraction between alkaline proteins and aluminum hydroxide adjuvants can be achieved by lowering the isoelectric point and changing the charge.

[0045] 2. Study on adsorption mechanism

[0046] 2.1 Study on the adsorption mechanism of basic proteins (COVID-19 antigen OD-RBD)

[0047] 2.1.1 Test results are shown in Table 4. Figures 1 to 3 .

[0048] Table 4 Test results of vaccines 1-1 to 1-5

[0049]

[0050] 2.1.2 Results Analysis

[0051] It can be seen from the test results in Table 4 that the isoelectric point of aluminum hydroxide adjuvant will gradually decrease with the increase of phosphate concentration of the adjuvant diluent. When the phosphate concentration of the adjuvant diluent is between 80 mM and 100 mM, the isoelectric point of aluminum hydroxide adjuvant tends to be stable and no longer decreases. At this time, the isoelectric points of aluminum hydroxide adjuvant are 3.47 and 3.44, respectively, and aluminum hydroxide adjuvant carries a negative charge.

[0052] In a buffer system (pH 5.6±0.1), the antigen and adjuvant carry the same (+) charge, and like charges repel each other, resulting in weak adsorption of alkaline protein to aluminum hydroxide adjuvant. However, the addition of phosphate buffer to the aluminum hydroxide adjuvant lowers the isoelectric point of the aluminum hydroxide adjuvant, shifting its charge from positive to negative. The alkaline protein carries a positive charge, while the adjuvant and antigen have opposite charges, leading to mutual adsorption, consistent with the test results. As the isoelectric point of the aluminum hydroxide adjuvant decreases, the electrostatic interaction between the adjuvant and the antigen gradually increases. When the phosphate concentration is between 80 mM and 100 mM, the antigen adsorption rate reaches 95% and stabilizes.

[0053] 2.2 Study on the adsorption mechanism of acidic protein (Norovirus GI.1 antigen)

[0054] 2.2.1 Test results are shown in Table 5. Figure 4 .

[0055] Table 5 Test results of vaccines 2-1 to 2-5

[0056]

[0057] 2.2.2 Results Analysis

[0058] It can be seen from the test results in Table 5 that with the continuous increase of phosphate concentration, the pH value of the vaccine increased slightly and then tended to remain unchanged, and the vaccine adsorption rate continued to decrease, indicating that the addition of phosphate may change the isoelectric point of the aluminum hydroxide adjuvant in the buffer system, thereby weakening the positive and negative charge potential energy of the antigen and adjuvant, and then weakening the electrostatic attraction between the two, resulting in a decrease in the adsorption rate.

[0059] Table 5 testing result confirms, and along with the continuous rising of phosphate concentration, the isoelectric point of aluminum hydroxide adjuvant declines gradually, and illustrates that phosphate can reduce the isoelectric point of aluminum hydroxide adjuvant, consistent with testing result.Along with the reduction gradually of the isoelectric point of aluminum hydroxide adjuvant, the electrostatic interaction between adjuvant and the antigen weakens, when the phosphate final concentration is 20mM, the adjuvant isoelectric point and the stoste isoelectric point are closer, now in buffer system (pH value is 5.6 ± 0.1), antigen and adjuvant are with identical charge (+), like sex repels, therefore phosphate concentration is when greater than 16mM (phosphorus aluminum ratio is 0.8649), antigen adsorption rate obviously declines (down to 88% from 97%), this phenomenon is consistent with theoretical adsorption rate variation result, therefore for guaranteeing adsorption rate greater than 95%, phosphorus aluminum ratio should be not more than 0.9 in sample system.

[0060] 2. Study on the effect of sodium chloride on adsorption rate

[0061] 1. Research on factors affecting the adsorption rate of basic proteins

[0062] 1.1 Basic protein 1 (DO-RBD stock solution)

[0063] Table 6 Effect of sodium chloride concentration on adsorption rate

[0064]

[0065]

[0066] 1.1.1 Experimental Results

[0067] The vaccine was tested in terms of osmotic pressure molar concentration, appearance, pH value and adsorption rate after being mixed with different stock solution diluents. The specific results are shown in Table 7 and Figure 5 .

[0068] Table 7 Summary of the selection and optimization test results of the stock solution dilution

[0069]

[0070] 1.1.2 Result Analysis

[0071] The above test results show that when NaCl is present in the stock solution diluent, the vaccine adsorption rate gradually increases as the NaCl concentration decreases. When the NaCl concentration drops to 0.1%, the adsorption rate reaches over 90%. Therefore, NaCl is a key factor affecting the antigen adsorption rate, and removing NaCl can increase the antigen adsorption rate to over 90%.

[0072] 1.2 Basic protein 2 (OD-RBD stock solution)

[0073] Table 8 Grouping table for confirmation test of antigen electrostatic interaction mode

[0074]

[0075]

[0076]

[0077] 1.2.1 Test results

[0078] The vaccine was prepared by mixing the stock solution with different ionic strengths and the antigen adsorption rate of the vaccine was tested. The specific results are shown in Table 9 and Figure 6 .

[0079] Table 9 OD-RBD antigen electrostatic interaction mode confirmation test results

[0080]

[0081]

[0082]

[0083] In summary, when NaCl is contained in the diluent component, as the NaCl concentration in the vaccine increases, the maximum adsorption capacity of the adjuvant for OD-RBD antigen gradually decreases and tends to be stable, indicating that the increase in ionic strength will significantly affect the adsorption capacity of the adjuvant for the antigen.

[0084] 2. Study on the factors affecting the adsorption rate of acidic protein

[0085] The test results are shown in Tables 10, 11, Figure 7 .

[0086] Table 10 Acidic protein (GI.1 antigen) maximum adsorption capacity test results

[0087]

[0088] Table 11 Test results of the effect of sodium chloride concentration on adsorption rate at maximum adsorption capacity of acidic protein (GI.1 antigen)

[0089]

[0090] In summary, when the diluent component contains NaCl, as the NaCl concentration in the vaccine increases, the maximum adsorption capacity of the adjuvant for acidic protein (GI.1 antigen) gradually decreases and tends to be stable, indicating that the increase in ionic strength will significantly affect the adsorption capacity of the adjuvant for acidic protein.

[0091] In summary, phosphate can significantly reduce the isoelectric point of aluminum hydroxide adjuvant. In the buffer system with the same pH value, the potential energy of aluminum hydroxide adjuvant is reduced, the adsorption effect with acidic protein is weakened, and the adsorption rate decreases.

[0092] 3. Preparation Prescription Optimization

[0093] 1. Dilution Optimization

[0094] This study intends to replace the osmotic pressure regulator sodium chloride with anhydrous glucose. The specific experimental design is shown in Table 12.

[0095] Table 12 Selection and optimization of stock solution dilution

[0096]

[0097] 1.1 Experimental Results

[0098] After being mixed with different stock solution diluents, the vaccine was tested in terms of osmotic pressure molar concentration, appearance, pH value and adsorption rate. The specific results are shown in Table 13.

[0099] Table 13 Summary of the selection and optimization test results of the stock solution dilution

[0100]

[0101] 1.2 Results Analysis

[0102] Analysis of the above test results shows that when anhydrous glucose (Glu) is used instead of sodium chloride in the stock solution diluent component, the adsorption rate remains unchanged with increasing Glu concentration and remains >90%. When the anhydrous glucose concentration is 3.0-4.0%, the osmotic pressure molarity is 267mOsmol / kg-328mOsmol / kg, which meets the requirements. Therefore, it is feasible to use anhydrous glucose (Glu) instead of sodium chloride in this vaccine to improve the adsorption rate and maintain its osmotic pressure.

[0103] IV. Conclusion

[0104] 1. Basic Protein Antigens: The adsorption of basic protein antigens to aluminum hydroxide adjuvants primarily relies on electrostatic attraction. In a neutral buffer system, basic protein antigens carry a positive charge, so phosphate buffer is required to lower the isoelectric point of the aluminum hydroxide adjuvant. When the phosphate concentration is between 80mM and 100mM and the P / Al ratio is between 0.5435 and 0.5990, the charge carried by the adjuvant changes from positive to negative. The adjuvant and antigen have opposite charges, resulting in mutual adsorption. The antigen adsorption rate reaches 95% and tends to stabilize. Ionic strength affects electrostatic interactions, so organic sugars should be used instead of sodium chloride solution as osmotic pressure regulators.

[0105] 2. Acidic protein antigen: The adsorption of acidic protein antigen and aluminum hydroxide adjuvant mainly relies on electrostatic attraction. In a neutral buffer system, the acidic protein antigen is negatively charged and the aluminum hydroxide adjuvant is positively charged, and they attract each other by electrostatic attraction. Phosphate buffer can also be used for the adsorption of acidic protein antigen and aluminum hydroxide adjuvant. When the final phosphate concentration is less than 16mM and the P / Al ratio is less than 0.8649, the antigen adsorption rate reaches 95%. At the maximum adsorption capacity of aluminum hydroxide adjuvant, the ionic strength will affect antigen adsorption. If sodium chloride is used as an osmotic pressure regulator for acidic protein, it should be used when the amount of antigen is lower than the maximum adsorption capacity of the adjuvant.

[0106] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a vaccine, characterized in that: The raw materials for preparing the vaccine include: a) a second antigen solution: an alkaline protein antigen solution containing a phosphate buffer; b) an aluminum adjuvant; and c) a solution containing phosphate ions; The preparation method includes: mixing an aluminum adjuvant with a solution containing phosphate ions so that the phosphorus-aluminum molar ratio is 0.4~0.6, and then mixing it with a second antigen solution to obtain a basic protein vaccine; the basic protein antigen is a new coronavirus antigen, and the new coronavirus antigen is an OD-RBD protein.

2. The preparation method according to claim 1, characterized in that The second antigen solution does not contain sodium ions and / or chloride ions.

3. The preparation method according to any one of claims 1-2, characterized in that The second antigen solution also contains organic sugars and histidine.

4. The preparation method according to claim 3, characterized in that The final concentration of the organic sugar is 1.8-3.0% w / v, and the final concentration of the histidine is 24 mM.

5. The vaccine prepared by the preparation method according to any one of claims 1 to 4.

Images