Desorption agent for aluminum adjuvant adsorbed novel coronavirus omicron strain inactivated vaccine and application thereof

Abstract

The application provides a desorption agent for an aluminum adjuvant adsorbed novel coronavirus Omicron strain inactivated vaccine and an application thereof, and belongs to the technical field of biological medicines. The desorption agent is composed of 1-2% Triton X-100, 0.05-0.2 mol / L EDTA and 0.8-1 mol / L phosphate buffer. The use of the desorption agent can effectively separate the antigen in the aluminum adjuvant adsorbed novel coronavirus Omicron strain inactivated vaccine from the aluminum adjuvant, can solve the problem of unstable biological structure of the novel coronavirus Omicron strain inactivated vaccine in the desorption process, and can realize the purposes of efficient recovery and accurate determination of the antigen in the novel coronavirus Omicron strain inactivated vaccine, thereby providing an important tool for quality control of the Omicron strain inactivated vaccine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a desorbent for aluminum adjuvant-adsorbed inactivated COVID-19 Omicron strain vaccine and its application. Background Technology

[0002] The Omicron variant of the novel coronavirus is a newly emerging variant of concern (VOC) following the Alpha, Beta, Gamma, and Delta variants. The Omicron antigen, particularly the receptor-binding domain (RBD) of the spike protein, exhibits significantly more amino acid mutations than other VOCs, leading to a decrease in the protective efficacy of vaccines against the original strain. Therefore, it is necessary to advance the development of monovalent and multivalent vaccines against the Omicron variant. The Omicron variant antigen is the main active ingredient in the Omicron variant inactivated vaccine, and its effective content is directly related to the protective effect after vaccination. Therefore, its antigen content and relative in vitro potency are important indicators for the quality control of the Omicron variant inactivated vaccine.

[0003] Aluminum adjuvants have been used in inactivated vaccines for many years. Aluminum adjuvants covalently or coordinately bind to charged groups on the surface of the antigen, encapsulating the antigen in a particulate manner. This forms large, soluble particles with the antigen, increasing the complexity of the viral structure and thus making the complex more immunogenic. Vaccines using aluminum compounds as adjuvants can effectively enhance the immune response. However, the binding of aluminum adjuvants to the Omicron strain antigen of the novel coronavirus blocks the specific antibody binding sites on the antigen surface. During ELISA (Enzyme-Linked Immunosorbent Assay) detection of the antigen, it cannot bind to the specific antibodies on the enzyme-linked reaction plate or enzyme-labeled antibody. Consequently, the antigen cannot be effectively detected in in vitro relative potency tests, which is a key technical bottleneck in antigen quality control during the development of the Omicron strain inactivated vaccine.

[0004] Therefore, it is necessary to develop a desorbent to target the adsorption between the Omicron strain antigen of the novel coronavirus and aluminum adjuvant, so as to disrupt the adsorption force of aluminum adjuvant and separate the antigen from the aluminum adjuvant without destroying the structure and specific epitopes of the antigen. This would enable the antigen to be quantified by enzyme-linked immunosorbent assay (ELISA) and achieve effective detection of the relative efficacy of the Omicron strain inactivated vaccine of the novel coronavirus in vitro. Summary of the Invention

[0005] To address the aforementioned problems, the first objective of this invention is to provide a desorbent for use in aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of the Omicron strain. This desorbent can desorb antigens in the aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of the Omicron strain without damaging the antigen's structure and specific epitopes, thereby enabling quantitative determination of the antigen using enzyme-linked immunosorbent assay (ELISA) and achieving effective quality control of the COVID-19 vaccine of the Omicron strain.

[0006] Specifically, the desorbent of the present invention for the aluminum adjuvant-adsorbed inactivated vaccine of the novel coronavirus Omicron strain consists of 1% to 2% Triton X-100, 0.05 to 0.2 mol / L EDTA, and 0.8 to 1 mol / L phosphate buffer.

[0007] In a preferred embodiment, the volume percentage of the Triton X-100 is 1%.

[0008] In a preferred embodiment, the concentration of EDTA is 0.1 mol / L.

[0009] In a preferred embodiment, the concentration of the phosphate buffer is 1 mol / L.

[0010] In a preferred embodiment, the EDTA is provided by Na2EDTA or Na4EDTA. In a further preferred embodiment, the Triton X-100 has a volume percentage of 1%, the EDTA concentration is 0.1 mol / L, and the phosphate buffer concentration is 1 mol / L.

[0011] A second objective of this invention is to provide the use of the desorbent described above, applied to an aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of the Omicron strain, in the preparation of a product for antigen dissociation in the aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of the Omicron strain.

[0012] In a preferred embodiment, the antigen-dissociating product is a kit for antigen dissociation in an aluminum adjuvant-adsorbed inactivated COVID-19 Omicron strain vaccine.

[0013] The third objective of this invention is to provide a method for desorbing antigens in an aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of Omicron strain using the aforementioned desorbent, comprising the following steps: centrifuging the aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of Omicron strain, discarding the supernatant, adding the desorbent, mixing well, desorbing at a constant temperature below 40°C for more than 0.5 hours, and then centrifuging, the resulting supernatant being the aluminum adjuvant-adsorbed COVID-19 vaccine of Omicron strain antigen desorbent solution.

[0014] In a preferred embodiment, the antigen concentration in the aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of Omicron strain is 800 AU / mL, and the final concentration of aluminum adjuvant is 1 mg aluminum hydroxide / mL; the volume ratio of the desorbent to the aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of Omicron strain is 4:5.

[0015] In a preferred embodiment, the aluminum adjuvant is aluminum hydroxide adjuvant.

[0016] In a preferred embodiment, the isothermal desorption refers to desorption at 25℃ to 37℃ for more than 0.5 hours.

[0017] In a further preferred embodiment, the isothermal desorption refers to desorption at 25℃ to 37℃ for more than 1 hour.

[0018] In a further preferred embodiment, the isothermal desorption refers to desorption at 30°C for more than 2 hours or at 37°C for more than 1 hour.

[0019] In a preferred embodiment, centrifugation refers to centrifugation at 4000 rpm for 15 minutes.

[0020] The beneficial effects of this invention are as follows: When the desorbent provided in this invention is used for the desorption of antigens from aluminum adjuvant-adsorbed inactivated COVID-19 Omicron strain vaccines, it can effectively separate the antigens from the aluminum adjuvant, solving the problem of biological structural instability during the desorption process of the COVID-19 Omicron strain inactivated vaccine and achieving the goal of efficiently recovering the COVID-19 Omicron strain inactivated vaccine antigens. Therefore, it also enables the accurate determination of the antigen content adsorbed onto the aluminum adjuvant in the vaccine, providing an important tool for the quality control of aluminum adjuvant-adsorbed COVID-19 Omicron strain inactivated vaccines. Detailed Implementation

[0021] The following description, in conjunction with embodiments, clearly and completely illustrates the technical solutions of the present invention, enabling those skilled in the art to fully understand the invention. Obviously, the described embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of the present invention.

[0022] The novel coronavirus Omicron strain used in the following examples has the accession number EPI_ISL_7138045 on the NCBI website.

[0023] Example 1

[0024] This embodiment provides a desorbent for an aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of strain Omicron, comprising 1% (volume percentage) Triton X-100, 0.1 mol / L Na2EDTA (disodium ethylenediaminetetraacetate), and 1 mol / L phosphate buffer.

[0025] The desorbent is prepared as follows:

[0026] (1) Measure 1 mL of Triton X-100 and add it to 9 mL of purified water. Mix well to obtain a Triton X-100 solution with a volume percentage of 10%.

[0027] (2) Weigh 2.964g sodium dihydrogen phosphate dihydrate, 11.098g disodium hydrogen phosphate dodecahydrate, and 1.86g Na2EDTA·2H2O. Measure 5mL of 10% (v / v) Triton X-100 solution, mix them, and make up to 50mL with purified water. Shake well to obtain the desorbent.

[0028] Example 2

[0029] This embodiment provides a method for using the desorbent prepared in Example 1 to desorb (or dissociate) antigens in an aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of strain Omicron, and the antigen content after desorption was determined.

[0030] The preparation method of the aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of Omicron strain is as follows: Using conventional techniques in the art, the COVID-19 Omicron strain is prepared by inoculating Vero cells, culturing, harvesting, inactivating the virus, concentrating, and purifying to obtain the Omicron strain inactivated vaccine stock solution. This stock solution is then prepared to a target antigen concentration of 800 AU / mL, diluted proportionally, and aluminum hydroxide adjuvant is added to prepare an aluminum adjuvant-adsorbed COVID-19 Omicron strain inactivated vaccine with a final antigen concentration of 400 AU / dose (0.5 mL per dose) and a final aluminum hydroxide adjuvant concentration of 0.5 mg / dose (0.5 mL per dose).

[0031] The desorption method includes the following steps:

[0032] Take 500 μL of the sample to be tested (aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of Omicron strain) and centrifuge at 4000 rpm for 15 min. Discard 400 μL of supernatant, add 400 μL of the desorbent prepared in Example 1, mix well, and desorb at 37 °C for 1 h. Then centrifuge at 4000 rpm for 15 min and take the supernatant, which is the aluminum adjuvant-adsorbed COVID-19 vaccine of Omicron strain antigen desorption solution.

[0033] The antigen content in the desorbed solution of the aluminum adjuvant-adsorbed novel coronavirus Omicron strain was determined using ELISA. The specific procedure was as follows:

[0034] Rabbit anti-novel coronavirus Omicron polyclonal antibody (obtained by immunizing Japanese white rabbits with inactivated novel coronavirus Omicron strain as antigen according to standard procedures) was diluted to 1:1000 with 0.05M carbonate buffer (pH=9.6) and coated onto microplates at 100 μL / well, incubating overnight at 4°C. After washing three times with PBST (containing 0.05% Tween 20, pH=7.2), blocking buffer (PBST containing 1% BSA) was added at 200 μL / well, and blocking was performed at 37°C for 1 h. The blocking buffer was discarded, and the plate was dried at 37°C for 2 h, then coated onto microplates again. Add the aluminum adjuvant-adsorbed novel coronavirus Omicron strain antigen desorption buffer and reference material (the preparation method of the reference material is the same as the preparation method of the aforementioned Omicron strain inactivated vaccine stock solution, batch number: 202201001) to the plate after a 2-fold serial dilution, 100 μL / well, and incubate at 37°C for 1 h; wash the plate 5 times with PBST, add enzyme-labeled antibody (1:2000 dilution) (enzyme-labeled antibody is 1G8-HRP disclosed in CN115838418A), 100 μL / well, and incubate at 37°C for 1 h; develop color with 1.25 mmol / L TMB (3,3',5,5'-tetramethylbenzidine) for 30 min, and terminate the reaction with 2 mol / L H2SO4. The OD value of each well was read using 450 nm as the detection wavelength and 630 nm as the reference wavelength. The antigen content in the desorbed solution of the aluminum adjuvant-adsorbed novel coronavirus Omicron strain was calculated using the double parallel line method based on the standard content, and the result was 772 AU / mL.

[0035] Example 3

[0036] This embodiment investigates the effect of desorption conditions on the desorption effect of the desorbent using the desorption method described in Example 2. The desorbent from Example 1 was used for desorption at different desorption temperatures and times (see Table 1). The antigen content in the desorbed solution was determined using the ELISA method described in Example 2, and the results are shown in Table 1.

[0037] Table 1. Desorption effects under different desorption conditions

[0038]

[0039] As shown in Table 1, desorption at 37℃ for 0.5 h or at 25℃ for 1 h both resulted in antigen recovery rates exceeding 80%. Desorption at 30℃ for 1 h or at 25℃ for 2 h both resulted in antigen recovery rates exceeding 85%. Desorption at 30℃ for 2 h resulted in antigen recovery rates exceeding 90%. When the desorption temperature was 37℃ and the desorption time was ≥1 h, the antigen recovery rate reached over 95%. With prolonged time, the improvement in antigen recovery rate was not significant. Therefore, considering time costs in practice, a desorption temperature of 37℃ and a desorption time of 1 h were chosen.

[0040] Example 4

[0041] In this embodiment, the effect of different desorbent compositions (see Table 2) on the desorption effect was studied according to the desorption method in Example 2. The desorption temperature was 37℃ and the desorption time was 1h. The content of antigen in the desorption solution was determined according to the ELISA method in Example 2. The results are shown in Table 2.

[0042] Table 2. Effects of different desorbent compositions on desorption efficiency

[0043] 1 1％ 0.1 1 97％ 2 2％ 0.05 0.8 92％ 3 1.5％ 0.2 0.8 91％

[0044] As can be seen from Table 2, when the volume percentage of Triton X-100 is 1% to 2%, the concentration of EDTA is 0.05 to 0.2 mol / L, and the concentration of phosphate buffer is 0.8 to 1 mol / L, the antigen recovery rate can reach more than 90% after desorption at 37℃ for 1 h.

[0045] Example 5

[0046] This embodiment studies the repeatability and intermediate precision of the desorbent prepared in Example 1 when using the desorption method in Example 2. The specific method is as follows: desorption experiments are conducted at different times, and two experimenters independently conduct 6 experiments each; the experimental results are shown in Table 3.

[0047] Table 3. Repeatability and precision test results of desorbent and desorption method

[0048]

[0049]

[0050] In Table 3, CV represents the coefficient of variation. In ELISA experiments, a CV ≤ 15% is considered acceptable. " / " in Table 3 indicates that data is not calculated. As shown in Table 3, after two independent researchers performed six repeated measurements, the CV values ​​were 6.64% and 7.39%, respectively, indicating good repeatability of the desorption method in Example 2. The combined mean of the two researchers' test results was 793, and the combined CV value was 6.85%, indicating good intermediate precision of the desorption method in Example 2.

[0051] Comparative Example 1

[0052] This comparative example provides a lysis buffer (see CN112379087B) for an aluminum adjuvant-adsorbed inactivated COVID-19 vaccine, comprising 0.3% (v / v) diethanolamine, 1% (v / v) Triton X-100, 10% (w / v) sodium citrate, 1 mol / L urea, and 0.05 mol / L LEDTA, prepared with 0.01 mol / L PBS buffer.

[0053] Following the desorption method in Example 2, the desorbents prepared in Example 1 and the comparative example were used to independently desorb six aluminum adjuvant-adsorbed inactivated COVID-19 Omicron strain vaccines with a theoretical antigen content of 800 AU / mL (preparation method see Example 2). The antigen content of each sample after desorption was then determined using the ELISA method in Example 2, and the results are shown in Table 4.

[0054] Table 4. Effects of different COVID-19 vaccine desorbents on the dissociation of antigens in Omicron strain inactivated vaccine.

[0055]

[0056] As shown in Table 4, when the desorbent in Comparative Example 1 was used to dissociate the antigen in the aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of the Omicron strain, the antigen recovery rate was significantly lower than that of the desorbent in Example 1. This indicates that the existing lysis buffer of the aluminum adjuvant-adsorbed inactivated COVID-19 vaccine has poor desorption effect on the aluminum adjuvant-ad ...

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Various modifications and variations can be made to the present invention by any person skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection of the present invention and the content of the specification should be included within the scope of protection of the present invention.

Claims

1. A desorbent for use in aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of the Omicron strain, characterized in that, The desorbent consists of 1% to 2% Triton X-100, 0.05 to 0.2 mol / L EDTA, and 0.8 to 1 mol / L phosphate buffer.

2. The desorbent for the aluminum adjuvant-adsorbed inactivated COVID-19 Omicron strain vaccine according to claim 1, characterized in that, The volume percentage of the Triton X-100 is 1%.

3. The desorbent according to claim 1 for use in an aluminum adjuvant-adsorbed inactivated COVID-19 Omicron strain vaccine, characterized in that, The concentration of EDTA is 0.1 mol / L.

4. The desorbent according to claim 1 for use in an aluminum adjuvant-adsorbed inactivated COVID-19 Omicron strain vaccine, characterized in that, The concentration of the phosphate buffer solution is 1 mol / L.

5. The use of the desorbent according to any one of claims 1 to 4 in the preparation of a product for antigen dissociation in an aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of strain Omicron.

6. The application according to claim 5, characterized in that, The product is a kit for antigen dissociation in an aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of the Omicron strain.

7. A method for using the desorbent according to any one of claims 1 to 4 for antigen desorption in an aluminum adjuvant-adsorbed inactivated COVID-19 Omicron strain vaccine, characterized in that, Includes the following steps: Centrifuge the aluminum adjuvant-adsorbed novel coronavirus Omicron strain inactivated vaccine, discard the supernatant, add the desorbent, mix well, and desorb at a constant temperature below 40°C for more than 0.5 hours. Then centrifuge, and the supernatant obtained is the aluminum adjuvant-adsorbed novel coronavirus Omicron strain antigen desorbent solution.

8. The method for antigen desorption according to claim 7, characterized in that, The antigen concentration in the aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of Omicron strain is 800 AU / mL, and the final concentration of aluminum adjuvant is 1 mg aluminum hydroxide / mL; the volume ratio of the desorbent to the aluminum adjuvant-adsorbed inactivated COVID-19 vaccine of Omicron strain is 4:

5.

9. The method for antigen desorption according to claim 7, characterized in that, The isothermal desorption refers to desorption at 25℃~37℃ for more than 0.5 hours; or / and the centrifugation refers to centrifugation at 4000 rpm for 15 minutes.

10. The method for antigen desorption according to claim 7, characterized in that, The aluminum adjuvant is aluminum hydroxide adjuvant.

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