Analytical methods for adjuvant-adjuvant and adjuvant-antigen interactions

Abstract

The application belongs to the technical field of vaccines, and discloses an analysis method for interaction of adjuvants and antigens, adjuvants and adjuvants, including analysis methods for interaction of antigens and CpG adjuvants, antigens and aluminum adjuvants, antigens and complex adjuvants, and CpG adjuvants and aluminum adjuvants. The analysis method is novel, the analysis result is accurate, the analysis content is more comprehensive, and the analysis method can provide technical support for determining the best vaccine preparation form, clarifying how the adjuvant affects the response of the immune system, and developing more effective vaccines.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, and specifically relates to an analytical method for the interaction between adjuvants and antigens, and between adjuvants. Background Technology

[0002] Adjuvants are an important auxiliary immune component in vaccines. According to relevant industry guidelines on adjuvants (Announcement No. 90 of 2019 issued by the National Medical Products Administration on the Technical Guidelines for Prophylactic Aluminum-Containing Adjuvant Vaccines; Committee for Medicinal Products for Human Use. Guideline on adjuvants in vaccines for human use[J]. Reproduction, 2005; WHO. Guidelines on the Nonclinical Evaluation of Vaccine Adjuvants and Adjuvanted Vaccines; TRS 987, Annex 2[EB / OL], 2014-10-18), sufficient research should be conducted on adjuvants and adjuvant-antigen compatibility formulations to support the necessity of adjuvant addition.

[0003] Human adjuvants are primarily single aluminum adjuvants, with research mainly focusing on their production and quality. In recent years, however, the emergence and application of novel single and compound adjuvants such as CpG, AS01, and nanoparticles have continuously improved vaccine development efficiency and protective efficacy. However, due to the diversity of new adjuvants and their different mechanisms of action, there is a lack of clear methods for studying adjuvant-antigen formulations. Furthermore, it is difficult to characterize and study the interactions between antigens and adjuvants, and between adjuvants themselves, using conventional methods. This aspect has always been a challenge in the research of novel adjuvant vaccine formulations, and a unified research methodology has yet to be established. The lack of research on adjuvant-antigen and adjuvant-adjuvant interactions is a significant factor affecting the quality of adjuvant-antigen complexes and finished vaccines. Summary of the Invention

[0004] This invention provides analytical methods for the interaction of adjuvants with antigens and between adjuvants. These methods include analytical methods for the interaction of antigens with CpG adjuvants, antigens with aluminum adjuvants, antigens with combined adjuvants, and CpG adjuvants with aluminum adjuvants.

[0005] In a first aspect, the present invention provides an analytical method for the interaction between an antigen and a CpG adjuvant, comprising the following steps: mixing the antigen with a CpG adjuvant, detecting at least one of the following: the molecular weight of the antigen before and after mixing, the migration ability of the antigen, the isoelectric point band range of the antigen, and the affinity between the antigen and the CpG adjuvant molecule, and determining whether the antigen and the CpG adjuvant interact.

[0006] In some embodiments, the antigen is the varicella-zoster virus glycoprotein E antigen protein.

[0007] In some embodiments, the CpG adjuvant is a BCG-CpG-DNA adjuvant.

[0008] In some embodiments, the analytical method further includes: mixing the antigen with an aluminum adjuvant and immunizing animals, and detecting the results of the immunogenicity test.

[0009] In some implementations, the antigen and CpG adjuvant are considered not to interact when the following conditions are met simultaneously: a1, there is no intermolecular affinity between the antigen and the CpG adjuvant; a2, the molecular weight of the antigen before and after mixing does not change; a3, the isoelectric point band of the antigen before and after mixing does not change; a4, the migration ability of the antigen before and after mixing does not change.

[0010] In some implementations, an interaction between the antigen and the CpG adjuvant is determined when at least one of the following conditions is met: b1, the antigen and the CpG adjuvant have intermolecular affinity; b2, the molecular weight of the antigen changes before and after mixing; b3, the isoelectric point band of the antigen changes before and after mixing; b4, the migration ability of the antigen changes before and after mixing.

[0011] In some implementations, surface plasmon resonance is used to detect the affinity between the antigen and CpG adjuvant molecules.

[0012] In some implementations, multi-angle light scattering size exclusion chromatography and / or non-reducing capillary gel electrophoresis are used to detect the molecular weight of the antigen before and after mixing;

[0013] In some implementations, imaging capillary isoelectric focusing electrophoresis is used to detect the isoelectric point zones of the antigens before and after mixing.

[0014] In some implementations, sodium dodecyl sulfate capillary gel electrophoresis is used to detect the migration ability of the antigen before and after mixing.

[0015] Secondly, the present invention provides an analytical method for the interaction between antigen and aluminum adjuvant, comprising the following steps: mixing antigen and aluminum adjuvant, detecting the particle size distribution of antigen before and after mixing, and determining whether the antigen and aluminum adjuvant interact.

[0016] In some embodiments, the antigen is the varicella-zoster virus glycoprotein E antigen protein.

[0017] In some embodiments, the aluminum adjuvant is aluminum hydroxide.

[0018] In some embodiments, the analytical method further includes: mixing the antigen with an aluminum adjuvant and immunizing animals, and detecting the results of the immunogenicity test.

[0019] In some implementations, if the particle size distribution of the antigen does not change before and after mixing, it is determined that the aluminum adjuvant does not interact with the antigen.

[0020] In some implementations, if the particle size distribution of the antigen changes before and after mixing, it is determined that the aluminum adjuvant has interacted with the antigen.

[0021] In some implementations, dynamic light scattering is used to detect the particle size distribution of the antigen before and after mixing.

[0022] Thirdly, the present invention provides an analytical method for the interaction between CpG adjuvant and aluminum adjuvant, comprising the following steps: mixing CpG adjuvant and aluminum adjuvant, detecting the nucleic acid content of CpG adjuvant before and after mixing, and determining whether CpG adjuvant and aluminum adjuvant interact.

[0023] In some embodiments, the CpG adjuvant is a BCG-CpG-DNA adjuvant.

[0024] In some embodiments, the aluminum adjuvant is aluminum hydroxide.

[0025] In some implementations, if the nucleic acid content of the CpG adjuvant does not change before and after mixing, it is determined that the CpG adjuvant and the aluminum adjuvant do not interact.

[0026] In some implementations, if the nucleic acid content of the CpG adjuvant changes before and after mixing, it is determined that the CpG adjuvant and the aluminum adjuvant have interacted.

[0027] Fourthly, the present invention provides an analytical method for the interaction between antigen and compound adjuvant, comprising the following steps: mixing antigen with CpG adjuvant and aluminum adjuvant, detecting at least one of the following before and after mixing: particle size distribution of antigen, antigen content, CpG adjuvant nucleic acid content, CpG nucleic acid molecular weight and distribution, and CpG effective content, to determine whether the antigen and compound adjuvant interact.

[0028] In some embodiments, the antigen is the varicella-zoster virus glycoprotein E antigen protein.

[0029] In some embodiments, the CpG adjuvant is a BCG-CpG-DNA adjuvant.

[0030] In some embodiments, the aluminum adjuvant is aluminum hydroxide.

[0031] In some embodiments, the analytical method further includes: mixing the antigen with CpG adjuvant and aluminum adjuvant and immunizing animals, and detecting the results of the immunogenicity test.

[0032] In some implementations, the antigen and adjuvant are considered not to have interacted when the following conditions are met simultaneously: c1, the particle size distribution of the antigen before and after mixing does not change; c2, the content of the antigen before and after mixing does not change; c3, the nucleic acid content of the CpG adjuvant does not change before and after mixing; c4, the molecular weight and distribution of the CpG nucleic acid do not change; c5, the effective content of CpG does not change.

[0033] In some implementations, the interaction between the antigen and the adjuvant is determined when at least one of the following conditions is met: d1, the particle size distribution of the antigen changes before and after mixing; d2, the content of the antigen changes before and after mixing; d3, the nucleic acid content of the CpG adjuvant changes before and after mixing; d4, the molecular weight and distribution of the CpG nucleic acid change; d5, the effective content of CpG changes.

[0034] The beneficial effects of this invention are as follows:

[0035] This invention provides a novel analytical method for analyzing the interactions between antigens and CpG adjuvants, antigens and aluminum adjuvants, and antigens and combined adjuvants. For the first time, this method determines whether an interaction occurs between the antigen and each adjuvant based on one or more of the following criteria selected before and after mixing: whether one or more physicochemical characteristics of the antigen (particle size distribution, molecular weight, isoelectric point) change; whether the adjuvant content changes; and whether intermolecular affinity exists between the antigen and adjuvant. The analytical method for analyzing the interaction between CpG adjuvants and aluminum adjuvants, also for the first time, determines whether an interaction occurs between the CpG adjuvant and aluminum adjuvant based on whether the nucleic acid content of the CpG adjuvant changes before and after mixing. This invention offers novel analytical methods and highly accurate results.

[0036] Based on the analytical methods for the interaction between antigen and CpG adjuvant, antigen and aluminum adjuvant, antigen and combined adjuvant, and CpG adjuvant and aluminum adjuvant provided by this invention, combined with the immunogenicity comparison of each combination with a single antigen, it can provide technical support for determining the optimal vaccine formulation, elucidating how adjuvants affect the immune system response, and developing more effective vaccines. Attached Figure Description

[0037] Figure 1A This is a particle size distribution of the gE protein solution.

[0038] Figure 1BThis is a particle size distribution of the gE protein and aluminum adjuvant mixture.

[0039] Figure 1C The particle size distribution of the gE protein and aluminum adjuvant mixture after desorption is shown in the diagram.

[0040] Figure 1D This is a particle size distribution of the gE protein and BCO2 adjuvant mixture.

[0041] Figure 1E The particle size distribution of the gE protein and BCO2 adjuvant mixture after desorption is shown in the diagram.

[0042] Figure 2 A spectrum showing the affinity of gE protein for BCG-CpG-DNA adjuvant SPR.

[0043] Figure 3A MALS detection map of gE protein.

[0044] Figure 3B MALS detection pattern for BCG-CpG-DNA adjuvant.

[0045] Figure 3C MALS detection pattern of gE protein and BCG-CpG-DNA adjuvant mixture.

[0046] Figure 4A The image shows the CE-SDS detection pattern of gE protein.

[0047] Figure 4B The CE-SDS detection spectrum for BCG-CpG-DNA adjuvant.

[0048] Figure 4C The image shows the CE-SDS detection pattern of the mixture of gE protein and BCG-CpG-DNA adjuvant.

[0049] Figure 5A The iCIEF detection map of gE protein.

[0050] Figure 5B The iCIEF detection map for BCG-CpG-DNA adjuvant.

[0051] Figure 5C The iCIEF detection spectrum is shown for a mixture of gE protein and BCG-CpG-DNA adjuvant.

[0052] Figure 6A This is a map showing the number of gE protein-specific IFN-γ cells (SFC).

[0053] Figure 6B A spectrum of gE protein-specific IL-2 cell counts (SFCs). Detailed Implementation

[0054] General definitions and terms

[0055] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from this specification and the accompanying drawings. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention, the following terms are defined below.

[0056] Antigen: A substance that can stimulate the body's immune system to produce antibodies or sensitize immune activity and can specifically bind to them, including but not limited to proteins. For example, a target antigen gene of a certain bacterium or virus is constructed in an expression vector, transformed into bacteria, yeast, or mammalian or insect cells, and expressed and purified.

[0057] gE protein: gE protein is one of the major glycoproteins on the envelope of the VZV virus, and the immune response induced by it plays an important role in limiting viral spread and recovery from infection. gE protein can be obtained commercially or prepared using genetic engineering techniques known in the art.

[0058] Adjuvants: Adjuvants are substances that can assist antigen responses and regulate the intensity and type of immune responses. The functions of adjuvants include increasing the immunogenicity of antigens, altering the nature of immune responses, reducing the amount of antigen and the number of immunizing agents required for successful immunization, and enhancing the immune response in immunocompromised individuals.

[0059] CpG adjuvants generally refer to DNA containing unmethylated cytosine-guanine dinucleotide (CpG) and its specific flanking sequences, which have immunostimulatory effects. Their structural characteristic is 5'-purine-purine-CpG-pyrimidine-pyrimidine-3'. They mainly include synthetically produced CpG-ODN adjuvants and biologically extracted CpG-DNA adjuvants.

[0060] BCG-CpG-DNA adjuvant: The BCG-CpG-DNA referred to in this invention is a double-stranded DNA fragment extracted from BCG, which contains a large number of unmethylated CpG sequences, and is therefore simply referred to as "BCG-CpG-DNA".

[0061] Compound adjuvants: scientific and organic combination of two or more adjuvants to enhance / promote antigen-specific or comprehensive immune responses through synergistic / additive effects, maximizing the adjuvant effect and reducing the occurrence of side effects, including but not limited to compound adjuvants formed by CpG adjuvants and aluminum adjuvants.

[0062] BC02 Adjuvant: A composite adjuvant system composed of BCG-CpG-DNA adjuvant and aluminum hydroxide adjuvant. The BCG-CpG-DNA adjuvant can activate immune cells and induce a strong Th1 immune response, while the aluminum hydroxide adjuvant plays an adjuvant role by slowly releasing antigens. The combination of the two can synergistically enhance the immune protection effect of the vaccine.

[0063] Interaction: Generally refers to a form of manifestation of the connection between substances or internal factors of substances, including but not limited to the physical or chemical connection between substances.

[0064] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. The experimental materials involved in the following embodiments are specifically as follows:

[0065] 1. Experimental animals: SPF-grade BALB / c mice, female, 6 - 8 weeks old, purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd., animal license number: SCXK(Shandong)20140007.

[0066] 2. Antigens and adjuvants: gE protein: Prepared by culturing and purifying the extracellular sequence of gE expressed by recombinant CHO cells as an antigen; BCG-CpG-DNA adjuvant: Prepared by crushing and extracting nucleic acids from BCG cultures and then purifying, sterilizing, and filtering (patent number: CN100379454C); Aluminum adjuvant: The imported supplier is Croda Denmark, product code: AJV3012.

[0067] 3. Main reagents: TMB chromogenic solution, termination solution, coating solution, and washing solution were purchased from Solarbio Science & Technology Co., Ltd.; gE protease-labeled secondary antibody was purchased from Thermo Fisher Scientific; Bovine serum albumin and Concanavalin A were purchased from Sigma; Mouse lymphocyte separation solution was purchased from Dakewe Biotech Co., Ltd.; HBS buffer and mouse lymphocyte separation solution were purchased from Cytiva; 1640 medium and PBS were purchased from HyClone; Fetal bovine serum (FBS) was purchased from Gibco; IFN-γ and IL-12 pre-coated ELISPOT kits were purchased from Mabteck AB, Sweden.

[0068] 4. Major Equipment: HR40-ⅡB2 biosafety cabinet purchased from Qingdao Haier Special Electric Appliances Co., Ltd.; Forma3111 CO2 incubator purchased from Thermo Fisher; JA3103N electronic balance purchased from Shanghai Minqiao Precision Scientific Instruments Co., Ltd.; YXQ-LS-100SⅡ vertical pressure steam sterilizer purchased from Shanghai Boxun Industrial Co., Ltd.; Heraeus Multifuge XIR benchtop high-speed refrigerated centrifuge purchased from Thermo Fisher, USA; iSpot enzyme-linked immunosorbent assay (ELISA) spectrometer purchased from Germany; UV spectrophotometer purchased from Shimadzu; nanoparticle size counter purchased from Malvern; Biacore T200 purchased from Cytiva; high performance liquid chromatograph purchased from Waters; laser scattering detector and differential refractive index detector purchased from WYATT; capillary isoelectric focusing instrument purchased from Maurice, etc.

[0069] Table 1 Summary of Abbreviations and Meanings

[0070]

[0071]

[0072] Example 1: Interaction analysis between gE protein and aluminum hydroxide

[0073] 1.1 Sample preparation: (1) gE protein: Take 0.1 ml of gE protein stock solution (protein concentration 5.4 mg / ml), add 0.1 ml of PBS buffer, and mix well; (2) gE protein and aluminum hydroxide mixture: Take 0.2 ml of gE protein (protein concentration 5.4 mg / ml), add 0.25 ml of aluminum hydroxide (aluminum hydroxide concentration 10 mg / ml), and mix well; (3) Desorption of gE protein and aluminum hydroxide mixture: Take 0.1 ml of gE protein and aluminum hydroxide mixture, add 0.1 ml of desorption solution (measure 0.625 ml of 20% diethanolamine + 0.1 ml of 10% Triton X-100 and mix well), and incubate at 37℃ for 30 min.

[0074] 1.2 Sample Testing: Add 100 μl of sample to the sample cell of the nanoparticle size counter, and place the sample cell into the instrument for testing. The instrument parameters are set as follows: temperature 25℃, equilibration time 120s, sample cell type ZEN 0040, and number of repetitions 3.

[0075] 1.3 Results:

[0076] Table 2. Main particle size distribution of the test samples

[0077]

[0078] like Figure 1A , 1B As shown in Figures 1C, the DLS particle size study results show that the gE protein particle size is 12.78 nm. The response intensity of the gE protein particle size peak in the gE protein and aluminum hydroxide mixture decreased significantly from 98.94% to 7.70%, and there were two particle sizes, 9.18 nm and 1252 nm, respectively. After desorption of the gE protein and aluminum hydroxide mixture, the average proportion of the gE protein particle size peak response intensity was 83.96%, which was significantly higher than 7.70% before desorption. However, the gE protein particle size was 11.13 nm, which was basically consistent with the gE protein original solution particle size of 12.78 nm.

[0079] In summary, aluminum hydroxide has an adsorption effect on gE protein, indicating that the two interact, but it has little effect on the protein particle size.

[0080] Example 2: Interaction analysis of gE protein with BCG-CpG-DNA adjuvant

[0081] 2.1 Surface Plasmon Resonance (SPR) Study

[0082] 2.1.1 Immobilization of adjuvant: Dilute BCG-CpG-DNA adjuvant to 200 μg / mL with acetate (pH 4.0), and add 200 μL of the diluted sample to the surface of the metal sensor chip of the device to complete the immobilization.

[0083] 2.1.2 Detection: Kinetic parameters were set. The gE protein sample was adjusted to 8000 nM using HBS buffer (pH 7.4), and then diluted to 4000, 2000, 1000, 500, 250, and 125 nM before detection. Instrument parameters were set as follows: flow rate 30 μl / min, binding time 60 s, dissociation time 90 s, and reaction temperature 25℃ (default).

[0084] 2.1.3 Results: (e.g.) Figure 2 As shown, the results of the SPR affinity study indicate that there was no significant change in resonance signal after the gE protein was mixed with the BCG-CpG-DNA adjuvant, suggesting that no affinity was detected between the gE protein and the BCG-CpG-DNA adjuvant.

[0085] 2.2 Multi-angle light scattering size exclusion chromatography (SEC-MALS) study

[0086] 2.2.1 Sample preparation: (1) gE protein: dilute gE protein stock solution to 100 μg / ml with PBS buffer; (2) BCG-CpG-DNA adjuvant: dilute BCG-CpG-DNA adjuvant stock solution to 200 μg / ml with PBS buffer; (3) gE protein + BCG-CpG-DNA adjuvant mixture: take 10 μl of gE protein stock solution, add 100 μl of BCG-CpG-DNA adjuvant stock solution, and then add 3900 μl of PBS buffer and mix well.

[0087] 2.2.2 The test sample was separated using a high-performance liquid chromatography (HPLC) system. The HPLC detection parameters were as follows: Zenix-CSEC column 7.8mm×300mm, 3μm, column temperature 25℃, detection wavelength 280nm, injection volume 100μg, mobile phase 10mM phosphate buffer, flow rate 0.5mL / min, and acquisition time 30min.

[0088] The test sample was separated by chromatographic column and then analyzed by MALS. The relevant parameters are as follows: flow cell temperature: 25℃, Light Scattering Instrument: wyatt heleos, Cell Type: Fused Silica, Wavelength: 660.0nm, Calibration Constant: 3.2339×10-5l / (V cm).

[0089] 2.2.3 Results:

[0090] Table 3. Statistical analysis of SEC-MALS results for gE protein and BCG-CpG-DNA adjuvant.

[0091]

[0092] The results are as follows Figure 3A , 3B As shown in Table 3 and 3C, using the SEC-MALS method, the retention time and molecular weight of gE protein in the mixture of gE protein and BCG-CpG-DNA adjuvant were basically consistent with the results of gE protein detection in a single sample. Therefore, in this system, the mixing of BCG-CpG-DNA adjuvant and gE protein did not affect the molecular weight of gE protein.

[0093] 2.3 Non-reducing capillary gel electrophoresis (CE-SDS) study

[0094] 2.3.1 Sample preparation: (1) gE protein: Take 0.1 ml of gE protein stock solution (protein concentration 5.4 mg / ml); (2) BCG-CpG-DNA adjuvant: Take 0.1 ml of BCG-CpG-DNA adjuvant stock solution (nucleic acid concentration 1.0 mg / ml); (3) gE protein + BCG-CpG-DNA adjuvant mixture: Take 10 μl of gE protein stock solution and add 100 μl of BCG-CpG-DNA adjuvant stock solution and mix well before testing.

[0095] 2.3.2 Sample Detection: The prepared sample was mixed with 1×Sample Buffer and iodoacetamide, reacted at 70℃ for 5 minutes, cooled to room temperature, and centrifuged at 13400g for 5 minutes. Then, 80 μl of the mixture was transferred to a 96-well plate for instrument analysis. The instrument parameters were set as follows: Sample Load, 20 sec 4600 Volts; Separation, 35.0 min 5750 Volts; Temperature 15℃.

[0096] 2.3.3 Results:

[0097] Table 4. Statistical analysis of CE-SDS results of the test samples

[0098]

[0099] The results are as follows Figure 4A , 4B As shown in Tables 4 and 4, the relative migration time (RMT) of the gE protein peak in the mixture of gE protein and BCG-CpG-DNA adjuvant was 2.654, which was basically consistent with the RMT of gE protein (2.668). The RMT of the BCG-CpG-DNA adjuvant peak in the mixture of gE protein and BCG-CpG-DNA adjuvant was 2.925, which was basically consistent with the RMT of BCG-CpG-DNA adjuvant (2.918). Therefore, in this system, the mixing of BCG-CpG-DNA adjuvant and gE protein did not affect their respective relative migration times, indicating that the mixing of BCG-CpG-DNA adjuvant and gE protein did not affect the migration ability of gE protein.

[0100] 2.4 Imaging capillary isoelectric focusing electrophoresis (iCIEF) study

[0101] 2.4.1 Sample preparation: (1) gE protein: Take 0.1 ml of gE protein stock solution (protein concentration 5.4 mg / ml); (2) BCG-CpG-DNA adjuvant: Take 0.1 ml of BCG-CpG-DNA adjuvant stock solution (nucleic acid concentration...

[0102] 1.0 mg / ml); (3) gE protein + BCG-CpG-DNA adjuvant mixture: Take 10 μl of gE protein stock solution and add 100 μl of BCG-CpG-DNA adjuvant stock solution and mix evenly, then test.

[0103] 2.4.2 Sample Testing: After appropriate ultrafiltration, the pretreated sample was prepared into a 100 μl composition according to the table below. Replace "Sample" with water (100 μl total volume). After preparation, mix thoroughly and centrifuge. Add the top 80 μl of the centrifuged sample to a 96-well plate, avoiding air bubbles. Briefly centrifuge the 96-well plate before loading it into the instrument. The sample system is as follows: 35 μl of 1% methylcellulose, 4 μl of amphoteric electrolyte, 1 μl of PI Marker 3.38, 1 μl of PI Marker 7.05, and 200 mM IDA. The total composition volume is 100 μl. If the volume is less than 100 μl after adding the sample, adjust with water. Instrument parameters are set as follows: Focus Period 1 1500V for 1.0 min, Focus Period 2 3000V for 5.0 min, Sample Load 55 Seconds, Temperature 10℃.

[0104] 2.4.3 Results: (e.g.) Figure 5A , 5B As shown in Figure 5C, the measured isoelectric point range of gE protein is between 4.0 and 6.0; the measured isoelectric point range of the mixture of gE protein and BCG-CpG-DNA adjuvant is also between 4.0 and 6.0; the test sample BCG-CpG-DNA adjuvant showed no significant response value in the iCIEF capillary method. Therefore, in this system, the mixing of BCG-CpG-DNA adjuvant with gE protein did not affect the isoelectric point range of gE protein.

[0105] 2.5 Conclusion: Based on the previous results, no affinity was detected between gE protein and BCG-CpG-DNA adjuvant. The mixture of BCG-CpG-DNA adjuvant and gE protein did not affect the molecular weight of gE protein, the migration ability of gE protein, or the isoelectric point range of gE protein. Based on the above results, it is concluded that there is no interaction between BCG-CpG-DNA adjuvant and gE protein.

[0106] Example 3: Interaction between BCG-CpG-DNA adjuvant and aluminum hydroxide

[0107] 3.1 Accurately measure 7.75 ml of PBS buffer, add 2 ml of BCG-CpG-DNA adjuvant stock solution (1 mg / ml), and then add 0.25 ml of aluminum hydroxide adjuvant stock solution (10 mg / ml). Mix thoroughly. Centrifuge at 6500 g for 5 min and collect the supernatant. Accurately take 1.0 ml of the test sample supernatant, dilute it 10 times with PBS buffer, and measure the absorbance at 260 nm using UV-Vis spectrophotometry. The absorption coefficient is expressed as E. 1％ 1cm =200, calculate the nucleic acid content in the supernatant of the test sample, and denot it as Cs.

[0108] Separately, accurately measure 8 ml of PBS buffer and 2 ml of BCG-CpG-DNA adjuvant stock solution (1 mg / ml), and mix well. Measure the absorbance at 260 nm using UV-Vis spectrophotometry, according to E... 1％ 1cm =200 to calculate the nucleic acid content of the test sample, denoted as Ct.

[0109] The adsorption rate was calculated using the following formula, and the interaction between BCG-CpG-DNA adjuvant and aluminum hydroxide was analyzed using the nucleic acid adsorption rate as an indicator. The formula for calculating the nucleic acid adsorption rate is: P(%) = (1 - Cs / Ct) × 100%.

[0110] In the formula, P is the adsorption rate (%); Cs is the nucleic acid content of the supernatant of the test sample (μg / ml); and Ct is the total nucleic acid content of the test sample (μg / ml).

[0111] 3.2 Results:

[0112] Table 5. Results of adsorption rate detection of BCG-CpG-DNA adjuvant and aluminum hydroxide.

[0113]

[0114] The results showed that the nucleic acid contents of the three batches of BCG-CpG-DNA adjuvant after dilution were 202.10 μg / ml, 214.58 μg / ml, and 188.32 μg / ml, respectively. The average nucleic acid contents of the supernatant after mixing BCG-CpG-DNA with aluminum hydroxide were 159.70 μg / ml, 165.58 μg / ml, and 141.38 μg / ml, respectively. Substituting the measured nucleic acid contents into the adsorption rate calculation formula, the nucleic acid adsorption rates of the three batches of BCG-CpG-DNA adjuvant mixed with aluminum hydroxide were 20.98%, 22.84%, and 24.93%, respectively. Under the experimental conditions, the nucleic acid adsorption rate of the mixture of BCG-CpG-DNA adjuvant and aluminum hydroxide was (22.92±1.98)%, indicating that aluminum hydroxide has a certain adsorption capacity for BCG-CpG-DNA adjuvant, causing a decrease in nucleic acid content after mixing, indicating an interaction between the two.

[0115] Example 4: Interaction analysis of gE protein with BCO2 adjuvant

[0116] The effect of mixing 4.1gE protein with BCO2 adjuvant on the content of BCG-CpG-DNA nucleic acid.

[0117] Processing and content detection of the supernatant sample after adsorption: Redissolve the lyophilized sample containing BCG-CpG-DNA adjuvant and gE antigen protein in 250 μg / ml aluminum hydroxide diluent. After mixing, the BCG-CpG-DNA adjuvant content is 200 μg / ml, and the gE antigen protein content is 100 μg / ml. Centrifuge the sample at 6500g for 5 min and collect the supernatant. Accurately take 1.0 ml of the supernatant, dilute it 10 times with PBS buffer, and measure the absorbance at 260 nm using UV-Vis spectrophotometry. 1％ 1cm =200 Calculate the BCG-CpG-DNA nucleic acid content in the supernatant of the test sample, and record it as the BCG-CpG-DNA nucleic acid content Cs of the supernatant of the test sample.

[0118] Preparation and content determination of aluminum-free test samples: Reconstitute the lyophilized sample containing BCG-CpG-DNA adjuvant and gE antigen protein with sterile water for injection. After mixing, the BCG-CpG-DNA adjuvant content should be 200 μg / ml, and the gE antigen protein content should be 100 μg / ml. Accurately take 1.0 ml of the mixed sample, dilute it 10-fold with PBS buffer, and measure the absorbance at 260 nm using UV-Vis spectrophotometry. 1％ 1cm =200 Calculate the BCG-CpG-DNA nucleic acid content of the test sample and record it as the BCG-CpG-DNA nucleic acid content Ct of the test sample.

[0119] Calculation of BCG-CpG-DNA adjuvant nucleic acid adsorption rate: The BCG-CpG-DNA adjuvant nucleic acid adsorption rate was calculated according to the following formula. This rate was used as an indicator to analyze the interaction between the BCG-CpG-DNA adjuvant and the gE protein containing aluminum adjuvant. Formula for calculating BCG-CpG-DNA nucleic acid adsorption rate: P(%) = (1 - Cs / Ct) × 100%.

[0120] In the formula, P is the adsorption rate, %; Cs is the BCG-CpG-DNA nucleic acid content of the supernatant of the test sample, μg / ml; and Ct is the BCG-CpG-DNA nucleic acid content of the test sample, μg / ml.

[0121] result:

[0122] Table 6 Results of BCG-CpG-DNA adjuvant adsorption rate detection

[0123]

[0124] The results showed that the nucleic acid contents of the three batches of BCG-CpG-DNA adjuvant after dilution were 203.5 μg / ml, 202.5 μg / ml, and 198.0 μg / ml, respectively. The average nucleic acid contents of the supernatant after mixing BCG-CpG-DNA with aluminum hydroxide and gE protein were 140.0 μg / ml, 136.0 μg / ml, and 137.50 μg / ml, respectively. Substituting the measured nucleic acid contents into the adsorption rate calculation formula, the nucleic acid adsorption rates of the three batches of BCG-CpG-DNA adjuvant mixed with aluminum hydroxide were 31.20%, 32.84%, and 30.56%, respectively. Under the conditions of this experiment, the nucleic acid adsorption rate of the mixture of BCG-CpG-DNA adjuvant and aluminum hydroxide was (31.53±1.18)%. It can be seen that aluminum hydroxide and gE protein adsorbed BCG-CpG-DNA adjuvant, which reduced the content of BCG-CpG-DNA nucleic acid after the gE protein was mixed with BCO2 adjuvant, thus causing its alteration.

[0125] 4.2 Effect of mixing gE protein with BCO2 adjuvant on gE protein content

[0126] Processing and content detection of the supernatant after adsorption of the test sample: Redissolve the lyophilized sample containing both BCG-CpG-DNA adjuvant and gE antigen protein in 250 μg / ml aluminum hydroxide diluent. After mixing, the BCG-CpG-DNA adjuvant content is 200 μg / ml, and the gE antigen protein content is 100 μg / ml. Centrifuge the sample at 6500g for 5 min and collect the supernatant. Accurately take 1.0 ml of the test sample supernatant, dilute it 800 times with PBS buffer, and detect the gE protein antigen content in the supernatant using ELISA. Calculate the gE protein antigen content in the test sample supernatant and record it as the gE protein antigen content Cs.

[0127] Preparation and content determination of aluminum-free test samples: The lyophilized sample containing BCG-CpG-DNA adjuvant and gE antigen protein was reconstituted with sterile water for injection. After mixing, the BCG-CpG-DNA adjuvant content was 200 μg / ml, and the gE antigen protein content was 100 μg / ml. 1.0 ml of the reconstituted sample was accurately taken, diluted 800-fold with PBS buffer, and the gE protein antigen content in the supernatant was detected using ELISA. The gE protein antigen content in the supernatant was calculated and recorded as the gE protein antigen content (Ct) of the test sample.

[0128] Calculation of gE protein antigen adsorption rate: The protein antigen adsorption rate was calculated according to the following formula, and the interaction between gE protein and aluminum adjuvant and BCG-CpG-DNA adjuvant was analyzed using the protein antigen adsorption rate as an indicator. Formula for calculating protein antigen adsorption rate: P(%) = (1-Cs / Ct) × 100%.

[0129] In the formula, P is the adsorption rate, %; Cs is the gE protein antigen content of the supernatant of the test sample, U / ml; and Ct is the gE protein antigen content of the test sample, U / ml.

[0130] result:

[0131] Table 7 Results of gE protein adsorption rate detection

[0132]

[0133] The results showed that the protein antigen contents of the three batches of gE protein after dilution were 7.81 U / ml, 7.94 U / ml, and 8.29 U / ml, respectively. The average protein antigen contents of the supernatant after mixing gE protein with aluminum hydroxide and BCG-CpG-DNA adjuvant were 5.82 U / ml, 5.53 U / ml, and 6.00 U / ml, respectively. Substituting the measured protein antigen contents into the adsorption rate calculation formula, the protein adsorption rates of the three batches of gE protein mixed with aluminum hydroxide and BCG-CpG-DNA adjuvant were 25.48%, 30.35%, and 27.62%, respectively. Under the conditions of this experiment, the protein adsorption rate of gE protein mixed with aluminum hydroxide and BCG-CpG-DNA adjuvant was (27.82±2.44)%. It can be seen that aluminum hydroxide and BCG-CpG-DNA adjuvant adsorbed gE protein, resulting in a decrease in gE protein content after mixing with BCG adjuvant, thus altering its composition.

[0134] 4.3 Effect of mixing gE protein with BCO2 adjuvant on gE protein particle size distribution

[0135] Sample preparation: (1) gE protein: Take 0.1 ml of gE protein stock solution (protein concentration 5.4 mg / ml), add 0.1 ml of PBS buffer, and mix well; (2) gE protein and BCO2 adjuvant mixture: Take 250 μg / ml aluminum hydroxide diluent to reconstitute the lyophilized sample containing BCG-CpG-DNA adjuvant and gE antigen protein, mix well, and the BCG-CpG-DNA adjuvant content in the sample is 200 μg / ml, and the gE antigen protein content is 100 μg / ml; (3) Take 0.1 ml of the mixed solution, add 0.1 ml of desorption solution (measure 0.625 ml of 20% diethanolamine + 0.1 ml of 10% Triton X-100 and mix well), and incubate at 37℃ for 30 min.

[0136] Sample detection: Add 100 μl of sample to the sample cell of the nanoparticle size counter, and place the sample cell into the instrument for dynamic light scattering detection. The instrument parameters are set as follows: temperature 25℃, equilibration time 120s, sample cell type ZEN 0040, and number of repetitions 3.

[0137] result:

[0138] Table 8. Main particle size distribution of the test samples

[0139]

[0140] like Figure 1A , 1DAs shown in Figure 1E, the DLS particle size study results show that the gE protein particle size is 12.78 nm. The response intensity of the gE protein particle size peak in the gE protein and BCO2 adjuvant solution decreased significantly from 98.94% to 41.78%, and there were two particle sizes, 8.59 nm and 424.40 nm, respectively. After desorption of the gE protein and BCO2 adjuvant mixture, the average proportion of the gE protein particle size peak response intensity was 63.51%, which was higher than the 41.78% before desorption. The gE protein particle size was 11.07 nm, which was basically consistent with the gE protein stock solution particle size of 12.78 nm.

[0141] In summary, BCO2 adjuvant has an adsorption effect on gE protein, which changes the particle size distribution of gE protein after mixing with BCO2 adjuvant.

[0142] Effects of 4.4gE protein mixed with BCO2 adjuvant on the molecular weight and distribution of BCG-CpG-DNA adjuvant nucleic acids

[0143] 4.4.1 Sample Preparation

[0144] (1) BCG-CpG-DNA sample: Take 0.1 ml of BCG-CpG-DNA adjuvant stock solution (nucleic acid concentration 1.0 mg / ml), dilute it with PBS buffer to about 5 μg / ml and inject.

[0145] (2) gE protein and BCO2 adjuvant mixture: Redissolve the lyophilized sample containing BCG-CpG-DNA adjuvant and gE antigen protein in 250 μg / ml aluminum hydroxide diluent. After mixing, the BCG-CpG-DNA adjuvant content in the sample is 200 μg / ml, and the gE antigen protein content is 100 μg / ml. Extract and purify the BCG-CpG-DNA in the mixture using phenol-chloroform-isoamyl alcohol organic solvents. Then dilute the purified sample to about 5 μg / ml for loading.

[0146] 4.4.2 Testing of the test sample

[0147] Using the matching agarose gel (Separation Buffer) and dilution buffer as the separation system, the injection voltage was 4kV and the analysis voltage was 4kV. A processing method was established based on the corresponding peak time to establish the relationship between the standard marker fragment size and the retention time, thereby obtaining the molecular weight fragment range of the test sample. The distribution ratio of different molecular weights was obtained by the peak area ratio of each molecular weight range.

[0148] 4.4.3. Research Results

[0149] Table 9. Results of the study on molecular weight and corresponding distribution ratio

[0150]

[0151] The results showed that the overall proportion of 3-10kb molecular weight in the molecular weight distribution of BCG-CpG-DNA adjuvant nucleic acid and the molecular weight distribution of nucleic acid in the mixture of gE protein and BCO2 adjuvant was higher than 60%. The overall proportion of 3-10kb molecular weight and the distribution proportions of each segment, <3k, and >10kb were basically the same in both cases, indicating that the mixture of gE protein and BCO2 adjuvant had no significant effect on the molecular weight and distribution of BCG-CpG-DNA nucleic acid.

[0152] Effect of 4.5g E protein mixed with BCO2 adjuvant on the effective CpG content of BCG-CpG-DNA adjuvant

[0153] 4.5.1 Sample Preparation

[0154] (1) BCG-CpG-DNA sample: Dilute the BCG-CpG-DNA adjuvant stock solution to about 5 μg / ml using PBS buffer before injection.

[0155] (2) gE protein and BCO2 adjuvant mixture: Redissolve the lyophilized sample containing both BCG-CpG-DNA adjuvant and gE antigen protein in 250 μg / ml aluminum hydroxide diluent. After mixing, the BCG-CpG-DNA adjuvant content in the sample is 200 μg / ml, and the gE antigen protein content is 100 μg / ml. Extract and purify the BCG-CpG-DNA in the mixture using phenol-chloroform-isoamyl alcohol organic solvents. Then, hydrolyze the purified sample with methyltransferase before loading.

[0156] 4.5.2 Detection of effective content

[0157] In this invention, the CpG content of BCG-CpG-DNA can be obtained by high-performance liquid chromatography (HPLC). For example, the reverse-phase high-performance liquid chromatography (RP-HPLC) method described in ZL200410033878.1 can be used. The cytosine (dC) of CpG dinucleotides is modified to 5-methylcytosine (m5-dC) using a specific methyltransferase SssI. The DNA is hydrolyzed into single deoxyribonucleosides using nuclease P1 and bacterial alkaline phosphatase (BAP). The difference in the amount of m5-dC detected in the hydrolyzed DNA samples of modified and unmodified DNA is used to quantify CpG by RP-HPLC.

[0158] 4.5.3 Research Results

[0159] Table 10 Results of the study on the effective content of CpG

[0160]

[0161] The results are shown in the table above. The effective CpG content of the BCG-CpG-DNA adjuvant was 27% ± 0.58%, and the effective CpG content of the gE protein and BCO2 adjuvant mixture was 28% ± 0.58%. The two were basically consistent, both within the range of 26% to 29%, indicating that the gE protein and the compound adjuvant mixture had no significant effect on the effective CpG content in BCG-CpG-DNA.

[0162] 4.6 Conclusion: Based on the previous results, mixing gE protein with BCO2 adjuvant altered the content of BCG-CpG-DNA nucleic acid, gE protein content, and gE protein particle size distribution, but had no significant effect on the molecular weight of BCG-CpG-DNA nucleic acid or the effective CpG content. Based on these findings, gE protein interacts with BCO2 adjuvant.

[0163] Example 5: Immunogenicity evaluation of gE protein in combination with different adjuvants

[0164] 5.1 Experimental Grouping:

[0165] The experimental animal groups included a PBS group, a gE protein group, a gE protein and aluminum hydroxide adjuvant group (gE / Al), a gE protein and BCG-CpG-DNA adjuvant group (gE / CpG), and a combined adjuvant group of gE protein, aluminum hydroxide, and BCG-CpG-DNA (gE / BC02). In the gE / Al, gE / CpG, and gE / BC02 groups, 0.0067M PBS was used as the carrier solution. The gE protein was directly mixed with different adjuvants to prepare the vaccine, ensuring that the dose of gE protein in the vaccine was 5 μg, the dose of BCG-CpG-DNA was 10 μg, and the dose of aluminum hydroxide was 12.5 μg. Immunogenicity was evaluated by immunizing the animals.

[0166] 5.2 Experimental Methods:

[0167] Different groups of vaccine samples were used to immunize BALB / c mice. Two intramuscular immunizations were administered, with an interval of 28 days between the two injections. The immunization volume was 50 μL per injection.

[0168] Four weeks after the final immunization, immunized mice were sacrificed on the operating table by blood collection, and their spleens were removed. The spleens were placed on a sieve and ground with a 5 mL syringe. The sieve was then rinsed with approximately 5–6 mL of mouse spleen lymphocyte separation solution to ensure as many cells as possible were incorporated into the liquid. The liquid was then aspirated into a 15 mL centrifuge tube using a Pasteur tube, and the top layer was covered with 500 μL of RPMI 1640 medium. Centrifugation was performed at room temperature, 800 g, for 30 min, with the centrifugation speed set to level 3. After centrifugation, the lymphocyte layer was aspirated using a Pasteur pipette and added to 8 mL of RPMI 1640 medium, followed by inverted washing. Centrifugation was performed at room temperature, 250 g, for 10 min, with the centrifugation speed set to level 3. The supernatant was discarded, and the cell pellet was resuspended in 1 mL of RPMI 1640 complete medium (containing 10% FBS and 1% penicillin-dextrose antibiotics). Based on the cell count results, the spleen cell concentration was adjusted to 2.5 × 10⁶ cells / mL using RPMI 1640 complete medium. 6 per mL.

[0169] Remove the ELISPOT plate from the kit and wash 5 times with 200 μL PBS per well. Drain the solution and add 200 μL of complete culture medium per well, incubating at room temperature for at least 30 minutes. Detect the number of mouse IFN-γ and mouse IL-2 cell spots specific to the secretory antigen. Discard the cell suspension, wash 5 times with 200 μL PBS buffer per well, and blot dry. Dilute the detection antibody 1000-fold with PBS containing 0.5% FBS, add 100 μL / well to a 96-well plate, and incubate at room temperature for 2 h. Discard the antibody solution, wash 5 times with PBS buffer at 200 μL / well, and blot dry. Dilute streptomycin-HRP 1000-fold with PBS containing 0.5% FBS, add 100 μL / well to a 96-well plate, and incubate at room temperature for 1 h. Discard the solution, wash 5 times with PBS buffer at 200 μL / well, and blot dry. Add 100 μL / well of universal substrate solution filtered through a 0.45 μm filter, and develop the color until distinguishable spots appear. Rinse with purified water to terminate the color development reaction. After air drying, count the spots using an ELISPOT plate reader. The antigen-specific SFC count = average number of spots in antigen-stimulated wells - average number of spots in unstimulated wells.

[0170] 5.3 Experimental Results:

[0171] Table 1. Data on the detection of 11gE protein-specific IFN-γ and IL-2 cell numbers (SFCs)

[0172] PBS group gE group gE / Al group gE / CpG group gE / BC02 group IFN-γ 19±10 14±7 118±56 132±48 250±59 IL-2 22±9 88±44 112±51 130±45 326±55

[0173] like Figure 6A , 6BAs shown in Table 11, the number of cells secreting gE-specific IFN-γ and IL-2 in the gE protein and adjuvant combination group (gE / BC02) was significantly higher than that in the PBS group, gE protein group, gE protein and aluminum adjuvant group (gE / Al), and gE protein and BCG-CpG-DNA adjuvant group (gE / CpG), with significant differences (p<0.001). This suggests that the combined adjuvant vaccine has a better cellular immune effect, which can significantly stimulate and induce antigen-induced high-titer cellular immunity and play a role in preventing herpes zoster.

[0174] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of 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 protection scope of the present invention.

Claims

1. An analytical method for the interaction between an antigen and a CpG adjuvant, characterized in that, Includes the following steps: The antigen is mixed with CpG adjuvant, and at least one of the following is detected before and after mixing: the molecular weight of the antigen, the migration ability of the antigen, the isoelectric point range of the antigen, and the affinity between the antigen and CpG adjuvant molecules, to determine whether the antigen interacts with the CpG adjuvant.

2. The analytical method according to claim 1, characterized in that, The antigen is the varicella-zoster virus glycoprotein E antigen protein.

3. The method according to any one of claims 1-2, characterized in that, The CpG adjuvant is a BCG-CpG-DNA adjuvant.

4. The analytical method according to any one of claims 1-3, characterized in that, The analytical method further includes: mixing the antigen with CpG adjuvant and immunizing animals, and detecting the results of the immunogenicity test.

5. The analytical method according to any one of claims 1-4, characterized in that, The antigen and CpG adjuvant are considered not to interact when the following conditions are met simultaneously: a1. There is no intermolecular affinity between the antigen and the CpG adjuvant; a2. The molecular weight of the antigen did not change before and after mixing; a3. The isoelectric point zone range of the antigens before and after mixing did not change; a4. The migration ability of the antigens before and after mixing did not change.

6. The analytical method according to any one of claims 1-4, characterized in that, An interaction between an antigen and a CpG adjuvant is determined when at least one of the following conditions is met: b1. There is an intermolecular affinity between the antigen and the CpG adjuvant; b2. The molecular weight of the antigen changes before and after mixing; b3. The isoelectric point zone range of the antigen changes before and after mixing; b4. The migration ability of antigens changes before and after mixing.

7. An analytical method for the interaction between an antigen and an aluminum adjuvant, characterized in that, Includes the following steps: The antigen was mixed with aluminum adjuvant, and the particle size distribution of the antigen before and after mixing was detected to determine whether the antigen and aluminum adjuvant interacted.

8. The analytical method according to claim 7, characterized in that, The antigen is the varicella-zoster virus glycoprotein E antigen protein.

9. The analytical method according to any one of claims 7-8, characterized in that, The aluminum adjuvant is aluminum hydroxide.

10. The analytical method according to any one of claims 7-9, characterized in that, The analytical method further includes: mixing the antigen with aluminum adjuvant and immunizing animals, and then detecting the results of the immunogenicity test.

11. The analytical method according to any one of claims 7-10, characterized in that, If the particle size distribution of the antigen does not change before and after mixing, it is determined that the aluminum adjuvant does not interact with the antigen.

12. The analytical method according to any one of claims 7-10, characterized in that, If the particle size distribution of the antigen changes before and after mixing, it is determined that the aluminum adjuvant has interacted with the antigen.

13. An analytical method for the interaction between CpG adjuvant and aluminum adjuvant, characterized in that, Includes the following steps: CpG adjuvant was mixed with aluminum adjuvant, and the nucleic acid content of CpG adjuvant before and after mixing was detected to determine whether CpG adjuvant and aluminum adjuvant interacted.

14. The analytical method according to claim 13, characterized in that, The CpG adjuvant is a BCG-CpG-DNA adjuvant.

15. The analytical method according to any one of claims 13-14, characterized in that, The aluminum adjuvant is aluminum hydroxide.

16. The analytical method according to any one of claims 13-15, characterized in that, If the nucleic acid content of the CpG adjuvant does not change before and after mixing, it is determined that the CpG adjuvant and the aluminum adjuvant do not interact.

17. According to any one of the analytical methods of claims 13-15, if the nucleic acid content of the CpG adjuvant changes before and after mixing, it is determined that the CpG adjuvant and the aluminum adjuvant have interacted.

18. An analytical method for the interaction between an antigen and a complex adjuvant, characterized in that, The process includes the following steps: mixing the antigen with CpG adjuvant and aluminum adjuvant, and detecting at least one of the following before and after mixing: particle size distribution of the antigen, antigen content, CpG adjuvant nucleic acid content, CpG nucleic acid molecular weight and distribution, and CpG effective content, to determine whether the antigen and the compound adjuvant interact.

19. The analytical method according to claim 18, characterized in that, The antigen is the varicella-zoster virus glycoprotein E antigen protein.

20. The analytical method according to any one of claims 18-19, characterized in that, The CpG adjuvant is a BCG-CpG-DNA adjuvant.

21. The analytical method according to any one of claims 18-20, characterized in that, The aluminum adjuvant is aluminum hydroxide.

22. The analytical method according to any one of claims 18-21, characterized in that, The analytical method further includes: mixing the antigen with CpG adjuvant and aluminum adjuvant and immunizing animals, and detecting the results of the immunogenicity test.

23. The analytical method according to any one of claims 18-22, characterized in that, The antigen and the adjuvant do not interact when the following conditions are met simultaneously: c1, the particle size distribution of the antigen before and after mixing does not change; c2. The antigen content remained unchanged before and after mixing; c3. The CpG adjuvant nucleic acid content did not change before and after mixing; c4. The molecular weight and distribution of CpG nucleic acid did not change; c5. The effective CpG content did not change.

24. The analytical method according to any one of claims 18-22, characterized in that, An interaction between the antigen and the adjuvant is determined when at least one of the following conditions is met: d1, the particle size distribution of the antigen changes before and after mixing; d2. The antigen content changes before and after mixing; d3. The CpG adjuvant nucleic acid content changed before and after mixing; d4. The molecular weight and distribution of CpG nucleic acid changed; d5. The effective CpG content changed.

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