RECOMBINANT VARICELLA-ZOSTER VIRUS (VZV) VACCINE

Abstract

The present disclosure discloses a recombinant varicella-zoster virus (VZV) vaccine, including a fusion protein formed by an amino acid sequence of an extracellular domain of a recombinant glycoprotein gE of a live attenuated VZV strain (OKA strain) gene and an Fc fragment of human immunoglobulin. The present disclosure further provides preparation and use of the fusion protein, a corresponding recombinant gene, a eukaryotic expression vector, etc. The fusion protein of the present disclosure has prominent immunogenicity and can induce the high-level expression of neutralizing antibodies in serum.

Claims

1. A recombinant varicella-zoster virus (VZV) vaccine preparation, comprising a fusion protein formed by an amino acid sequence of an extracellular domain of a recombinant glycoprotein gE of a live attenuated VZV strain (OKA strain) gene and an Fc fragment of human immunoglobulin, wherein the fusion protein has an amino acid sequence shown in SEQ ID No. 1.

2. The vaccine preparation according to claim 1, further comprising a vaccine adjuvant.

3. The vaccine preparation according to claim 2, wherein the vaccine adjuvant is an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant, or a mixture of aluminum hydroxide and aluminum phosphate adjuvants.

4. The vaccine preparation according to claim 1, wherein each dosage unit of the vaccine preparation comprises 5 μg to 200 μg of the fusion protein.

5. The vaccine preparation according to claim 4, wherein each dosage unit of the vaccine preparation comprises 10 μg to 100 μg of the fusion protein.

6. The vaccine preparation according to claim 5, wherein each dosage unit of the vaccine preparation comprises 20 μg to 60 μg of the fusion protein.

7. The vaccine preparation according to claim 1, further comprising other substances that can enhance immunogenicity, wherein the other substances that can enhance immunogenicity comprise, but are not limited to: phosphatidylcholine (PC), lecithin, 3D-MPL, long-chain fatty acid (ester), mineral oil, vegetable oil, sodium methylcellulose (MC-Na), sodium carboxymethylcellulose (CMC-Na), and cholesterol-containing liposome.

8. The vaccine preparation according to claim 1, wherein the vaccine preparation is a lyophilized preparation.

9. The vaccine preparation according to claim 8, wherein the lyophilized preparation is dissolved by an aluminum hydroxide adjuvant suspension before use, and then a resulting mixture is thoroughly mixed and injected intramuscularly or subcutaneously.

10. A recombinant gene capable of expressing the fusion protein according to claim 1, wherein the recombinant gene has a DNA sequence shown in SEQ ID No. 2.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0049] FIG. 1 shows an agarose gel electrophoresis result of enzyme digestion products of the VZV gE-Fc plasmids,

[0050] where lane 1: DL10000 DNA Marker (10,000 bp, 7,000 bp, 4,000 bp, 2,000 bp, 1,000 bp, 500 bp, and 250 bp),

[0051] lanes 2 and 3: enzyme digestion products of the plasmid pUC57-gE-Fc, and

[0052] lanes 4 to 5: enzyme digestion products of the plasmid pXC-K383L.

[0053] FIG. 2 shows positive clones obtained by colony PCR screening of the VZV gE-Fc recombinant plasmids,

[0054] where lane 1: DL10000 DNA Marker (10,000 bp, 7,000 bp, 4,000 bp, 2,000 bp, 1,000 bp, 500 bp, and 250 bp),

[0055] lanes 2 to 7: 6 clones gE-Fc-1 to 6 obtained by colony PCR screening.

[0056] FIG. 3 shows an agarose gel electrophoresis result of linearization enzyme digestion products of the plasmid expression vector,

[0057] where lane 1: DL10000 DNA Marker (10,000 bp, 7,000 bp, 4,000 bp, 2,000 bp, 1,000 bp, 500 bp, and 250 bp),

[0058] lane 2: the plasmid pXC4-VZV gE-Fc, and

[0059] lane 3: the linearized plasmid VZV gE-Fc-straight.

[0060] FIG. 4 shows an HPLC-SEC chromatogram of a recombinant VZV gE protein purified by affinity chromatography.

[0061] FIG. 5 shows an HPLC-SEC chromatogram of a recombinant VZV gE protein.

[0062] FIG. 6 shows the titer (geometric mean titer (GMT)) of serum antibodies produced in mice immunized with a VZV gE vaccine including an aluminum adjuvant at various dosages.

[0063] FIG. 7 shows the titer determination results of serum neutralizing antibodies produced in BALB/C mice immunized with a recombinant VZV gE vaccine including an aluminum adjuvant for adsorption.

[0064] FIG. 8 shows the GMT of neutralizing antibodies produced in BALB/C mice immunized with a VZV gE vaccine including an aluminum adjuvant at different dosages.

DETAILED DESCRIPTION

[0065] The present disclosure is further illustrated through the following examples, but the examples are not intended to limit the present disclosure.

Example 1. Construction of Plasmid Expression Vector

[0066] 1. Source of Gene Sequence

[0067] The VZV gE of the present disclosure was an extracellular domain (ECD, 31-546 aa) of the gE, with a total of 516 amino acids. The Fc fragment was human IgG1 Fc, with a total of 232 amino acids (Appendix: Amino Acid Sequence 1). A gene of the VZV gE and a gene of the Fc of human IgG1 were linked in tandem (Appendix: DNA sequence 2). The Nanjing Genscript Biotechnology Co., Ltd was entrusted to synthesize the gene sequence of the VZV gE-Fc fusion protein and insert the gene sequence into a pUC57-1.8K vector, and a synthesized gene included enzyme digestion site, Kozak sequence, signal peptide, target gene (2,244 bp), and stop codon, with a total length of 2,355 bp. Codon optimization was conducted when the recombinant gene was synthesized to facilitate the expression in CHO cells Cricetulus griseus.

[0068] 2. Construction of Expression Plasmids Carrying the VZV gE-Fc Gene

[0069] A glycerol-preserved strain with the recombinant gene provided by the Nanjing Genscript Biotechnology Co., Ltd was inoculated into an LB (Amp.sup.+) medium and cultivated at 37° C. and 180 rpm for 15 h, and the TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 was used to extract the plasmid pUC57-gE-Fc; the plasmid pUC57-gE-Fc was digested with HindIII and EcoR enzymes to obtain a target gene fragment gE-Fc-H/E (with a size of about 2,300 bp); and the mammalian expression plasmid pXC-K383L was digested to obtain a vector fragment pXC-H/E (with a size of about 7,000 bp). The agarose gel electrophoresis result of the enzyme digestion products was shown in FIG. 1.

[0070] The TaKaRa MiniBest Agarose Gel Extraction Kit was used to recover the target fragment (shown by an arrow in FIG. 1). With sticky-end ligation technology, recovered digestion products gE-Fc-H/E and pXC-H/E were subjected to ligation at 16° C. for 6 h through TaKaRa DNA Ligation Kit LONG (TaKaRa), and then a ligation product was transformed into competent DH5αand subjected to inverted cultivation at 37° C. for 15 h; two clones gE-Fc-1 and gE-Fc-2 were screened out by colony PCR screening (FIG. 2); with pXC-F and pXC-R as primers and the plasmid gE-Fc-1 as a template, the target gene was amplified by PCR, with a size of about 2,400 bp; an amplification product was sequenced by Beijing Huada Gene, and the whole sequence was determined by adding two additional reactions; and a sequencing result was analyzed by the software BioEdit7.0.9.0, and it was found that the sequence of the colon gE-Fc-1 was exactly the same as the designed sequence. Sequencing primers:

TABLE-US-00001 pXC-F: 5′-TAACAGACTGTTCCTTTCCATG-3′ pXC-R: 5′GTAAAACCTCTACAAATGTGGT-3′ 1-F: 5′-AGCACATCTGCCTGAAGC-3′ 1-F1: 5′-GCTTATTGTCTGGGCATCT-3′

[0071] The clone gE-Fc-1 was inoculated into 300 ml of an LB (Amp.sup.+) medium and cultivated at 37° C. and 180 rpm for 16 h; the plasmid pXC-VZV gE-Fc was extracted using a large-quantity/large plasmid extraction kit (Beijing Biomed Gene Technology Co., Ltd.); and the plasmid was linearized by endonuclease PvuI (TaKaRa), purified by extraction with phenol/chloroform/isoamyl alcohol, precipitated with ethanol, and re-dissolved in 1 ml of sterile TE Buffer (TaKaRa). The gel electrophoresis result of the plasmid pXC-VZV gE-Fc and the linearized plasmid VZV gE-Fc-straight was shown in FIG. 3.

Example 2. Establishing and Screening of Stable Cloned Strains

[0072] In a sterile laminar flow bench, a perforation voltage of a gene pulse generator Xcell (Bio-Rad) was set as follows: 300 V, 900 μF single pulse, and infinite resistance; a disposable electroporation cuvette (Bio-Rad) with a gap of 4 mm was taken out and added with 40 μg of a linearized plasmid DNA (100 μ1) and 0.7 ml of a CHO K1 cell suspension (1.5 ×10.sup.7 cells/ml), and the linearized plasmid VZV gE-Fc-straight was directly transfected into CHO K1 cells by electrotransfection; the cells in the electroporation cuvette were transferred into a triangular culture flask, 30 ml of a CD CHO medium (GIBCO) was added, and the cells were cultivated in a shaker at 36° C. to 37° C., 5% CO2, and 135 rpm for 24 h; then the cells were collected by low-speed centrifugation and inoculated into a 50 μM MSX-containing CD CHO medium (without glutamine) instead; a resulting cell suspension was transferred into a 96-well flat-bottom culture plate by limiting dilution, and the culture plate was incubated in a 37° C. and 10% CO.sub.2 incubator; the cells were observed under an inverted microscope, and monoclonal cell wells were marked; then the monoclonal lines with high expression were screened out by ELISA (goat anti-human IgG +expression product VZV gE-Fc+goat anti-human IgG-HRP) and protein A HPLC; the screened lines were continuously subcultivated and tested, and finally 3 cell clone lines with high expression of the target gene were obtained, with clone numbers of 5B3, 8D8, and 12C3; and an expression level of the recombinant protein in the culture supernatant was detected according to the feed test and HPLC, and the clone line 5B3 was selected for scale-up experiment.

Example 3. Expression and Purification of a Target Product

[0073] The obtained clone line 5B3 was inoculated into a 2 L triangular flask with 500 ml of a CD CHO medium, a cap of the breathable flask was tightened, and then the clone line was cultivated in a rotating shaker at 36° C. to 37° C., 5% CO.sub.2, and 135 r/min for 4 d; the 5B3 cells were transferred into a 5 L full-automatic bioreactor with 2.0 L of a CD Opti-CHO medium, and cultivation was conducted under the following parameters: rotational speed: 60 r/min, temperature: 36.5° C., pH: 7.0 to 7.4, and dissolved oxygen (DO): 40% to 60%; a sample was collected every day to detect cell viability, cell density, and glucose content; 4 d after the cultivation (when a viable cell density increased to 5×10.sup.6 to 7×10.sup.6 cells/ml), 200 ml to 300 ml of CD Efficient Feed C was supplemented, and then CD Efficient Feed C was supplemented once every other day; the glucose content in the culture was determined every day, where if the glucose content was lower than 11.1 mmole/L, a 40% glucose solution was supplemented to the full-automatic bioreactor through a peristaltic pump until the glucose content reached 22 mmole/L. L; 12 d to 14 d after the cultivation (when a proportion of viable cells decreased to 60% to 70%), the cultivation was stopped; and a culture was centrifuged at 12,000 r/min (or using a 3 M depth filter) to remove cells and cell debris and collect a cell culture supernatant. The culture supernatant was filtered through a 0.45 μm filter membrane, and a filtrate was allowed to pass through a Protein A gel chromatography column (Mabselect™ Sure, MabSelect™ Sure LX, MabCapture™ A, AT Besterose A, etc.) pre-equilibrated with 40 mM PBS (pH 7.4, 150 mM NaCl); the column was rinsed with 2 to 4 column volumes of 40 mM PBS until A.sub.280 returned to a baseline level, and then a 100 mM glycine-hydrochloric acid buffer (or citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, PH: 3.0 to 4.0) was used instead to elute a conjugated substance; and a collected eluate was placed at room temperature (18° C. to 25° C.) for 30 min (low pH for virus inactivation), then a pH was adjusted to 7.4 to 8.0 with 0.2 M Na.sub.2HP0.sub.4, and a resulting mixture was filtered through a 0.45 μm filter membrane to further remove insoluble particles. A protein solution obtained from purification by Protein A affinity chromatography was assayed by Shimadzu LC-20AT HPLC (BioCore SEC-500, 7.8 mm×30 cm, Suzhou Nanowin Science and Technology Co., Ltd). The main peak (dimer) accounted for about 65% to 78%, the monomer accounted for about 20% to 30%, and there were still some polymer products before the main peak, generally accounting for less than 10%. The chromatogram was shown in FIG. 4.

[0074] Then the obtained protein solution with the target product was loaded into DEAE Sepharose 4 Fast Flow (or Q Sepharose 4 Fast Flow, NanoGel 50Q, Besterose DEAE, Besterose Q, POROS Q, POROS XQ, etc.) equilibrated with 20 mM PB (pH: 7.4 to 8.0) buffer; after the loading was completed, the chromatography column was rinsed with 20 mM PB buffer until A.sub.280 returned to the baseline level; then NaCl solutions at different concentrations were used for gradient elution, and the target protein VZV gE was collected; and the anion exchange chromatography column was rinsed with 1 column volume of a 1.0 M NaCl solution for regeneration and finally equilibrated with 20 mM PB (pH: 7.4 to 8.0) buffer for later use.

[0075] The collected VZV gE was purified by Sephacryl S400 HR or another suitable molecular sieve chromatographic column, and a target product was collected, sterilized by filtration, and stored at 2° C. to 8° C.

[0076] Purified VZV gE was tested for purity by Shimadzu LC-20AT HPLC under the following conditions: mobile phase: 40 mM PBS (including 0.5 M Na.sub.2SO.sub.4, pH 7.5), flow rate: 0.750 ml/min, and analytical column: BioCore SEC500 (7.8×300 mm, Nanowin Science and Technology Co., Ltd; or TSK 5000 SWxl, Toyo Soda), and the A.sub.280 test results showed that the VZV gE had a purity of more than 98% (as shown in FIG. 5) and a relative molecular weight of about 400 KDa.

Example 4. Formaldehyde Inactivation for the Target Product

[0077] The recombinant VZV gE obtained in Example 3 was diluted to 100 μg/ml to 1,000 μg/ml with 20 mM PBS (pH: 7.2 to 8.0, 135 mM NaCl); then a 38% formaldehyde solution was added to a final concentration of 0.1% (v/v) of a total volume of a resulting mixture, and the mixture was placed at 37° C. for 72 h, during which period, the mixture was shaken twice every day for thorough mixing; and then the mixture was placed at 2° C. to 8° C.

Example 5. β-propiolactone Inactivation for the Target Product

[0078] The recombinant VZV gE solution obtained in Example 3 was cooled to 2° C. to 8° C. and weighed, then β-propiolactone was added to a final concentration of 0.1% to 0.01% of a weight of the solution, and a resulting mixture was placed at 2° C. to 8° C. for 72 h, during which period, the mixture was shaken twice every day for thorough mixing. 72 h later, the VZV gE solution was heated to 37° C. and kept at the temperature for 4 h such that the β-propiolactone was completely converted into lactic acid, and then the solution was placed at 2° C. to 8° C.

Example 6. Removal of Formaldehyde or β-propiolactone from the Target Product

[0079] The protein solution with the recombinant VZV gE obtained in Example 5 or 6 was appropriately diluted with 20 mM PB (pH: 7.2 to 8.0) or a 20 mM Tris-HCl solution (pH: 7.2 to 8.0) until a NaCl concentration in the solution was lower than 50 mM; then the VZV gE-containing solution was allowed to pass through a DEAE Sepharose 4FF chromatography column equilibrated with 20 mM PB (or 20 mM Tris-HCl, pH: 7.2 to 8.0); then the column was rinsed with a 20 mM PB solution (or a 20 mM Tris-HCl solution, PH: 7.2 to 8.0) until A.sub.280 completely returned to the baseline level, and then further rinsed with 4 column volumes of the solution; an eluent with 0.4 M NaCl (a solution with 20 mM PB or 20 mM Tris-HCl, pH: 7.5) was used to elute the VZV gE conjugated on the gel; and a solution with the target product was collected and filtered with a 0.2 μm sterilization filter membrane to obtain a filtrate, which was a vaccine stock solution.

Example 7. Preparation of a Vaccine with an Aluminum Adjuvant

[0080] The vaccine stock solution obtained in Example 6 was diluted with 20 mM Tris-HCl (pH: 7.2 to 7.5, including 135 mM to 150 mM NaCl) to 10 μg/ml to 800 μg/ml, a resulting solution was thoroughly mixed with an equal volume of an aluminum hydroxide adjuvant suspension (aluminum content: 0.2 mg/ml to 1.5 mg/ml) at room temperature, and a resulting mixture was placed at 2° C. to 8° C.

[0081] The vaccine solution with an aluminum adjuvant was taken out from the 2° C. to 8° C. environment and dispensed into 2 ml vials (or pre-filled glass syringes) under aseptic conditions, with 0.5 ml (or 1.0 ml) per vial, and then the vials were sealed and stored at 2° C. to 8° C. in the dark.

[0082] In the table below, the preparation of 1,000 ml vaccines with different VZV gE contents was taken as an example (the first column from the left showed an antigen content in 1 ml of a prepared vaccine, and the first column from the right showed an antigen content in 0.5 ml of a vaccine for routine intramuscular injection). The vaccine stock solution with a VZV gE concentration of 800 μg/ml was used to prepare the vaccines with an aluminum adjuvant, and a preparation method was as follows:

TABLE-US-00002 TABLE 1 Preparation of VZV gE vaccine solutions including an aluminum adjuvant with different antigen contents VZV gE Stock solution 20 mM Aluminum VZV gE content (800 μg/ml) Tris-HCl adjuvant content (μg/ml) volume (ml) (ml) (ml) (μg/0.5 ml)  10 12.5 487.5 500 5  20 25 475 500 10  40 50 450 500 20 100 125 375 500 50 200 250 250 500 100 400 500 0 500 200

Example 8. Lyophilization of the Recombinant VZV gE Fusion Protein

[0083] The vaccine stock solution obtained in Example 6 was diluted to 40 μg/ml to 800 μg/ml with 20 mM Tris-HCl (pH: 7.2 to 7.5, including 135 mM NaCl), then a 10% sucrose (or 10% trehalose, 10% mannitol, and 10 lactose) solution was added to a final concentration of 3%, and a VZV gE concentration in the solution to be dispensed was adjusted to 20 μg/ml (or 50 μg/ml, 80 μg/ml, 100 μg/ml, 200 μg/ml, and 400 μg/ml); a resulting mixture was thoroughly mixed and then dispersed into 2 ml tube-like bottles, with 1.0 ml per bottle, and the bottles were partially stoppered with butyl rubber stoppers and then placed in a lyophilization bin; with a pre-freezing temperature set to −40° C. to −45° C., the vaccine solution was frozen for 4 h, and then vacuum pumping was conducted for lyophilizing, where an automatic temperature rise program was adopted for temperature control: increasing for 6 h from −40° C. to −25° C., increasing for 4 h from −25° C. to −5° C., holding at 0° C. to 5° C. for 1 h, holding at 25° C. for 1 h, and holding at 35° C. for 6 h to 8 h; and then the butyl rubber stoppers were tightly pressed down under vacuum (or introduced with high-purity nitrogen or argon for pressing).

[0084] The stoppered bottles were taken out from the lyophilization bin and sent to an automatic capping machine to tighten the aluminum caps. Then the bottles were stored in a cold storage at 2° C. to 8° C.

[0085] Before use, 1.0 ml of water for injection or an aluminum hydroxide adjuvant suspension was drawn with a disposable sterile syringe and injected into a bottle with lyophilized VZV gE, and a resulting mixture was mixed gently for about 5 min to obtain a vaccine without visible particles. The vaccine should be used immediately after dissolution, or should be used within 30 minutes after dissolution at latest. This vaccine should be used for subcutaneous or intramuscular injection and is prohibited from being used for intravenous injection.

Example 9. Animal Immunization Experiment

[0086] The vaccine that included 100 μg/ml of VZV gE and an aluminum adjuvant for adsorption was taken out from the cold storage at 2° C. to 8° C. and diluted with 20 mM Tris-HCl to obtain solutions with 8 μg/ml and 2 μg/ml of VZV gE, and then an equal volume of an aluminum adjuvant was added to obtain aluminum adjuvant-containing vaccines with 4 μg/ml and 1 μg/ml of VZV gE (or 2 μg/ml and 0.5 μg/0.5 ml of VZV gE) for the animal experiment.

[0087] 4 to 6 week-old female BALB/C mice were randomly divided into 5 groups, 8 in each group. Each mouse in the control group was intraperitoneally injected with 0.5 ml of an aluminum adjuvant. Mice in the 4 experimental groups were intraperitoneally injected with 0.5 ml of the aluminum adjuvant-containing vaccine at VZV gE dosages of 50 μg, 10 μg, 2 μg, and 0.5 μg. 8 mice were used for each dosage group. After the initial immunization, immunization was conducted once every two weeks, with a total of 4 immunizations. Blood was collected from the tail vein 7 d after the second and third immunizations, and serum was isolated and cryopreserved at −70° C. Blood was collected from the heart 7 d after the fourth immunization, and serum was isolated and cryopreserved at −70° C.

[0088] Antibody titer determination by ELISA: The recombinant VZV gE-His protein was diluted to 1 μg/ml with a carbonate buffer, coated on a 96-well microplate (Costar) at 100 μl/well, placed at 37° C. for 1 h, and then placed overnight at 2° C. to 8° C.; the liquid in the 96-well plate was discarded, and then the plate was washed 3 times with 20 mM PBS; 200 μl of a blocking solution (2% bovine serum albumin (BSA) and component V) was added to each well, and blocking was conducted at room temperature for 60 min; the solution in the wells was removed, and the plate was washed 3 times with a 20 mM PBS-T solution; mouse serum pre-diluted at 1:50 (or 1:500 or 1:1,000) was added to wells in the first column on the 96-well microplate, and then 2-fold serial dilution was conducted, where only mouse serum (1:100) immunized intraperitoneally with an aluminum adjuvant was used for the negative control; reaction was conducted at 37° C. for 60 min, then the solution in the wells was removed, and the plate was washed 3 times with a 20 mM PBS-T solution; the goat anti-mouse IgG-HRP conjugate was taken and diluted with an enzyme conjugate diluent at 1:100 (the diluent included 1 mg/ml of human IgG), pre-reacted for 30 min at room temperature, and then added to the 96-well microplate at 100 μl/well; reaction was conducted at 37° C. for 30 min, the blocking solution in the wells was removed, and then the plate was washed 3 times with a 20 mM PBS-T solution; 100 μl of a TMB chromogenic solution was added to each well, and 10 min later, 50 μl of a stop solution was added to stop the reaction; then the TECAN Infinit 200 microplate reader was used to determine A.sub.450 absorbance values; and a value 3 times a A.sub.450 value of the mixed serum in the negative control was taken as a Cut-Off value (if the A.sub.450 value in the negative control was lower than 0.100, it was counted as 0.100) to determine the titer of immunized serum. The geometric mean and standard deviation of the anti-VZV gE antibody titers in serum of mice in each experimental group were shown in Table 2 below. After the second immunization, except that 1 mouse in the lowest-dosage group (0.5 μg) was not positive for the serum antibody, all mice were positive for the serum antibody. The statistical analysis of the antibody titers determined for experimental mice in each group showed that, after the third immunization, the serum antibody titer of experimental mice in each dosage group was significantly higher than that after the second immunization; after the fourth immunization, the serum antibody titer was partially increased, which was insignificant; and after the second, third, and fourth immunizations, there was no significant difference in the serum antibody titer among mice in the three high-dosage groups. The serum antibody titers of BALB/C mice immunized with VZV gE vaccines with an aluminum adjuvant at various dosages were shown in FIG. 6.

[0089] Serum neutralizing antibody titer determination: VZV is a virus that can cause cell fusion lesions on human embryonic lung diploid cells. Therefore, the virus plaque reduction neutralization test can be used to test the ability of antibodies with different serum dilutions to neutralize the virus, and a serum titer at which the number of plaques is reduced by 50% is calculated. The virus neutralization test with serum antibodies is the most direct test to detect whether there are antibodies that can neutralize VZV in immunized serum. This test has disadvantages such as cumbersome operations, low sensitivity, high manpower cost, large time consumption, and inability to use equipment for automatic interpretation, and the calculation of ED.sub.50 requires professional data processing software. Due to the above problems, few people use this test to determine serum neutralizing antibodies. Due to the large consumption of serum in this test, the neutralizing antibody determination was conducted only on mouse serum collected after the last immunization.

[0090] This test was a VZV serum neutralization test conducted on a flat-bottomed 96-well microplate, and MRC-5 cells sensitive to VZV (purchased from ATCC) were used as a cell matrix. The serum frozen in a refrigerator at −70° C. was taken out and thawed at room temperature, diluted at 1:10 with a 10% FBS-containing 199 medium (GIBCO) in a sterile clean bench, and then 4-fold diluted serially in a 96-well plate, with a total of 7 dilutions and two wells for each dilution. The rabbit anti-VZV gE serum was adopted as the positive control serum, and mixed serum of mice immunized with an aluminum adjuvant (diluted at 1:10) was adopted as the negative control serum. On each 96-well plate, 6 virus solution control wells and 6 cell control wells were set. The diluted serum to be tested was mixed with a specified amount of OKA virus (purchased from ATCC in the United States), and then the 96-well microplate was covered. The microplate was shaken on a shaker for 30 s and then placed in a 10% CO.sub.2 and 37° C. incubator (10%), and reaction was conducted for 30 min.

[0091] A culture flask with a single layer of confluent MRC-5 cells (ATCC, generation 25 to 38) was taken out from the CO.sub.2 incubator, the medium in the T75 culture flask (Corning) was removed in a hundred-level clean bench, and 5 ml of a 0.25% trypsin (GIBCO) solution was added to digest the single layer of MRC-5 cells; a resulting mixture reacted for 3 min at room temperature, then the trypsin solution was removed, and 10 ml of a cell culture medium was added; and an inner surface of the cell culture flask was gently rinsed to disperse the MRC-5 cells, and a specified volume of cell culture medium was added to obtain a cell suspension. The 96-well microplate with the serum to be tested was taken out from the incubator, the cell suspension was added with a multi-channel pipette, and the plate was covered; the microplate was shaken on a shaker for 30 s, and then incubated in a 37° C. CO.sub.2 incubator (10%) for 3 d to 4 d; and 48 h later, the cytopathic effect in each well was observed with an inverted microscope every day, and the number of plaques in each well was accurately counted and recorded. 96 h later, a 12-channel pipette was used to transfer the liquid in each well into a waste liquid tank with 0.1% sodium hypochlorite, 0.1% crystal violet was added to stain for 1 h, and after destaining, the microplate was placed on absorbent paper in an inverted manner and dried at room temperature.

[0092] The number of plaques in each serum sample at each dilution was entered into an EXCEL 2016 table. With an average number of plaques in the virus solution wells (average of 6 wells, 25 to 30 per well) as 100%, the reduced number of plaques in serum at each dilution was calculated and converted into a percentage, and then the ED.sub.50 value was calculated from the data with Prism 5.0 software. The ED.sub.50 value is the serum titer at which the number of plaques is reduced by 50%. The serum neutralizing antibody titer ED.sub.50 determination curve for each mouse in the four dosage groups was shown in FIG. 7, and the GMT of serum neutralizing antibodies and the distribution of neutralizing antibody titers in each dosage group were shown in Table 3. The comparison of GMT of VZV serum neutralizing antibodies among the dosage groups was shown in FIG. 8, and it can be seen from the figure that the neutralizing antibody titer of the lowest dosage group was significantly lower than that of the other three groups, and the serum neutralizing antibody titers of the other three groups all reached a high level.

TABLE-US-00003 TABLE 2 Serum antibody titers of BALB/C mice immunized with a VZV gE vaccine including an aluminum adjuvant at different dosages (ELISA) Adminis- tration Number of Lower Upper group immunizations Minimum quartile Median quartile Maximum GMT SD 50 μg 2 15996 15996 31989 63973 63973 31989 1.90 3 255859 255859 255859 511682 511682 331894 1.43 4 511682 511682 1023293 1023293 1023293 788860 1.43 10 μg 2 15996 31989 31989 53827 127938 38019 1.85 3 127938 152055 255859 255859 255859 215278 1.38 4 255859 511682 511682 1023293 1023293 609537 1.63 2 μg 2 500 9506 22646 31989 31989 13459 4.15 3 63973 127938 127938 255859 255859 152055 1.63 4 127938 152055 255859 511682 511682 279254 1.78 0.5 μg 2 1 100 2000 2000 3999 474 16.98 3 2000 9506 45290 63973 255859 29376 4.49 4 31989 38019 90573 215278 255859 90573 2.29

TABLE-US-00004 TABLE 3 Serum neutralizing antibody titers of BALB/C mice immunized with a VZV gE vaccine including an aluminum adjuvant for adsorption at different dosages Immunization dosage group Statistics 50 μ/mouse 10 μg/mouse 2 μg/mouse 0.5 μg/mouse Minimum 488 470 84 23 Lower 1213 665 175 59 quartile Median 1321 867 428 88 Upper 1660 1262 859 124 quartile Maximum 2188 1698 957 377 GMT 1294 899 378 87 SD 1.55 1.51 2.38 2.20