Recombinant Herpes Zoster Vaccine Composition and Application Thereof

Abstract

Disclosed in the present invention are a recombinant herpes zoster vaccine composition and an application thereof. Compared with other combinations of antigens and adjuvants, the novel vaccine composition provided by the present invention has a more beneficial immune effect.

Claims

1. A recombinant herpes zoster vaccine composition, comprising gE protein of varicella zoster virus (VZV) and a combined adjuvant.

2. The vaccine composition according to claim 1, wherein the vaccine composition further comprises a pharmaceutically acceptable combined adjuvant.

3. The vaccine composition according to claim 2, wherein the pharmaceutically acceptable combined adjuvant is an aluminum adjuvant combined with a CpG adjuvant.

4. The vaccine composition according to claim 3, wherein a content of the gE protein is 10-100 μg.

5. The vaccine composition according to claim 3, wherein a content of the aluminum adjuvant is approximately 100-600 μg.

6. The vaccine composition according to claim 3, wherein a content of the CpG adjuvant is approximately 50-1200 μg.

7. The vaccine composition according to claim 3, wherein a content of the gE protein is 10-100 μg, a content of the aluminum adjuvant is approximately 100-600 μg, and a content of the CpG adjuvant is approximately 50-1200 μg.

8. The vaccine composition according to claim 3, wherein a content of the gE protein is 50-100 μg, a content of the aluminum adjuvant is approximately 225-600 μg, and a content of the CpG adjuvant is approximately 300-900 μg.

9. A method for preventing diseases associated with herpes zoster virus infection, comprising administrating the recombinant herpes zoster vaccine composition according to claim 1.

10. A kit, comprising the recombinant herpes zoster vaccine composition according to claim 1, wherein the kit comprises a vaccine administration device, and the administration device comprises a needle device, a liquid ejection device, a powder device, and a spray device.

11. The vaccine composition according to claim 4, wherein the content of the gE protein is 50-100 μg.

12. The vaccine composition according to claim 5, wherein the content of the aluminum adjuvant is 225-600 μg.

13. The vaccine composition according to claim 6, wherein the content of the CpG adjuvant is 300-900 μg.

14. The kit according to claim 10, wherein the vaccine composition further comprises a pharmaceutically acceptable combined adjuvant.

15. The kit according to claim 14, wherein the pharmaceutically acceptable combined adjuvant is an aluminum adjuvant combined with a CpG adjuvant.

16. The kit according to claim 15, wherein a content of the gE protein is 10-100 μg.

17. The kit according to claim 15, wherein a content of the aluminum adjuvant is approximately 100-600 μg.

18. The kit according to claim 15, wherein a content of the CpG adjuvant is approximately 50-1200 μg.

19. The kit according to claim 15, wherein a content of the gE protein is 10-100 μg, a content of the aluminum adjuvant is approximately 100-600 μg, and a content of the CpG adjuvant is approximately 50-1200 μg.

20. The vaccine composition according to claim 3, wherein the CpG adjuvant is CPG 7909.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1: Preparation of Vaccine Composition Containing VZV gE Antigen Protein

[0028] In order to study the technical effects of the vaccine composition provided by the present disclosure, the inventors of the present disclosure prepared the following vaccine composition formulations (0.5 ml/dose), each of which to contains VZV gE protein, an aluminum adjuvant, and a CpG ODN adjuvant. The specific preparation method included: firstly adsorbing a gE antigen stock solution onto an aluminum adjuvant (aluminum phosphate adjuvant) to prepare adsorption samples with different ratios of gE antigen/aluminum adjuvant (w/w) (where the aluminum adjuvant content was substantially the content of aluminum element); and then adding CpG ODN samples (CpG 7909) of different concentrations to the gE antigen/aluminum adsorption samples. After the above formulations were thoroughly mixed, if not administrated immediately, they were stored at 4° C. The specific ratios were as follows.

TABLE-US-00001 TABLE 1 Aluminum CpG ODN gE adjuvant adjuvant Volume No. (μg) (μg) (μg) (μl) 1 10 600 300 500 2 10 600 600 500 3 10 600 900 500 4 10 600 1200 500 5 50 600 300 500 6 50 600 600 500 7 50 600 900 500 8 50 600 1200 500 9 100 600 300 500 10 100 600 600 500 11 100 600 900 500 12 100 600 1200 500 13 0 600 1200 500 14 10 225 600 500 15 50 225 600 500 16 100 225 600 500

Example 2

[0029] For the obtained recombinant herpes zoster vaccine composition to be evaluated of Example 1, the inventors carried out immunogenicity studies using C57BL/6 mice as animal models. The immunogenicity of the vaccine composition provided by the present disclosure was investigated using gE protein as antigen and an aluminum adjuvant and CpG ODN as adjuvants. 6-8 weeks old C57BL/6 mice were randomly divided into groups, 10 mice in each group. The vaccine prepared using gE protein in combination with adjuvants is intramuscularly injected. A vaccine group and an adjuvant group were set. Immunization was performed at weeks 0 and 3, and blood and spleens were collected at week 5. The ELISA method was used to detect the titer of the anti-VZV gE protein binding antibody in serum, namely total IgG, and the ELISPOT method was used to detect the cellular immunity level, mainly expression of IFN-γ, in spleen cells. It should be noted that in addition to the above-mentioned 16 groups of experiments, parallel experiments with the same ratios were conducted in the present disclosure without changing other parameters and procedures to verify the effect of the vaccine composition to composed of 1018 of CpG ODN, VZV gE protein, and an aluminum adjuvant. The data finally obtained showed that the IgG (GMT) value thereof is substantially the same as the value in the case of CpG 7909, and the IFN-γ value thereof is slightly lower than the value in the case of CpG 1018, but there is no significant difference. For example, for group 3, when CpG ODN is CpG 7909, the IFN-γ value is 24, for group 4, the IFN-γ value is 30, and for groups 9-11, the IFN-γ values are 120, 125, and 102.

[0030] The evaluation method for immunogenicity is a conventional technical means in the art. As an example, the more specific operations are as follows.

[0031] 1. Animal Experiment of Recombinant Herpes Zoster Vaccine

[0032] 6-8 weeks old C57BL/6 mice were selected and randomly divided into groups, 10 mice in each group. Vaccines of different dosages (Table 1) were intraperitoneally injected with an injection volume of 0.05 ml. Immunization was performed at weeks 0 and 3, and blood and spleens were collected at week 5. Serum was separated for ELISA to detect binding antibody titer, and splenic lymphocytes were separated for ELISPOT analysis.

[0033] 2. Binding Antibody Titer Detection

[0034] Serum was collected 2 weeks after the second immunization of mice, and the titer of anti-gE protein binding antibody was detected.

[0035] (1) The antigen gE stock solution was diluted with PBS to 1 μg/ml, and 100 μl of the diluted stock solution was added to each well of an ELISA plate. The plate was stored at 4° C. overnight and washed by a plate washer.

[0036] (2) 5% skimmed milk was prepared with PBS, and 100 μl of skimmed milk to was added to each well of the ELISA plate. The plate was maintained at 37° C. for 2 hours and washed by a plate washer.

[0037] (3) 2% skimmed milk was prepared with PBS, the serum to be tested was gradiently diluted, and 100 μl of diluted serum was added to each well of the ELISA plate. The plate was maintained at 37° C. for 1 hour and washed by a plate washer.

[0038] (4) The goat anti-mouse antibody was diluted with 2% skimmed milk in PBS at a ratio of 1:10000, and 100 μl of secondary antibody diluted with skimmed milk was added to each well of the ELISA plate. The plate was maintained at 37° C. for 1 hour and washed by a plate washer.

[0039] (5) A chromogenic solution was prepared in a proportion of 9 ml of chromogenic buffer, 1 ml of TMB, and 10 μl of 3% H.sub.2O.sub.2. 100 μl of the chromogenic solution was added to each well of the ELISA plate. The plate was maintained at 37° C. for 10 min and washed by a plate washer.

[0040] (6) Reading is performed at 450 nm/620 nm.

[0041] 3. Cellular Immunoassay

[0042] At two weeks after the second immunization of mice, spleens were taken from each group of mice, and lymphocytes were separated. The level of IFN-γ expressed by mouse splenic lymphocytes (n=2) was assessed by ELISPOT.

[0043] (1) ELISPOT Plate Coating (Sterile Operation, Performed the Day Before Removing the Spleen)

[0044] The ELISPOT plate was wetted with 35% alcohol, wherein an amount of 15 μl/well of alcohol was added to a 96-well ELISPOT plate and maintained for a retention time of no more than 1 minute. 200 μl/well sterile water was added to wash the plate 5 times. 150 μl of IFN-γ coating antibody was added to 10 ml PBS, mixed thoroughly, and filtered by a 0.2-μm filter membrane. The diluted coating antibody solution was added to the 96-well ELISPOT plate in an amount of 100 μl/well and stored at 4° C. overnight.

[0045] (2) ELISPOT Plate Sealing (Sterile Operation)

[0046] The coating antibody was discarded, 200 μl/well of sterile PBS was added to wash the plate 5 times. A 1640 complete medium (containing 10% FBS) was added to the 96-well ELISPOT plate in an amount of 200 μl/well, and the plate was sealed at room temperature for more than 30 minutes. Liquid was discarded, and water was removed as much as possible by inverting the plate on a sterilized gauze to avoid air bubbles when a medium was added in a subsequent step.

[0047] (3) Lymphocyte Preparation (Sterile Operation)

[0048] Mice were sacrificed and immersed in 75% ethanol. The mouse spleen was taken out in a clean bench. A burned 200-mesh copper screen was placed into a 35 mm culture dish, 1 ml of a lymphocyte separation solution was added, and grinding is performed with a plunger of a 1-ml syringe. The separation solution in which spleen cells are suspended was filtered through the burned 200-mesh copper screen and transferred to a 15-ml centrifuge tube, the lymphocyte separation solution was added therein to 4 ml, and 0.5 ml of RPM11640 basal medium was added to cover the liquid surface. It was room temperature, 800 g, acceleration 3, centrifugation 30 minutes. The lymphocyte to layer was sucked out, and then added with 10 ml RPM11640 basal medium, and subjected to washing, at room temperature, with centrifugation of 250 g, 10 minutes. The supernatant was discarded, 2 ml RPM11640 complete medium was added to resuspend the cells, and the cells were counted.

[0049] (4) Sample Addition (Sterile Operation)

[0050] Addition of cells: the cells were diluted with a complete medium to 6×10.sup.6/ml according to cell count results, and meanwhile, 1000 times dilution of mAb CD28-A was added to the cell suspension. 100 μl/well of the cell suspension was added to the ELISPOT plate. Positive control: 1 μl of ConA stimulant was added, and a stimulation concentration was 5 μg/ml. Sample to be tested: stimulant gE protein peptide library diluted with serum-free medium was added to a final concentration of 2 μg/ml, Negative control: neither ConA stimulant nor stimulant short peptide was added. They were incubated at 37° C. and 5% CO.sub.2 for 24 hours, during which the culture plate should not be moved to avoid changes in cell position and the resulting blur of ELISPOT spots.

[0051] (5) Spot Detection

[0052] The cell suspension was discarded, and 200 μl/well of sterile PBS was added to wash the plate 5 times. 50 μl of biotin-labeled detection antibody was added to 10 ml diluent (PBS+0.1% BSA), mixed thoroughly, and filtered by a 0.2-μm filter membrane. 100 μl of the detection antibody dilution was added to each well and incubated at 37° C. for 2 hours. The biotin-labeled detection antibody dilution was discarded, and 200 μl/well of sterile PBS was added to wash the plate 5 times. The antibody was diluted with a diluent (PBS+0.1% BSA), 50 μl of the antibody was added with 10 ml of the diluent, to mixed thoroughly, and filtered by a 0.2-μm filter membrane. 100 μl of the dilution was added to each well and incubated at 37° C. for 1 hour. The operations from this step should be conducted in a dark place. The antibody dilution was discarded, and 200 μl/well of sterile PBS was added to wash the plate 5 times. Fluorescence enhancer-II was added to the 96-well ELISPOT plate in an amount of 50 μl/well, and incubation was performed at 37° C. for 15 minutes. The liquid in the plate was discarded, the plate was inverted on absorbent paper and patted to remove the small drops of water. The protective layer was removed, the plate was placed in an electric thermostat incubator, and the film was dried at 37° C. in a dark place. The ELISPOT plate was placed in CTL-ImmunoSpot® S5 VersC CnClyzer, an enzyme-linked spot image automatic analyzer, for which parameters were appropriately adjusted for performing spot counting.

[0053] The specific results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Summary of results of investigation on proportions of antigen, aluminum adjuvant, and CpG ODN adjuvant (7909) Alumi- CpG IFN-γ (Number num ODN Injec- of positive adju- adju- tion cells per gE vant vant volume IgG million No. (μg) (μg) (μg) (μl) (GMT) spleen cells) 1 10 600 300 50 1:4095000 73 2 10 600 600 50 1:1236000 43 3 10 600 900 50 1:2297000 72 4 10 600 1200 50 1:2178000 54 5 50 600 300 50 1:2702000 128 6 50 600 600 50 1:2169000 139 7 50 600 900 50 1:2862000 86 8 50 600 1200 50 1:2345000 91 9 100 600 300 50 1:4000000 157 10 100 600 600 50 1:2297000 175 11 100 600 900 50 1:2862000 183 12 100 600 1200 50 1:3194000 121 13 0 600 1200 50 1:100   3 14 10 225 600 50 1:1888000 61 15 50 225 600 50 1:2551000 100 16 100 225 600 50 1:1764000 152

TABLE-US-00003 TABLE 3 Summary of results of investigation on proportions of antigen, aluminum adjuvant, and CpG1018 Alumi- CpG IFN-γ (Number num 1018 Injec- of positive adju- adju- tion cells per gE vant vant volume IgG million No. (μg) (μg) (μg) (μl) (GMT) spleen cells) 1 10 600 300 50 1:1888000 80 2 10 600 900 50 1:2551000 24 3 10 600 1200 50 1:1764000 30 4 100 600 300 50 1:4095000 120 5 100 600 900 50 1:2491000 125 6 100 600 1200 50 1:1037000 102 7 0 600 1200 50 1:100   6

[0054] It can be seen from Tables 2-3 that the vaccine composition prepared according to the specific compositions and ratios provided by the present disclosure has good immune activity, and especially when the content of gE protein is 50-100 μg, the content of the aluminum adjuvant is 225-600 μg, and the content of the CpG ODN adjuvant is 300-900 μg, the vaccine composition to has excellent cellular immunity effect and can be used as a new generation of vaccine composition.

[0055] All the documents mentioned in the present disclosure are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present disclosure, those skilled in the art can make various changes or modifications to the present disclosure, and these equivalent forms also fall within the scope defined by the appended claims of the present application.