Herpes zoster vaccine composition and application thereof

Abstract

The invention belongs to the field of medicines, and particularly discloses a herpes zoster vaccine composition containing a composite adjuvant and application of the herpes zoster vaccine composition. The vaccine composition comprises an antigen and a composite adjuvant, the antigen is VZV gE protein, the amino acid sequence of the VZV gE protein is shown as SEQ ID No.1, and the composite adjuvant is composed of squalene, Tween 80, poloxamer and CpG oligonucleotide. According to the invention, VZV gE protein is adopted as an antigen, and an appropriate composite adjuvant is obtained through adjuvant screening and can induce a high-level neutralizing antibody. The vaccine disclosed by the invention can be used for immunizing a two-needle or three-needle immunizing procedure by adopting an immunizing dose of 1 * (namely 0.5 mL) in clinical application, and has an application value.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a varicella zoster vaccine composition, and more particularly to a varicella zoster vaccine composition containing a compound adjuvant and its application. Background Art

[0002] Varicella-zoster virus (VZV), also known as human herpesvirus 3 (HHV-3), is a member of the α-herpesvirus subfamily of the Herpesviridae family. VZV is an enveloped virus with a double-stranded DNA genome of approximately 125,000 nucleotides. The genome of VZV is surrounded by an icosahedral capsid. The viral tegument (the space between the capsid and the viral envelope) is a structure composed of virus-encoded proteins and enzymes. The viral envelope is derived from the host cell membrane and contains virus-encoded glycoproteins. After children are infected, they develop chickenpox. Due to the neurotropism of the virus, it can be latent in neurons of the dorsal root ganglia of the spinal nerves for a long time after infection. When the elderly have low resistance, are tired, infected, or have a cold, the virus can grow and reproduce again, and migrate along nerve fibers to the skin, causing strong inflammation in the affected nerves and skin.

[0003] So far, there is no specific treatment for herpes zoster, and vaccination is also the only effective means of prevention and control. Some vaccines against herpes zoster have been developed in the prior art, but there are problems such as low protein expression efficiency, poor protein activity, and unsatisfactory immune effects. Therefore, it is necessary to develop improved VZV vaccines in this field. Summary of the Invention

[0004] The purpose of the present invention is to provide a varicella zoster vaccine composition and its application.

[0005] The present invention provides a varicella zoster vaccine composition, which includes an antigen and a compound adjuvant. The antigen is VZV gE protein, and the compound adjuvant is composed of squalene, Tween 80, poloxamer, and CpG oligonucleotide.

[0006] Each 0.5 mL of the vaccine composition contains 10 - 100 μg of VZV gE protein, 5 - 10 mg of squalene, 0.5 - 1.5 mg of polysorbate 80, 1.0 - 5.0 mg of poloxamer, and 0.1 - 3.0 mg of CpG oligonucleotide.

[0007] Preferably, each 0.5 mL of the vaccine composition contains 30 - 60 μg of VZV gE protein, 8 - 9 mg of squalene, 0.8 - 1.2 mg of polysorbate 80, 2.0 - 4.0 mg of poloxamer, and 0.5 - 1.5 mg of CpG oligonucleotide.

[0008] Preferably, each 0.5 mL of the vaccine composition contains 50 μg of VZV gE protein, 8.6 mg of squalene, 0.96 mg of polysorbate 80, 2.5 mg of poloxamer, and 1.0 mg of CpG oligonucleotide.

[0009] Preferably, each 0.5 mL of the vaccine composition contains 50 μg of VZV gE protein, 8.6 mg of squalene, 0.964 mg of polysorbate 80, 2.5 mg of poloxamer, and 0.5 mg of CpG oligonucleotide.

[0010] Among them, the CpG oligonucleotide is CpG1018.

[0011] The poloxamer is poloxamer 188.

[0012] In a specific embodiment, the amino acid sequence of the VZV gE protein is as shown in SEQ ID No.1.

[0013] The present invention also provides a method for preparing the vaccine composition, which is to dissolve the VZV gE protein in a complex adjuvant solution to obtain the finished vaccine composition; or the VZV gE protein is stored in the form of a separate freeze-dried powder, and the VZV gE protein powder is dissolved in the complex adjuvant solution before the vaccine composition is injected.

[0014] Among them, the preparation method of the complex adjuvant includes the following steps: (1) Prepare an oil phase from squalene; prepare an aqueous phase by stirring poloxamer and Tween 80 in buffer PBS at 18 - 55 °C. (2) Slowly add the prepared aqueous phase to the prepared oil phase, make up the volume with buffer PBS, and then use a high-speed stirrer at room temperature to stir at a speed of 8000 - 20000 rpm for 15 - 30 minutes to obtain a primary emulsion solution. (3) Pour the primary emulsion solution into a microfluidic homogenizer, adjust the pressure to 15000 - 20000 psi, adjust the temperature of the refrigerator, control the outlet temperature of the emulsion at 20 - 30 °C, perform high-pressure homogenization cycling 3 - 10 times, and filter through a 0.22 μm sterilizing filter to obtain a refined emulsion solution; measure the diameter of the refined emulsion solution by a Malvern particle size detector to be between 100 nm and 200 nm, and detect the pH value of the refined emulsion solution to be between 6.3 - 6.7 or 7.0 - 7.4.

[0015] (4) Aspirate the CpG oligonucleotide into the above refined emulsion solution and mix well to obtain it.

[0016] The present invention further provides the use of the vaccine composition in the preparation of a drug for preventing herpes zoster disease or its complications.

[0017] The present invention uses the VZV gE protein as an antigen, and through adjuvant screening, a suitable composite adjuvant is obtained, which can induce a relatively high level of neutralizing antibodies. The vaccine of the present invention can adopt an immunization dose of 1× (i.e., 0.5 mL) in clinical applications, and an immunization schedule of two or three injections, and has application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Showing the titers of antibodies binding to the gE protein monomer and tandem dimer vaccines at 3 weeks after the first immunization.

[0019] Figure 2 Showing the titers of binding antibodies at 3 weeks after the second immunization.

[0020] Figure 3 Showing the detection of IFN-γ and IL-2 cytokines at 6 weeks after the second immunization. Wherein: * p<0.05, ** p<0.01, *** p<0.001.

[0021] Figure 4 Showing the results of humoral immunity detection.

[0022] Figure 5 Showing the neutralizing antibody titers (plaque method) in the study of immunogenicity and persistence in cynomolgus monkeys. Among them, the dotted line represents the initial serum dilution factor of 10; the error bar represents the 95% confidence interval; when the neutralizing antibody titer IC50 <10, it is plotted as 5; 3 weeks after the first immunization is 3 weeks after the first administration, 3 weeks after the second immunization is 6 weeks after the first administration, 4 weeks after the third immunization is 4 weeks of the recovery period, and 8 weeks after the third immunization is 8 weeks of the recovery period. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0024] Unless otherwise specified, the experimental materials, reagents and instruments used in the embodiments of the present invention can be obtained commercially. Unless specifically specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0025] Example 1. Antigen Preparation The VZV gE protein can be obtained by using conventional technical means of modern molecular biology. Typical methods include: the method of expressing the above gE protein in CHO cells, including the following steps: (1) Clone the VZV gE protein gene (whose nucleotide sequence is shown in SEQ ID No.2 and the expressed amino acid sequence is shown in SEQ ID No.1) into an expression vector; (2) Transfect the expression vector obtained in step (1) into CHO cells; (3) Through screening of the mixed clone cell population and monoclonal cell screening, obtain an engineered cell line stably expressing the gE protein; (4) Use the cell line described in step (3) for expression to obtain the VZV gE protein.

[0026] The host cell for the expression of the antigen protein of the recombinant varicella zoster vaccine (CHO cells) is CHO-K1 cells, that is, CHO-K1 cells carrying the Oka strain glycoprotein gE gene. The medium uses the medium of a commercial CHO-K1 cell line. After the cells are thawed and revived, they are amplified and cultured in a carbon dioxide shaker and a 50L WAVE reactor, and then inoculated into a 50L WAVE reactor for expression culture. Nutrients are supplemented during the culture process to provide sufficient nutrients for the cells to grow and express proteins. After 10-15 days of culture, the cell culture fluid is harvested and deeply filtered to obtain a cell culture product for the purification of the antigen protein.

[0027] The vaccine antigen protein of this product is the gE protein expressed extracellularly by CHO cells, and its purification process technical route is: first, use a composite anion exchange chromatography to enrich and preliminarily purify the target protein, then use hydroxyapatite chromatography and affinity chromatography for further purification, and finally use gel filtration chromatography to remove multimeric proteins and small molecule impurity proteins while changing the formulation of the stock solution.

[0028] Mix the VZV gE protein monomer or its tandem dimer with the AS01 adjuvant to prepare a vaccine sample. In each 0.5 mL vaccine sample, the gE protein monomer or its tandem dimer is 50 μg, and the AS01 adjuvant is 1.35 mg. This invention sets up three dose groups of 1 / 10×, 1 / 25×, and 1 / 50× human dose (0.5 mL) for immunization in a BALB / c mouse immunization experiment, and uses Singrix from GSK Company with the same antigen components as a positive control. Through the mouse immunization experiment, the immunization effects of the VZV gE protein monomer and tandem dimer were compared. The results of the combined antibody after the second immunization and 6 weeks are as Figure 1 shown. In the case of using the AS01 adjuvant, both the VZV gE protein monomer and tandem dimer can reach or exceed the immunization effect of Singrix. During the construction of the stable expression CHO cell line, it was found that the tandem dimer structure was severely broken, so finally the monomer gE protein was selected as the vaccine antigen.

[0029] Example 2. Adjuvant screening As shown in Table 1, using the commercially available vaccine Shingrix as a control, aluminum adjuvant (Al(OH) 3 ), oil-in-water adjuvant (MF59), AS01, and a series of composite adjuvants were selected and mixed with the VZV monomer gE protein prepared in Example 1 to prepare vaccine samples. The fixed antigen dose was 1 / 25× of the human dose (the human antigen dose was 50 μg), and a BALB / c mouse immunization test was carried out.

[0030] The preparation method of the vaccine sample involving the composite adjuvant is as follows: (1) Squalene was taken to prepare an oil phase; poloxamer 188 and Tween 80 were stirred in buffer PBS at 18 - 55 °C to prepare an aqueous phase; (2) The prepared aqueous phase was slowly added to the prepared oil phase, and after volume adjustment with buffer PBS at room temperature, a high-speed stirrer was used to stir at a speed of 8000 - 20000 rpm for 15 - 30 minutes to obtain a primary emulsion solution; (3) The primary emulsion solution was poured into a microfluidic homogenizer, the pressure was adjusted to 15000 - 20000 psi, the temperature of the refrigerator was adjusted, and the outlet temperature of the emulsion was controlled at 20 - 30 °C. The high-pressure homogenization was cycled 3 - 10 times, and it was filtered through a 0.22 μm sterilizing filter to obtain a refined emulsion solution; the diameter of the refined emulsion solution was measured to be between 100 nm and 200 nm by a Malvern particle size detector, and the pH value of the refined emulsion solution was detected to be between 6.3 - 6.7 or 7.0 - 7.4.

[0031] (4) Oligonucleotide CpG 1018 was aspirated into the above-mentioned refined emulsion solution and mixed evenly to obtain the product.

[0032] The VZV monomer gE protein prepared in Example 1 was dissolved in the above-mentioned emulsion-like composite adjuvant to obtain a finished vaccine.

[0033] Alternatively, the VZV monomer gE protein prepared in Example 1 can also be stored in the form of a separate lyophilized powder, and then dissolved in the above-mentioned emulsion-like composite adjuvant before injection of the vaccine composition.

[0034] Table 1. Ratios of Different Adjuvants to Antigen

[0035] The immunization procedure is as follows: Compare the enhancing effects of various adjuvants on the immunogenicity (humoral immunity, cellular immunity) of antigens in BALB / c mice. Immunize BALB / c mice twice at an interval of 3 weeks. Use the ELISA method to detect the binding antibody titer in the serum at 3 weeks after the second immunization to evaluate the humoral immunity level; when evaluating the cellular immunity level, take the splenocytes of the intermediate concentration group of various adjuvants at 6 weeks after the second immunization, stimulate them with VZV polypeptide, and use the ELISpot method to detect the number of cells secreting IFN-γ and IL-2. The results are as Figures 2 - 3 shown.

[0036] It can be seen from the results that the gE protein alone can induce a certain level of humoral immunity but cannot induce obvious cellular immunity. After using the adjuvant, the immunization effect of the antigen can be significantly improved.

[0037] When the antigen is formulated with the composite adjuvant, the highest level of cellular immunity is stimulated, which is higher than that of the commercial vaccine Shingrix. At the same time, it induces humoral immunity comparable to that of Shingrix. The overall immunization effect is better than that of the commercial vaccine Shingrix, especially the composite adjuvant 2 has the best effect.

[0038] Therefore, through a series of mouse immunization experiments, the present invention screened vaccine adjuvants and compared traditional aluminum hydroxide adjuvant, MF59 adjuvant, AS01 adjuvant, and a series of composite adjuvants. The results show that in order to achieve the required immunization effect, it is necessary to add an adjuvant to the vaccine of the present product. By comparison, the composite adjuvant of the present invention is adopted, and the composite adjuvant 2 of the present invention is preferred.

[0039] Example 3: Study on the immunogenicity and immunological persistence of mice In order to further optimize the dosage of CpG1018 in the above-mentioned composite adjuvant 2, as shown in Table 2, the antigen and adjuvant were used. Referring to the vaccine preparation method of Example 2, vaccine groups 1, 2, 3, and 4 were prepared. Using commercial Shingrix as a control (1 / 10× of the human dose), immunize C57BL / 6J mice at an interval of 3 weeks for two doses. Vaccine groups 1, 2, and 3 immunize C57BL / 6J mice with an antigen dose of 1 / 10× of the human dose, and vaccine group 4 immunizes C57BL / 6J mice with an antigen dose of 1 / 5× of the human dose; Blood was collected at 3, 4, 6, 11, 15, and 19 weeks after the first immunization, and the binding antibody titer was measured by ELISA to investigate the immunogenicity and immunological persistence of the vaccine in mice.

[0040] Table 2. Vaccine groups with different adjuvants

[0041] The detection results of humoral immunity are as Figure 4As shown, the levels of humoral immune responses induced by Vaccine Groups 1 to 4 were comparable to those of the commercially available vaccine Shingrix, and there were no significant differences among the groups. After immunizing mice with each group of vaccines, the level of binding antibodies reached a peak at 1 week after the second immunization (week 4), showed a downward trend at 3 weeks after the second immunization (week 6), and reached a plateau after 8 weeks of the second immunization (week 11) and was maintained until 16 weeks after the second immunization (week 19), which was consistent with the change trend of the binding antibody titers in the Shingrix vaccine group.

[0042] Therefore, the VZV vaccines (Vaccine Groups 1 to 4) produced by the present invention can induce strong humoral immunity in mice and can be maintained at a relatively high level for at least 16 weeks after the second immunization (week 19 in total).

[0043] Example 4. Immunogenicity and Immunological Persistence Study in Cynomolgus Macaques In the present invention, two dose groups, low dose group and high dose group, were set for Vaccine Group 2 in Example 4 above. Animals in the low dose group were respectively given the clinically proposed 1 human dose (1× (i.e., 0.5 mL)) and 4 human doses (4× (i.e., 2.0 mL)), and at the same time, a control group of 0.9% sodium chloride injection and an adjuvant group were established. Cynomolgus macaques (3 - 5 years old) were immunized at 3 - week intervals for three doses and an 8 - week recovery period was set. Humoral immune neutralizing antibodies were detected at 3 weeks, 6 weeks after the first administration and 4 weeks, 8 weeks during the recovery period. The results are shown in Figure 5 .

[0044] Two doses of the vaccine of the present invention can reach the peak level of neutralizing antibodies in cynomolgus macaques, and the antibody level did not increase further after three doses of immunization; the neutralizing antibody level in the high dose group was significantly higher than that in the low dose group after the second immunization, but with the passage of time, the difference between the groups gradually narrowed, suggesting that an immunization dose of 1× (i.e., 0.5 mL) can be selected for the vaccine of the present invention in practical applications.

[0045] Based on the comprehensive data, the recombinant varicella - zoster vaccine (CHO cell) of the present invention can induce a relatively high level of neutralizing antibodies in cynomolgus macaques after immunization. In clinical applications, the vaccine of the present invention can adopt an immunization dose of 1× (i.e., 0.5 mL) and an immunization schedule of two or three doses.

Claims

1. A herpes zoster vaccine composition, characterized in that: The method comprises an antigen and a composite adjuvant, wherein the antigen is VZV gE protein, and the composite adjuvant is composed of squalene, Tween 80, poloxamer and CpG oligonucleotide; Preferably, each 0.5 mL of the vaccine composition contains 10-100 μg of VZV gE protein, 5-10 mg of squalene, 0.5-1.5 mg of polysorbate 80, 1.0-5.0 mg of poloxamer, and 0.1-3.0 mg of CpG oligonucleotide.

2. The herpes zoster vaccine composition according to claim 1, characterized in that Each 0.5 mL of the vaccine composition contains 30-60 μg of VZV gE protein, 8-9 mg of squalene, 0.8-1.2 mg of polysorbate 80, 2.0-4.0 mg of poloxamer and 0.5-1.5 mg of CpG oligonucleotide.

3. The herpes zoster vaccine composition according to claim 2, characterized in that Each 0.5 mL of the vaccine composition contains 50 μg of VZV gE protein, 8.6 mg of squalene, 0.964 mg of polysorbate 80, 2.5 mg of poloxamer and 1.0 mg of CpG oligonucleotide.

4. The herpes zoster vaccine composition according to claim 2, characterized in that Each 0.5 mL of the vaccine composition contains 50 μg of VZV gE protein, 8.6 mg of squalene, 0.964 mg of polysorbate 80, 2.5 mg of poloxamer and 0.5 mg of CpG oligonucleotide.

5. The herpes zoster vaccine composition according to any one of claims 1 to 4, characterized in that The CpG oligonucleotide is CpG1018.

6. The herpes zoster vaccine composition according to any one of claims 1 to 4, characterized in that The poloxamer is poloxamer 188.

7. The herpes zoster vaccine composition according to any one of claims 1 to 4, characterized in that The amino acid sequence of the VZV gE protein is shown in SEQ ID No.

1.

8. The method for preparing the vaccine composition according to any one of claims 1 to 7, characterized in that: The VZV gE protein is dissolved in a composite adjuvant solution to prepare a finished vaccine composition; or the VZV gE protein is stored in the form of a lyophilized powder alone, and the composite adjuvant solution is used to dissolve the VZV gE protein powder before the vaccine composition is injected.

9. The preparation method according to claim 8, characterized in that: The preparation method of the composite adjuvant comprises the following steps: (1) Squalene is prepared as an oil phase; poloxamer and Tween 80 are mixed in a buffer solution PBS and stirred at 18-55°C to prepare an aqueous phase; (2) Slowly adding the prepared aqueous phase to the prepared oil phase, diluting the volume with buffer solution PBS, and stirring at room temperature with a high-speed stirrer at a speed of 8000-20000 rpm for 15-30 minutes to prepare a colostrum solution; (3) Pour the colostrum solution into a microfluidizer, adjust the pressure to 15,000-20,000 psi, adjust the temperature of the refrigerator, control the outlet temperature of the emulsion at 20-30°C, cycle the high-pressure homogenization for 3-10 times, and filter with a 0.22 μm sterilizing filter to obtain a refined milk solution; the diameter of the refined milk solution is measured by a Malvern particle size detector to be between 100 nm and 200 nm, and the pH value of the refined milk solution is measured to be between 6.3-6.7 or 7.0-7.4; (4) Pipette CpG oligonucleotide into the above-mentioned refined milk solution and mix well.

10. Use of the vaccine composition according to any one of claims 1 to 7 in the preparation of a medicament for preventing herpes zoster disease or its complications.

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