CN108992667A - A kind of shingles zoster vaccine and preparation method thereof, application - Google Patents

Abstract

The invention discloses a herpes zoster vaccine, a preparation method and application thereof, and belongs to the field of vaccines. Aiming at the problem that existing herpes zoster vaccines have relatively high safety risks, the present invention provides a herpes zoster vaccine, which uses Poly(I:C) as an adjuvant. Poly(I:C), referred to as polyinosinic acid, is double-stranded RNA, and its safety has been confirmed in large-scale clinical use. Poly(I:C) in the present invention is a high-efficiency interferon-inducing agent, which can generate an immune response similar to viral infection in vivo, induce CD4 ⁺ and CD8 ⁺ T cells, and enhance cellular immunity and humoral immune response.

Description

A kind of herpes zoster vaccine and its preparation method and application

technical field

The invention belongs to the field of vaccines, and in particular relates to a herpes zoster vaccine and its preparation method and application.

Background technique

Herpes zoster (HZ) is an acute infectious skin disease caused by recurrent infection of varicella-zoster virus (VZV) latent in the sensory ganglion, clinically manifested as lesions on one side of the body Herpes, typically confined to a dermatome, is often accompanied by radicular pain. Patients experience significant pain and discomfort, which may last for weeks, months, or years in severe cases, reducing the quality of life. A common serious complication is post-herpetic neuralgia (Post-Herpetic Neuralgia, PHN). Data show that more than 90% of adults are at risk of herpes zoster, and the risk of herpes zoster infection in people over the age of 50 is significantly higher, which may be associated with previous infection with wild-type varicella virus (VZV) virus, and is prone to secondary herpes zoster It is related to age-related attenuation of cellular immune function, and about 20% of people over 50 years old will develop PHN, and the possibility of PHN is higher with age. The prevention and treatment of HZ has become an important global public health issue, and the development and popularization of HZ vaccines for middle-aged and elderly people has important clinical significance. my country is a country with a large population. With the advent of an aging society, the number of elderly people affected by herpes zoster is increasing year by year. It is urgent to develop a safe and effective herpes zoster vaccine with independent intellectual property rights. However, there is no herpes zoster vaccine on the market in China. vaccine.

There are currently only two herpes zoster vaccines approved for marketing in the world. One is Merck’s Zostavax vaccine, which was approved for marketing in 2006. This vaccine is a live vaccine with high viral titer; the other is Glaxo GSK's subunit adjuvant vaccine (HZ/SU), which was first approved for marketing in North America in 2017.

There have been clinical reports of Zostavax causing HZ. The reason may be that infected people will cause more serious lesions after vaccination with live vaccines, thus causing people's concerns about its safety. In addition, live attenuated vaccines may not be suitable for immunocompromised patients. Rare complications caused by the Oka strain of varicella-zoster virus include pneumonia, hepatitis, herpes zoster meningitis, recurrent herpes zoster, severe rash, and secondary VZV transmission, mainly in immunocompromised or Patients with other serious medical conditions that were not diagnosed at the time of vaccination. Immunocompromised individuals, including patients with hematologic malignancies, patients undergoing immunosuppressive therapy, patients undergoing hematopoietic stem cell transplantation (HCT) or solid organ transplantation (SOT), HIV-infected patients, and those with autoimmune disease patients, have a higher incidence of developing HZ relative to the general population.

Currently, there is only Shringix vaccine marketed by GSK, which is a recombinant subunit vaccine supplemented with AS01B adjuvant. According to the patent (CN 101189254 B), the subunit adjuvant vaccine (HZ/su) adjuvant AS01B, whose mechanism of action is the Toll4-like receptor pathway, can effectively stimulate cellular immunity and antibodies in vivo. However, the AS01B adjuvant used in the vaccine is in the form of liposomes containing MPLA and QS21. MPLA is mainly a TLR4 receptor agonist, and also has a slight TLR2 receptor agonist activity. MPLA is derived from LPS, which is purified by chromatography followed by acid and heat treatment to prepare natural MPLA (Fransen F. et al., 2007). LPS is the main component of natural endotoxin, and the human body is extremely sensitive to bacterial endotoxin, which often causes adverse reactions such as fever in the human body. Although MPLA has undergone a series of detoxification treatments, there are still risks in clinical use.

QS-21 is an HPLC-grade purified product of Quil A, a mixture of amphiphilic triterpene glycosides extracted from Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), which has been used as an adjuvant in veterinary medicine. The signaling pathway activated by QS-21 is still unclear. The main side effect of QS-21 is hemolytic activity, but in clinical practice, adverse reactions are mainly avoided by controlling the dosage and different preparation methods. QS-21 promotes antigen-specific antibody responses and also activates cytotoxic CD8 ⁺ T cells.

In summary, the existing herpes zoster vaccines have high safety risks.

Contents of the invention

1. Problems to be solved

Aiming at the problem that existing herpes zoster vaccines have relatively high safety risks, the present invention provides a herpes zoster vaccine, which uses Poly(I:C) as an adjuvant. Poly(I:C), referred to as polyinosinic acid, is double-stranded RNA. As the main active ingredient of polyinosinic acid injection, a prescription drug in my country, it has been developed since the 1960s and has been widely used clinically in my country. Its safety has been confirmed in large-scale clinical use. Poly(I:C) in the present invention is a high-efficiency interferon-inducing agent, which can generate an immune response similar to viral infection in vivo, induce CD4 ⁺ and CD8 ⁺ T cells, and enhance cellular immunity and humoral immune response. Poly(I:C) can effectively induce the production of IFN-γ in pigs, and at the same time induce the gene expression of monocyte MHC-II and CD80/CD86 and chemokines, TLR-5, TLR-9, IL- 12 and p35, proving that it can effectively enhance Th1-type responses.

2. Technical solution

In order to solve the above problems, the present invention adopts the following technical solutions.

A herpes zoster vaccine comprising the following components, antigen and Poly(I:C) adjuvant, said antigen being VZV gE protein, inactivated varicella zoster virus purified liquid and inactivated varicella zoster virus lysed and purified one of the liquids.

Preferably, the herpes zoster vaccine further includes kanamycin sulfate.

Preferably, the concentration range of the kanamycin sulfate is 500-5000 U/dose.

Preferably, the weight ratio of VZV gE protein to Poly(I:C) adjuvant in the antigen is (50-200):1.

Preferably, the effective concentration of the antigen is 50-100 μg/mL.

Application of any one of the above-mentioned herpes zoster vaccines in the preparation of medicines for preventing or improving herpes zoster and/or postherpetic neuralgia.

A preparation method of herpes zoster vaccine, comprising the steps of:

(1) subculture the MRC-5 cells, wherein the MRC-5 cells are host cells;

(2) inoculating inactivated varicella-zoster virus into host cells for infection, adding culture medium for culturing, harvesting varicella-zoster virus stock solution, inactivating, and purifying to obtain purified inactivated varicella-zoster virus;

(3) purify the inactivated varicella-zoster virus purified liquid by Q-Sepharose FF ion gel chromatography, collect the eluate, and obtain the lysed purified liquid of inactivated varicella-zoster virus;

(4) Poly(I:C) was added and freeze-dried to obtain a herpes zoster vaccine.

Preferably, before the Q-Sepharose FF ion gel column on the inactivated varicella-zoster virus purified solution in the step (3), the described inactivated varicella-zoster virus purified solution is first pre-treated. Treatment, the pretreatment is as follows: first add TritonX-100 to the inactivated varicella-zoster virus purification solution, make the final concentration of TritonX-100 0.6-1.0% by volume, and shake at room temperature for 4-6h .

Preferably, the eluent is a 0.01M PBS solution containing 1M sodium chloride, and the eluate is detected by ultraviolet light, and the eluate is collected when a peak emerges at 280nm.

A herpes zoster vaccine prepared by any one of the above preparation methods.

Merck's Zostavax vaccine is currently widely used as a live attenuated vaccine, and domestic similar products are only in the clinical research stage. The live attenuated herpes zoster vaccine uses the vOka live attenuated vaccine strain, which has been proved to be a mixed strain, and retains the ability of nerve infection and latent ability, which may cause relapse from the vaccine strain and cause herpes zoster. The oka strain has also been applied to the development of herpes zoster vaccine. It has been proven to reduce the incidence of herpes zoster in elderly patients by adopting a high-titer concentrated form to improve the immunogenicity of the vaccine. However, with the advent of an aging society, Addressing the potential risk of herpes zoster, including potential risks from existing vaccine strains, becomes a serious issue. Therefore, it is urgent to develop new, safer and more effective VZV preventive vaccines.

Currently, there is only Shringix vaccine marketed by GSK, which is a recombinant subunit vaccine supplemented with AS01B adjuvant. According to the patent (CN 101189254 B), the subunit adjuvant vaccine (HZ/su) adjuvant AS01B, whose mechanism of action is the Toll4-like receptor pathway, can effectively stimulate cellular immunity and antibodies in vivo. However, the AS01B adjuvant used in the vaccine is in the form of liposomes containing MPLA and QS21. MPLA is mainly a TLR4 receptor agonist, and also has a slight TLR2 receptor agonist activity. MPLA is derived from LPS, which is purified by chromatography followed by acid and heat treatment to prepare natural MPLA (Fransen F. et al., 2007). LPS is the main component of natural endotoxin, and the human body is extremely sensitive to bacterial endotoxin, which often causes adverse reactions such as fever in the human body. Although MPLA has undergone a series of detoxification treatments, there are still risks in clinical use.

QS-21 is an HPLC-grade purified product of Quil A, a mixture of amphiphilic triterpene glycosides extracted from Quil A (derived from the bark of the South American tree Quillaja Saponaria Molina), which has been used as an adjuvant in veterinary medicine. The signaling pathway activated by QS-21 is still unclear. The main side effect of QS-21 is hemolytic activity, but in clinical practice, adverse reactions are mainly avoided by controlling the dosage and different preparation methods. QS-21 promotes antigen-specific antibody responses and also activates cytotoxic CD8 ⁺ T cells

There are risks in the clinical use of live attenuated vaccines, and purely inactivated or subunit vaccines cannot produce sufficient immunity and clinical therapeutic effects, so it is necessary to choose an effective vaccine adjuvant system. The safety and effectiveness of the Poly(I:C) adjuvant to be selected in the present invention have been confirmed.

Poly(I:C), also known as polyinosinic acid, is double-stranded RNA. As the main active ingredient of polyinosinic acid injection, a prescription drug in my country, it has been developed since the 1960s and has been widely used clinically in my country. Its safety has been confirmed in large-scale clinical use.

Poly(I:C), as a broad-spectrum antiviral drug, is composed of polynucleotides, which can induce the production of interferon in the body, has a good curative effect on diseases caused by various viruses, and can promote the formation of antibodies and stimulate macrophages. Poly(I:C) in the present invention is a high-efficiency interferon-inducing agent, which can generate an immune response similar to viral infection in vivo, induce CD4 ⁺ and CD8 ⁺ T cells, and enhance cellular immunity and humoral immune response. Studies have found that Poly(I:C) can effectively induce the production of IFN-γ in pigs, and at the same time induce the gene expression of monocyte MHC-II and CD80/CD86 and chemokines, TLR-5, TLR-9 , IL-12 and p35 production, proving that it can effectively enhance Th1-type responses.

In the present invention, Poly(I:C) adjuvant is selected to assist recombinantly expressed VZV gE protein or inactivated VZV virus or split VZV virus subunit. Poly(I:C) adjuvant is a double-chain polyinosinic acid polymer, which can be recognized by Toll3 receptors and can produce an immune response similar to viral infection, so it can induce CD4 ⁺ and CD8 ⁺ T cells to produce cellular immunity and humoral immunity . Co-immunization of mice with Poly(I:C) and hepatitis B surface antigen can enhance specific humoral immunity and cellular immune response (Shen, CellMol Immunol, 2007).

3. Beneficial effects

Compared with the prior art, the beneficial effects of the present invention are:

(1) The adjuvant used in the vaccine of the present invention is Poly(I:C), and Poly(I:C) is called for short polyinosinic acid, which is double-stranded RNA, as the main effect of the prescription drug polyinosinic acid injection in my country The ingredients have been developed since the 1960s and are widely used clinically in my country. Its safety has been confirmed in large-scale clinical use. Poly(I:C) in the present invention is a high-efficiency interferon-inducing agent, which can generate an immune response similar to viral infection in vivo, induce CD4 ⁺ and CD8 ⁺ T cells, and enhance cellular immunity and humoral immune response. Poly(I:C) can effectively induce the production of IFN-γ in pigs, and at the same time induce the gene expression of monocyte MHC-II and CD80/CD86 and chemokines, TLR-5, TLR-9, IL- 12 and p35 production, which proves that it can effectively enhance Th1 type response;

(2) The adjuvant used in the vaccine of the present invention is Poly(I:C), and the antigen is one of VZV gE protein, inactivated varicella zoster virus purified liquid and inactivated varicella zoster virus lysed purified liquid, the antigen It works synergistically with adjuvants to greatly improve the immune effect;

(3) in the prior art, poly(I:C) hydrolysis is avoided by adding kanamycin and calcium chloride, and the vaccine of the present invention can avoid poly(I:C) hydrolysis as long as kanamycin sulfate is added;

(4) in a kind of preparation method of the vaccine that contains inactivated varicella-zoster virus lysing and purifying liquid of the present invention, use Q-Sepharose FF ion gel purification, greatly improved purity, and then improved immune effect, Increased the safety of clinical use.

Description of drawings

Fig. 1 is the electropherogram of different groups of embodiment 16.

Detailed ways

Embodiment 1 The preparation of the vaccine containing recombinant varicella zoster virus (VZV) gE protein

Experimental materials and sources:

CHO cell line, purchased from Thermo company;

CD CHO medium was purchased from Thermo Fisher.

Preparation:

1. The recombinant CHO cell line stably expressing the VZV gE protein adopts a well-known method, according to the amino acid sequence of the VZV gE protein found on the NCBI website, synthesizes the gene sequence, and transfects it into CHO cells according to the method described in the "Molecular Cloning Experiment Guide". Monoclonal cells were selected by the limiting dilution method, and after multiple rounds of screening, they were expanded and cultured in cell shake flasks and then frozen. After collecting the supernatant of the subcloned cells, samples were taken for Western Blot detection. According to the gray scale of the bands, the monoclonal cells with higher expression levels were compared, and finally selected as the dominant cell line.

2. Recombinant CHO cells are amplified by cell culture flasks after recovery, and finally subcultured to a bioreactor, cultured at 36°C for 7 days, and fed-batch or perfusion culture methods are used to supplement the nutrient solution, and the nutrient solution is CD CHO medium .

3. Take samples every day to detect the expression of VZV gE protein. When the expression reaches the maximum value and does not change anymore, collect the cell supernatant, which contains the VZV gE protein secreted by the recombinant CHO cells.

4. The collected supernatant culture solution is clarified, purified, adjusted to pH 3.5, and inactivated to obtain VZV gE protein with a purity greater than 95%.

5. Mix the purified VZV gE protein with Poly(I:C) adjuvant according to a certain ratio, so that the content of VZV gE protein is 50 μg/mL per dose, add lyoprotectant, and then aseptically subpackage, each dose 1.0mL, freeze-dried into a white loose body, which is the vaccine containing VZV gE protein. The vaccine is a clear liquid after reconstitution. The formula for this vaccine is as follows:

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use.

Embodiment 2 Preparation of vaccine containing recombinant varicella-zoster virus (VZV) gE protein

Experimental materials and sources:

CHO cell line, purchased from Thermo company;

CD CHO medium was purchased from Thermo Fisher.

Preparation:

1. The recombinant CHO cell line stably expressing the VZV gE protein adopts a well-known method, according to the amino acid sequence of the VZV gE protein found on the NCBI website, synthesizes the gene sequence, and transfects it into CHO cells according to the method described in the "Molecular Cloning Experiment Guide". Monoclonal cells were selected by the limiting dilution method, and after multiple rounds of screening, they were expanded and cultured in cell shake flasks and then frozen. After collecting the supernatant of the subcloned cells, samples were taken for Western Blot detection. According to the gray scale of the bands, the monoclonal cells with higher expression levels were compared, and finally selected as the dominant cell line.

2. Recombinant CHO cells are amplified by cell culture flasks after recovery, and finally subcultured to a bioreactor, cultured at 38°C for 10 days, and fed-batch or perfusion culture methods are used to supplement the nutrient solution, and the nutrient solution is CD CHO medium .

3. Take samples every day to detect the expression of VZV gE protein. When the expression reaches the maximum value and does not change anymore, collect the cell supernatant, which contains the VZV gE protein secreted by the recombinant CHO cells.

4. The collected supernatant culture solution is clarified, purified, adjusted to pH 3.5, and inactivated to obtain VZV gE protein with a purity greater than 95%.

5. Mix the purified VZV gE protein with Poly(I:C) adjuvant according to a certain ratio, so that the content of VZV gE protein is 100 μg/mL per dose, add lyoprotectant, and then aseptically subpackage, each dose 1.0mL, freeze-dried into a white loose body, which is the vaccine containing VZV gE protein. The vaccine is a clear liquid after reconstitution. The formula for this vaccine is as follows:

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use.

Embodiment 3 Preparation of vaccine containing recombinant varicella-zoster virus (VZV) gE protein

Experimental materials and sources:

CHO cell line, purchased from Thermo company;

CD CHO medium was purchased from Thermo Fisher.

Preparation:

1. The recombinant CHO cell line stably expressing the VZV gE protein adopts a well-known method, according to the amino acid sequence of the VZV gE protein found on the NCBI website, synthesizes the gene sequence, and transfects it into CHO cells according to the method described in the "Molecular Cloning Experiment Guide". Monoclonal cells were selected by the limiting dilution method, and after multiple rounds of screening, they were expanded and cultured in cell shake flasks and then frozen. After collecting the supernatant of the subcloned cells, samples were taken for Western Blot detection. According to the gray scale of the bands, the monoclonal cells with higher expression levels were compared, and finally selected as the dominant cell line.

2. Recombinant CHO cells are amplified by cell culture flasks after recovery, and finally subcultured to a bioreactor, cultured at 37°C for 8 days, and fed-batch or perfusion culture methods are used to supplement the nutrient solution, and the nutrient solution is CD CHO medium .

3. Take samples every day to detect the expression of VZV gE protein. When the expression reaches the maximum value and does not change anymore, collect the cell supernatant, which contains the VZV gE protein secreted by the recombinant CHO cells.

4. The collected supernatant culture solution is clarified, purified, adjusted to pH 3.5, and inactivated to obtain VZV gE protein with a purity greater than 95%.

5. Mix the purified VZV gE protein with Poly(I:C) adjuvant according to a certain ratio, so that the content of VZV gE protein is 75 μg/mL per dose, add lyoprotectant, and then aseptically subpackage, each dose 1.0mL, freeze-dried into a white loose body, which is the vaccine containing VZV gE protein.

Embodiment 4 The preparation of the vaccine containing recombinant varicella zoster virus (VZV) gE protein

Embodiment 4 is basically the same as embodiment 1, and difference is that the formula of vaccine is different, and the formula of this vaccine is as follows:

Element Quantity (per dose) VZV gE protein 50μg Sodium chloride (NaCl) 1600μg Disodium hydrogen phosphate (Na ₂ HPO ₄ ) 288μg Potassium dihydrogen phosphate (KH ₂ PO ₄ ) 48μg Potassium chloride (KCl) 40μg Poly(I:C) 0.5mg sucrose 20mg Kanamycin Sulfate 1500U water for injection Appropriate amount to 1mL

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use.

Example 5 Preparation of vaccine containing recombinant varicella-zoster virus (VZV) gE protein

Embodiment 5 is basically the same as Example 1, and the difference is that the formulation of the vaccine is different, and the formulation of the vaccine is as follows:

Element Quantity (per dose) VZV gE protein 100μg Sodium chloride (NaCl) 1600μg Disodium hydrogen phosphate (Na ₂ HPO ₄ ) 288μg Potassium dihydrogen phosphate (KH ₂ PO ₄ ) 48μg Potassium chloride (KCl) 40μg Poly(I:C) 0.5mg sucrose 20mg Kanamycin Sulfate 2000U water for injection Appropriate amount to 1mL

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use.

Embodiment 6 The preparation method of the vaccine containing inactivated varicella-zoster virus purified liquid

Experimental materials and sources:

MRC-5 cells and varicella-zoster virus were purchased from ATCC;

0.2%～0.5% human serum albumin, purchased from Tonglu Biopharmaceutical;

MEM medium, purchased from Thermo company;

PBS, homemade;

Ultrafiltration membrane bag, purchased from Millipore;

β-propiolactone, purchased from Serva company;

TritonX-100 was purchased from Merck.

Preparation:

1. Passage MRC-5 cells to 28 passages in cell culture flasks and cell factories, in which MRC-5 cells are host cells;

2. When the host cell is amplified to a fusion degree of more than 90%, the VZV virus is inoculated into the host cell for infection, and the M.O.I. is 0.001;

3. Add MEM medium containing 0.2% to 0.5% human serum albumin, and place it in a cell incubator at 34°C for cultivation;

4. Observe the degree of cell lesions every day. When the CPE of the cells reaches more than 50%, discard the medium and wash the cells with PBS;

5. Add 0.03% EDTA solution to elute the infected cells, collect the cell suspension in a centrifuge tube, centrifuge at 4000 rpm for 10 minutes at 4°C, remove the supernatant, add stabilizer to combine, mix well, and take a sample from a single bottle to make aseptic Detection, -80°C freezer;

6. Place the sample tested for sterility in an ultrasonic bottle for ultrasonic crushing, centrifuge at 8,000 rpm for 10 minutes at 4°C, and collect the supernatant, that is, the virus stock solution;

7. Use an ultrafiltration membrane bag with a molecular weight cut-off of 100KD to carry out 50-fold concentration; according to the 2015 edition of Pharmacopoeia IV, Lowry method method 1, the total protein content in the virus concentrate is 35mg/mL;

8. Mix β-propiolactone and virus concentrate at a volume ratio of 1:6000, inactivate at 3°C for 12 hours, and hydrolyze at 35°C for 2 hours;

9. Sepharose 4FF gel column chromatography purifies the virus inactivation solution, detects with ultraviolet light, collects the VZVgE protein peak at the absorption point of 280nm, and obtains the purified solution of inactivated varicella zoster virus.

10. Mix the inactivated varicella-zoster virus purified solution with Poly(I:C) adjuvant according to a certain ratio, so that the content of VZV gE protein is 50 μg/mL per dose, add lyoprotectant, and then aseptically subpackage , 1.0 mL per dose, lyophilized into a white loose body, which is the vaccine containing VZV gE protein. The vaccine is a clear liquid after reconstitution.

The formula for this vaccine is as follows:

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use. The 50 μg of inactivated varicella-zoster virus purified liquid refers to the content of VZV gE protein in the inactivated varicella-zoster virus purified liquid. The content of VZV gE protein was detected by ELISA method.

Example 7 The preparation method of the vaccine containing the purified liquid of inactivated varicella-zoster virus

Experimental materials and sources:

MRC-5 cells and varicella-zoster virus were purchased from ATCC;

0.2%～0.5% human serum albumin, purchased from Tonglu Biopharmaceutical;

MEM medium, purchased from Thermo company;

PBS, homemade;

Ultrafiltration membrane bag, purchased from Millipore;

β-propiolactone, purchased from Serva company;

TritonX-100 was purchased from Merck.

Preparation:

1. Passage MRC-5 cells to 28 passages in cell culture flasks and cell factories, in which MRC-5 cells are host cells;

2. When the host cell is amplified to a fusion degree of more than 90%, the VZV virus is inoculated into the host cell for infection, and the M.O.I. is 0.01;

3. Add MEM medium containing 0.2% to 0.5% human serum albumin, and place it in a cell incubator at 37°C for cultivation;

4. Observe the degree of cell lesions every day. When the CPE of the cells reaches more than 50%, discard the medium and wash the cells with PBS;

5. Add 0.03% EDTA solution to elute the infected cells, collect the cell suspension in a centrifuge tube, centrifuge at 4000 rpm for 10 minutes at 4°C, remove the supernatant, add stabilizer to combine, mix well, and take a sample from a single bottle to make aseptic Detection, -80°C freezer;

6. Place the sample tested for sterility in an ultrasonic bottle for ultrasonic crushing, centrifuge at 8,000 rpm for 10 minutes at 4°C, and collect the supernatant, that is, the virus stock solution;

7. Use an ultrafiltration membrane bag with a molecular weight cut-off of 100KD for 50-fold concentration; according to the 2015 edition of the Pharmacopoeia, the fourth Lowry method method 1, the total protein content in the virus concentrate is 95mg/mL;

8. Mix β-propiolactone and virus concentrate at a volume ratio of 1:6000, inactivate at 5°C for 36 hours, and hydrolyze at 35°C for 2 hours;

9. Sepharose 4FF gel column chromatography purifies the virus inactivation solution, detects with ultraviolet light, collects the VZVgE protein peak at the absorption point of 280nm, and obtains the purified solution of inactivated varicella zoster virus.

10. Mix the inactivated varicella-zoster virus purified solution with Poly(I:C) adjuvant according to a certain ratio, so that the content of VZV gE protein is 100 μg/mL per dose, add lyoprotectant, and then aseptically subpackage , 1.0 mL per dose, lyophilized into a white loose body, which is the vaccine containing VZV gE protein. The vaccine is a clear liquid after reconstitution. The formula for this vaccine is as follows:

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use. The content of VZV gE protein was detected by ELISA method.

The 100 μg of inactivated varicella-zoster virus purified solution means that the content of VZV gE protein in the inactivated varicella-zoster virus purified solution is 100 μg.

Example 8 The preparation method of the vaccine containing inactivated varicella-zoster virus purified liquid

Experimental materials and sources:

MRC-5 cells and varicella-zoster virus were purchased from ATCC;

0.2%～0.5% human serum albumin, purchased from Tonglu Biopharmaceutical;

MEM medium, purchased from Thermo company;

PBS, homemade;

Ultrafiltration membrane bag, purchased from Millipore;

β-propiolactone, purchased from Serva company;

TritonX-100 was purchased from Merck.

Preparation:

1. Passage MRC-5 cells to 28 passages in cell culture flasks and cell factories, in which MRC-5 cells are host cells;

2. When the host cell is amplified to a fusion degree of more than 90%, the VZV virus is inoculated into the host cell for infection, and the M.O.I. is 0.005;

3. Add MEM medium containing 0.2% to 0.5% human serum albumin, and place it in a cell incubator at 35°C for cultivation;

4. Observe the degree of cell lesions every day. When the CPE of the cells reaches more than 50%, discard the medium and wash the cells with PBS;

5. Add 0.03% EDTA solution to elute the infected cells, collect the cell suspension in a centrifuge tube, centrifuge at 4000 rpm for 10 minutes at 4°C, remove the supernatant, add stabilizer to combine, mix well, and take a sample from a single bottle to make aseptic Detection, -80°C freezer;

6. Place the sample tested for sterility in an ultrasonic bottle for ultrasonic crushing, centrifuge at 8,000 rpm for 10 minutes at 4°C, and collect the supernatant, that is, the virus stock solution;

7. Use an ultrafiltration membrane bag with a molecular weight cut-off of 100KD for 50-fold concentration; according to the 2015 edition of the Pharmacopoeia, the fourth Lowry method method 1, the total protein content in the virus concentrate is 65mg/mL;

8. Mix β-propiolactone and virus concentrate at a volume ratio of 1:6000, inactivate at 3-5°C for 24 hours, and hydrolyze at 35°C for 2 hours;

9. Sepharose 4FF gel column chromatography purifies the virus inactivation solution, detects with ultraviolet light, collects the VZVgE protein peak at the absorption point of 280nm, and obtains the purified solution of inactivated varicella zoster virus.

10. Mix the inactivated varicella-zoster virus purified solution with Poly(I:C) adjuvant according to a certain ratio, so that the content of VZV gE protein is 50 μg/mL per dose, add lyoprotectant, and then aseptically subpackage , 1.0 mL per dose, lyophilized into a white loose body, which is the vaccine containing VZV gE protein. The vaccine is a clear liquid after reconstitution. The formula for this vaccine is as follows:

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use. The content of VZV gE protein was detected by ELISA method.

The 50 μg of inactivated varicella-zoster virus purified liquid means that the content of VZV gE protein in the inactivated varicella-zoster virus purified liquid is 50 μg.

Example 9

Embodiment 9 is basically the same as Embodiment 8, except that the formulation of the vaccine is different, and the formulation of the vaccine is as follows:

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use. The content of VZV gE protein was detected by ELISA method.

The 100 μg of inactivated varicella-zoster virus purified solution means that the content of VZV gE protein in the inactivated varicella-zoster virus purified solution is 100 μg.

Example 10 Preparation method of vaccine containing inactivated varicella-zoster virus lysed and purified solution

Take the inactivated varicella-zoster virus purification solution obtained in Example 6, add TritonX-100 with a final concentration of 0.6% in proportion by volume, and shake at room temperature for 4 hours to obtain an inactivated varicella-zoster virus lysate. The herpes zoster virus lysate was purified by Q-sepharose FF ion gel chromatography, and the 0.01M PBS solution containing 1M sodium chloride was fully washed to remove foreign proteins. Ultraviolet detection, the VZVgE protein peak is collected at 280nm absorption, which is the lysed and purified liquid of inactivated varicella zoster virus.

Mix the inactivated varicella-zoster virus lysate and purification liquid with Poly(I:C) adjuvant according to a certain ratio, so that the content of VZV gE protein in the inactivated varicella-zoster virus lysis and purification liquid is 50 μg/mL per dose, add The freeze-dried protective agent is then aseptically divided into 1.0 mL per dose, and freeze-dried into a white loose body, which is the vaccine containing the lysed and purified solution of inactivated varicella-zoster virus. The vaccine is a clear liquid after reconstitution. The formula for this vaccine is as follows:

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use. The 50 μg of inactivated varicella-zoster virus lysate and purify liquid refers to the content of VZV gE protein in the inactivated varicella-zoster virus lyse-purify liquid. The content of VZV gE protein was detected by ELISA method.

Example 11 Preparation method of vaccine containing inactivated varicella-zoster virus lysed and purified liquid

Take the inactivated varicella-zoster virus purification solution obtained in Example 6, add TritonX-100 with a final concentration of 1.0% in proportion by volume, shake at room temperature for 6 hours, and obtain the inactivated varicella-zoster virus lysate, and inactivate the varicella-zoster virus The herpes zoster virus lysate was purified by Q-sepharose FF ion gel chromatography, and the 0.01M PBS solution containing 1M sodium chloride was fully washed to remove foreign proteins. Ultraviolet detection, the VZVgE protein peak is collected at 280nm absorption, which is the lysed and purified liquid of inactivated varicella zoster virus.

Mix the inactivated varicella-zoster virus lysate and purification liquid with Poly(I:C) adjuvant according to a certain ratio, so that the content of VZV gE protein in the inactivated varicella-zoster virus lysis and purification liquid is 100 μg/mL per dose, add The freeze-dried protective agent is then aseptically divided into 1.0 mL per dose, and freeze-dried into a white loose body, which is the vaccine containing the lysed and purified solution of inactivated varicella-zoster virus. The vaccine is a clear liquid after reconstitution. The formula for this vaccine is as follows:

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use. The 100 μg of inactivated varicella-zoster virus lysate and purify liquid refers to the content of VZV gE protein in the inactivated varicella-zoster virus lyse-purify liquid. The content of VZV gE protein was detected by ELISA method.

Example 12 Preparation method of vaccine containing inactivated varicella-zoster virus lysed and purified liquid

Take the purified solution of inactivated varicella-zoster virus obtained in Example 6, add TritonX-100 with a final concentration of 1.0% in proportion by volume, and shake at room temperature for 5 hours to obtain an inactivated varicella-zoster virus lysate. The herpes zoster virus lysate was purified by Q-sepharose FF ion gel chromatography, and the 0.01M PBS solution containing 1M sodium chloride was fully washed to remove foreign proteins. Ultraviolet detection, the VZVgE protein peak is collected at 280nm absorption, which is the lysed and purified liquid of inactivated varicella zoster virus.

Mix the inactivated varicella-zoster virus lysate and purification liquid with Poly(I:C) adjuvant according to a certain ratio, so that the content of VZV gE protein in the inactivated varicella-zoster virus lysis and purification liquid is 100 μg/mL per dose, add The freeze-dried protective agent is then aseptically divided into 1.0 mL per dose, and freeze-dried into a white loose body, which is the vaccine containing the lysed and purified solution of inactivated varicella-zoster virus. The vaccine is a clear liquid after reconstitution. The formula for this vaccine is as follows:

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use. The 100 μg of inactivated varicella-zoster virus lysate and purify liquid refers to the content of VZV gE protein in the inactivated varicella-zoster virus lyse-purify liquid. The content of VZV gE protein was detected by ELISA method.

Example 13 Preparation method of vaccine containing inactivated varicella-zoster virus lysed and purified liquid

Take the purified solution of inactivated varicella-zoster virus obtained in Example 6, add TritonX-100 with a final concentration of 1.0% in proportion by volume, and shake at room temperature for 5 hours to obtain an inactivated varicella-zoster virus lysate. The herpes zoster virus lysate was purified by Q-sepharose FF ion gel chromatography, and the 0.01M PBS solution containing 1M sodium chloride was fully washed to remove foreign proteins. Ultraviolet detection, the VZVgE protein peak is collected at 280nm absorption, which is the lysed and purified liquid of inactivated varicella zoster virus.

Mix the inactivated varicella-zoster virus lysate and purification liquid with Poly(I:C) adjuvant according to a certain ratio, so that the content of VZV gE protein in the inactivated varicella-zoster virus lysis and purification liquid is 100 μg/mL per dose, add The freeze-dried protective agent is then aseptically divided into 1.0 mL per dose, and freeze-dried into a white loose body, which is the vaccine containing the lysed and purified solution of inactivated varicella-zoster virus. The vaccine is a clear liquid after reconstitution. The formula for this vaccine is as follows:

Among them, the role of sucrose is to shape and protect the protein, and the role of inorganic salt is to ensure isotonicity and physiological pH. PBS is prepared according to the formula of phosphate buffered saline (PBS) in the "Molecular Cloning Experiment Guide". Adjust the pH value between 7.0 and 8.0 with 0.1M NaOH solution before use. The 100 μg of inactivated varicella-zoster virus lysate and purify liquid refers to the content of VZV gE protein in the inactivated varicella-zoster virus lyse-purify liquid. The content of VZV gE protein was detected by ELISA method.

Example 14

Immunological Study of Herpes Zoster Vaccine Containing Poly(I:C) Adjuvant

Experimental materials and sources:

SPF grade female BALB/c mice were purchased from Anhui Medical University;

Varicella virus, purchased from ATCC;

Poly(I:C), self-made.

Test design:

Female BALB/c mice were randomly divided into groups with 6 mice in each group, and the age and body weight of the mice were similar. Two times of immunization, each injection of 1 mL of the corresponding group of vaccines, the interval between the two doses was 2 weeks, blood was collected from the vein 4 weeks after the second dose, and the positive control was varicella virus liquid containing 20,000 PFU, simulating the reduction of varicella virus that was only marketed abroad. The immune effect of live herpes zoster vaccine; the negative control was only given phosphate buffered saline PBS.

The test groups are shown in the table below.

test results:

ELISA method was used to detect the level of VZV IgG in the serum. Compared with the serum before immunization, if the antibody titer increased by more than 4 times after immunization, it was regarded as seroconversion. The ELISPOT method was used to detect the cell levels mainly IL-2 and IFN-γ. The results are shown in the table below.

Note: * indicates p value <0.05, that is, compared with the phosphate buffer saline PBS group, the difference between the two is statistically significant. The results showed that the VZV gE protein containing Poly(I:C) adjuvant at a concentration of 50-100 μg could produce effective cellular immunity and humoral immunity. The results show that in BALB/c mice, the vaccine provided by the invention can significantly (p<0.05) increase the level of humoral immunity (VZV IgG antibody), improve the level of cellular immunity, and can significantly (p<0.05) increase IL-2 and IFN-γ levels.

The synergy of embodiment 15 antigen and adjuvant

Experimental material: with embodiment 14.

Experimental design: Female BALB/c mice were randomly divided into groups, with 6 mice in each group, and the age and body weight of the mice were similar. Two times of immunization, each injection of 1 mL of the corresponding group of vaccines, the interval between the two doses was 2 weeks, blood was collected from the vein 4 weeks after the second dose, and the positive control was varicella virus liquid containing 20,000 PFU, simulating the reduction of varicella virus that was only marketed abroad. The immune effect of live herpes zoster vaccine; the negative control was only given phosphate buffered saline PBS. Each group was compared with VZV gE containing 100 μg gE protein without adjuvant, inactivated varicella-zoster virus purified liquid, and inactivated varicella-zoster virus lysed purified liquid, and the synergistic effect of different combinations was observed.

The results are shown in the table below:

Wherein, the inactivated varicella-zoster virus purified liquid (100 μg) means that the content of VZV gE protein in the inactivated varicella-zoster virus purified liquid is 100 μg;

Wherein, the inactivated varicella-zoster virus lysate and purify solution (100 μg) means that the content of VZV gE protein in the inactivated varicella-zoster virus lyse-purify solution is 100 μg.

The experimental results show that: when VZV gE protein containing 0.5-1 mg Poly(I:C) adjuvant is at a concentration of 50-100 μg, the positive conversion rate of the antibody after immunization can reach 100%, which is consistent with the positive control. However, 100 μg VZV gE without adjuvant, inactivated varicella-zoster virus purified solution, and inactivated varicella-zoster virus lysed and purified solution cannot produce 100% seroconversion after immunization alone, indicating that the adjuvant is at a concentration of 0.5-1 mg It has good synergy with VZV gE protein.

The protective effect of embodiment 16 kanamycin sulfate containing Poly (I:C) vaccine

Experimental design: Vaccines of groups 1 to 6 were prepared respectively, stored at different temperatures for 24 hours, and the degradation of Poly(I:C) was compared by electrophoresis. Electrophoresis conditions: 1% agarose, sample volume 5ul, electrophoresis 30min@130V constant voltage. The experimental design is shown in the table below.

group drugs used temperature Group 1 Embodiment 1 gained vaccine does not add kanamycin sulfate 4°C Group 2 Embodiment 1 gained vaccine does not add kanamycin sulfate 25°C Group 3 Embodiment 1 gained vaccine does not add kanamycin sulfate 37°C Group 4 The vaccine obtained in embodiment 1 4°C Group 5 The vaccine obtained in embodiment 1 25°C Group 6 The vaccine obtained in embodiment 1 37°C

Among them, the medicine used in group 1 is the vaccine prepared according to the method of Example 1, except that kanamycin sulfate is not added during the preparation process, and the others are the same;

The medicine used in group 2 is the vaccine prepared according to the method of Example 1, except that kanamycin sulfate is not added during the preparation process, and the others are the same;

The medicine used in group 3 is the vaccine prepared according to the method of Example 1, except that kanamycin sulfate is not added during the preparation process, and the others are the same.

L1 refers to the lanes of group 1, L3 refers to the lanes of group 2, and L5 refers to the lanes of group 3.

L2 refers to the lane of group 4, L4 refers to the lane of group 5, and L6 refers to the lane of group 6.

The results show that Poly(I:C) without kanamycin sulfate has a decreasing molecular weight and an increasing degree of hydrolysis as the temperature increases, see lanes L1, L3, and L5 in the figure below; while adding kanamycin sulfate The molecular weight of Poly(I:C) of the protein decreased as the temperature increased, but compared with the group without kanamycin sulfate, the degree of hydrolysis was effectively reduced (lanes L2, L4, L6 in the figure below). Especially when stored at 4°C, Poly(I:C) containing kanamycin sulfate has the highest stability. The existing Poly(I:C) is double-stranded RNA, which is easily degraded when it exists alone, and is easily degraded by RNase after injection into the human body. In the prior art, in order to prevent the degradation of polyinosinic acid, the method of simultaneously adding calcium ions and kanamycin sulfate is adopted to protect the stability of RNA during transportation, storage and injection. The present invention only adds kanamycin sulfate, which can effectively ensure the storage of Poly(I:C) at different temperatures and avoid the hydrolysis of RNase.

Claims (10)

1. a kind of shingles zoster vaccine, which is characterized in that including following component: antigen and Poly (I:C) adjuvant, the antigen are One of VZV gE albumen, inactivation varicellazoster virus refined solution and inactivation varicellazoster virus cracking refined solution.

2. shingles zoster vaccine according to claim 1, which is characterized in that the shingles zoster vaccine further includes sulfuric acid card That mycin.

3. shingles zoster vaccine according to claim 2, which is characterized in that the concentration range of the kanamycin sulfate is 500~5000U/ agent.

4. shingles zoster vaccine according to claim 1, which is characterized in that VZV gE albumen and Poly in the antigen (I:C) weight ratio of adjuvant is (50~200): 1.

5. shingles zoster vaccine according to claim 1, which is characterized in that the effective concentration of the antigen is 50~100 μ g/mL。

6. shingles zoster vaccine described in claim 1-5 any one preparation for prevent or improve shingles zoster and/or Application in the drug of postherpetic neuralgia.

7. a kind of preparation method of shingles zoster vaccine, includes the following steps:

(1) MRC-5 cell is passed on, wherein MRC-5 cell is host cell；

(2) inactivation varicellazoster virus is inoculated into host cell and is infected, culture medium is added and is cultivated, harvests Varicellazoster virus stoste inactivates, purifying, obtains inactivation varicellazoster virus refined solution；

(3) inactivation varicellazoster virus refined solution is chromatographed with Q- Ago-Gel FF ionic gel and is purified, collect elution Liquid obtains inactivation varicellazoster virus cracking refined solution；

(4) Poly (I:C) is added, freeze-drying obtains shingles zoster vaccine.

8. the preparation method of shingles zoster vaccine according to claim 7, which is characterized in that will go out in the step (3) On varicellazoster virus refined solution living before Q- Ago-Gel FF ionic gel column, first to the band-like blister of inactivation varicella Exanthema virus refined solution carries out pre-treatment, and the pre-treatment is as follows: TritonX-100 is first added to the band-like blister of inactivation varicella In exanthema virus refined solution, by volume, make the final concentration of 0.6-1.0% of TritonX-100, room temperature shakes 4-6h.

9. the preparation method of shingles zoster vaccine according to claim 7, which is characterized in that the eluent is chlorine containing 1M Change the 0.01M PBS solution of sodium, the eluent of ultraviolet detection outflow collects eluent in 280nm appearance.

10. a kind of shingles zoster vaccine, which is characterized in that be prepared using claim 7-9 any one preparation method.