CN107174658B - Varicella virus inactivated vaccine for human and preparation method thereof - Google Patents
Varicella virus inactivated vaccine for human and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
- A61L2/0088—Liquid substances
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
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- A61L2202/00—Aspects relating to methods or apparatus for disinfecting or sterilising materials or objects
- A61L2202/20—Targets to be treated
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention relates to the technical field of biology, in particular to a varicella virus inactivated vaccine for human and a preparation method thereof, which comprises the following steps of mixing beta-propiolactone and a varicella virus concentrated solution according to the volume ratio of 1: 2000-1: 8000 mixing, inactivating for 12-36 h at 3-5 ℃, and hydrolyzing for 1.5-3 h at 34-37 ℃ to obtain inactivated virus liquid; the inactivated virus liquid is purified, subpackaged and freeze-dried to prepare the inactivated vaccine. The varicella virus inactivated vaccine prepared by the method is safe and effective, has slight inoculation reaction, and is more suitable for people with low immune function which are not suitable for attenuated live vaccines.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a varicella virus inactivated vaccine for human use and a preparation method thereof.
Background
The pathogens of Varicella and herpes zoster are both Varicella-zoster virus (VZV). VZV belongs to the family of herpesviridae and is a double-stranded DNA virus.
VZV is a highly contagious herpesvirus that causes both chickenpox and shingles (HZ); the former is commonly seen in childhood and young adults, while the latter is the result of latent VZV reactivation and replication. Following initial infection with VZV, the virus remains dormant in sensory ganglia, and such VZV reactivation is associated with decreased immune function, most often in elderly or immunocompromised individuals (Weinberg et al, Journal of Infectious Diseases (2009) 200: 1068-77). Some patients also experience a prolonged period of HZ-related pain after the HZ rash has healed.
The effect of using vaccines to prevent varicella has been recognized worldwide. Varicella vaccines based on live attenuated viruses (VZV Oka strain) were developed and clinically tested in the last 70 and 80 years. In 1984, the vaccine was first licensed to the market in germany and sweden. At present, a plurality of freeze-dried varicella attenuated live vaccines are sold on the market and can be refrigerated or frozen by a refrigerator. Some of these vaccines are monovalent vaccines (containing only VZV virus components), and some are combined with measles, mumps, and rubella triple vaccine (MMR).
Studies have shown that monovalent varicella live attenuated vaccines are well tolerated and that the most commonly reported adverse events in healthy children after vaccination with varicella vaccines are minor, including mild tenderness, redness and rash at the site of injection. However, live attenuated vaccines may not be suitable for patients with compromised immune function. The rare complications caused by the VZV Oka strain include pneumonia, hepatitis, herpes zoster meningitis, recurrent herpes zoster, severe rashes and secondary VZV transmission, mainly in immunocompromised patients or patients with other serious conditions that have not been diagnosed at the time of vaccination. Immunocompromised individuals, including patients with hematological malignancies, patients undergoing immunosuppressive therapy, patients receiving hematopoietic stem cell transplantation (HCT) or Solid Organ Transplantation (SOT), HIV-infected patients, and patients with autoimmune diseases, have a higher incidence of developing HZ relative to the general population. Furthermore, these patient populations are at increased risk of developing serious and life-threatening complications. At present, no specific medicine is available for treating varicella, the prevention is mainly performed, and the varicella vaccine can only be inoculated to prevent VZV virus infection, so that the safe and effective varicella vaccine has great significance for the immunity of high risk groups.
In order to adapt to the use of the vaccine by the immunocompromised people, researchers have researched and developed inactivated vaccines, virus-like particle vaccines, subunit vaccines and DNA vaccines. Safer vaccines for immunocompromised subjects can be developed by heat-inactivating VZV or by using subunit vaccines (Cohen, j.i., (2008) j.infect.dis.197(Suppl 2): S237-S241). Redman et al (J. infectious Diseases (1997)176:578-85) describe inactivated VZV vaccines inactivated by heating at 50 ℃ and resulting in an infectious virus content of <1.2pfu/0.5 mL. However, even this level of infectivity is at risk for immunocompromised patients. Us patent numbers 6214354 and 5997880 mention the potential to use inactivated VZV vaccines and disclose examples of producing and testing heat-treated vaccines; in the patent (CN 103167880A) of Moshadong, the inactivated varicella vaccine is produced by using gamma-radiation inactivated VZV virus. If a method of inactivating VZV were developed, the method would produce a VZV sample that is safe and effective (i.e., non-infectious but retaining its immunogenicity and antigenicity) in immunocompromised patients, which would satisfy a significant medical need.
Currently, methods for inactivation of varicella vaccines are known as heat inactivation and gamma ray inactivation. The heat inactivation is firstly proposed by Smith and the like when developing the cholera suis inactivated vaccine, and the method is simple and easy to implement. However, the method for killing microorganisms by heating is rough, protein denaturation is easily caused, and further immunogenicity is obviously affected, the method has the possibility of incomplete inactivation, and the residual virus content is still unsafe for patients with low immunity; in addition, there are studies showing that60Singlet oxygen and free radicals generated by Co gamma ray irradiation have great damage effect on protein, mainly manifested by peptide bond breakage on the primary structure of the protein, amino acid composition change, beta folding content reduction in space conformation, loose compact structure and obvious increase of irregular curling. Viruses that live in cellular systems are most radiation resistant, and double-stranded viruses are less susceptible to inactivation by ionizing radiation than single-stranded viruses. Poor control of dose uniformity during the irradiation process also affects vaccine quality.
Beta-propiolactone (BPL), also known as beta-hydroxypropanolide, is a heterocyclic compound (C)3H4O2) Has strong inactivation effect on viruses and is a good virus inactivator. BPL MieThe living mechanism acts on pathogen DNA or RNA, destroys the structure of nucleic acid by reacting with purine base (mainly guanine) of nucleic acid, but does not act directly on protein, thus maintaining good immunogenicity. BPL is extremely easy to hydrolyze, disappears after being hydrolyzed in water bath at 37 ℃ for 2h, and is hydrolyzed into non-toxic human fat metabolite beta-hydroxypropionic acid. In addition, because it can be completely hydrolyzed in the vaccine liquid, it is not necessary to consider the residues in the finished vaccine, and the vaccination reaction is also slight. Meanwhile, the time for inactivating the pathogen by the beta-propiolactone is short, thereby obviously shortening the production period of the vaccine and improving the economic benefit.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a varicella virus inactivated vaccine for human use and a preparation method thereof.
The invention is realized by the following technical scheme:
the preparation method of the varicella virus inactivated vaccine for human comprises the following steps:
(1) passaging human embryonic fibroblast (MRC-5) cells for no more than 34 passages;
(2) when the cells are expanded to the fusion degree of more than 90%, inoculating the varicella virus into the cells for infection;
(3) adding the cells infected with the varicella virus into a Minimum Essential Medium (MEM) containing 2-5% fetal bovine serum, and culturing in a cell culture box at 34-37 ℃;
(4) observing the degree of cytopathic effect, pouring out the culture medium when the cytopathic effect (CPE) reaches more than 50%, and washing the cells by Phosphate Buffer Solution (PBS);
(5) adding 0.02-0.05% of Ethylene Diamine Tetraacetic Acid (EDTA) solution to elute infected cells, centrifuging the cell suspension, removing supernatant, adding a stabilizer, mixing, uniformly mixing, sampling in a single bottle, and freezing at-65-minus 80 ℃;
(6) carrying out ultrasonic crushing and centrifugation on a sample for detecting sterility, and collecting supernatant, namely virus stock solution;
(7) carrying out ultrafiltration concentration on the virus stock solution by adopting an ultrafiltration membrane package;
(8) detecting the protein content and virus titer of the virus liquid after ultrafiltration concentration;
(9) mixing beta-propiolactone with a virus concentrate with the protein content of 35-95 mg/mL and the virus titer of not less than 4.0lgPFU/mL according to the volume ratio of 1: 2000-1: 8000 mixing, inactivating for 12-36 h at 3-5 ℃, and hydrolyzing for 1.5-3 h at 34-37 ℃ to obtain inactivated virus liquid;
(10) purifying the inactivated virus liquid by adopting column chromatography to obtain an inactivated virus purified liquid;
(11) the inactivated virus purified solution is subjected to blind passage for at least 3 generations in human embryonic fibroblast (MRC-5) cells, the virus titer is detected, and no plaque is formed in the result.
(12) Subpackaging and freeze-drying the inactivated virus purified solution to obtain the varicella virus inactivated vaccine.
Preferably, the multiplicity of infection (M.O.I.) for the inoculation of the virus into the cells for infection in step (2) is 0.01 to 0.1.
Preferably, the centrifugation condition in the step (5) is 3000-4000 rpm centrifugation for 10-15 minutes at 3-5 ℃.
Preferably, the centrifugation condition in the step (6) is 7000-8000 rpm centrifugation for 10-15 minutes at 3-5 ℃.
Preferably, the ultrafiltration concentration of the virus stock solution by using the ultrafiltration membrane package in the step (7) is 30-60 times of the ultrafiltration concentration of the virus stock solution by using the ultrafiltration membrane package with the molecular weight cut-off of 100KD or 300 KD.
Preferably, the protein content in step (8) is detected by Lowry method.
Preferably, the inactivation condition in the step (9) is that the volume ratio of beta-propiolactone to the virus concentrate is 1: 8000 mixing, inactivating at 4 deg.C for 24h, and hydrolyzing at 37 deg.C for 2 h.
Preferably, the purification mode of step (10) is to load the virus inactivation solution in batches by using a Sepharose4FF chromatographic column, and collect the varicella virus protein peak under the ultraviolet detection of 280nm, namely the virus inactivation purification solution.
Preferably, the virus titer in steps (8) and (11) is detected using plaque assay.
The invention also protects the varicella virus inactivated vaccine for human prepared by any one of the preparation methods.
The invention has the beneficial effects that:
the beta-propiolactone directly acts on pathogen DNA or RNA, destroys the structure of nucleic acid by reacting with purine base (mainly guanine) of nucleic acid, but does not directly act on protein, and has better immunogenicity and thorough inactivation effect compared with heat inactivation, gamma ray inactivation and formaldehyde inactivation. In addition, because the beta-propiolactone is extremely easy to hydrolyze, can be completely hydrolyzed in the vaccine liquid, and is hydrolyzed into the nontoxic human fat metabolite beta-hydroxypropionic acid, the residue in the finished vaccine is not needed to be considered, and the inoculation reaction is slight. Meanwhile, the time for inactivating the pathogen by the beta-propiolactone is short, thereby obviously shortening the production period of the vaccine and improving the economic benefit. The varicella virus inactivated vaccine prepared by the method is safe and effective, has slight inoculation reaction, and is more suitable for people with low immune function which are not suitable for attenuated live vaccines.
Detailed Description
The invention will now be further described with reference to the following examples, which are intended to illustrate, but not limit the invention.
EXAMPLE 1 method for preparing a varicella virus inactivated vaccine for human use
(1) Passage of MRC-5 cells to 28 passages in cell culture flasks and cell factories;
(2) when the cells are expanded to the fusion degree of more than 90%, inoculating the virus into the cells for infection, wherein the M.O.I. is 0.01-0.1;
(3) adding MEM culture medium containing 2-5% fetal calf serum, and culturing in a cell culture box at 34-37 ℃;
(4) observing the degree of cytopathic effect every day, pouring out the culture medium when the CPE of the cells reaches more than 50%, and washing the cells by PBS;
(5) adding 0.03% EDTA solution to elute infected cells, collecting the cell suspension in a centrifuge tube, centrifuging at 4000rpm at 4 ℃ for 10 minutes, removing supernatant, adding stabilizer, mixing, sampling in a single bottle, and freezing at-80 ℃;
(6) placing a sample for detecting sterility in an ultrasonic bottle for ultrasonic disruption, centrifuging at 8000rpm at 4 ℃ for 10 minutes, and collecting supernatant, namely virus stock solution;
(7) concentrating by 50 times with ultrafiltration membrane with molecular weight cutoff of 100 KD;
(8) the protein content detected by Lowry method is 52mg/mL, and the virus titer detected by plaque method is 4.9 lgPFU/mL;
(9) mixing beta-propiolactone and virus concentrated solution according to the volume ratio of 1: 6000, inactivating at 3-5 ℃ for 12-36 h, and hydrolyzing at 35 ℃ for 2 h;
(10) purifying the inactivated virus liquid by column chromatography, and collecting virus peaks according to the ultraviolet detection value to obtain an inactivated virus purified liquid;
(11) blind passage of the inactivated virus purified solution in MRC-5 cells for 3 generations, and detecting the virus titer by a plaque method;
(12) subpackaging and freeze-drying the inactivated virus purified solution to obtain the varicella inactivated vaccine;
example 2 selection of protein concentration of Virus concentrates
(1) Preparing 5 batches of inactivated virus solution according to the steps (1) to (10) of the preparation method of the inactivated vaccine for human using the poxvirus;
(2) the concentrations of the virus concentrates and infectivity after inactivation were measured as shown in the following table:
according to the results in the table, virus concentrated solution with protein content of 35-95 mg/ml is selected for inactivation.
Example 3 plaque assay for infectivity of inactivated vaccine against varicella virus
MRC-5 cells were plated in 6-well plates, 3 x 10 cells were plated per well5Single cell, 5% CO2And incubating the cells in the incubator at 34-37 ℃ for 3-4 days, and determining the titer of the varicella virus after the cells grow into a single layer. The culture medium is discarded, and the varicella virus inactivated vaccine is diluted to a proper concentration to infect cells in 5% CO2Adsorbing for 1-2 hours at 34-37 ℃ in an incubator, then supplementing 3mL of virus culture solution into each hole, and adding 5% CO2Culturing in an incubator at 34-37 ℃. And (5) carrying out dyeing after culturing for 7-10 days.Counting and taking an average value, multiplying the average value by a dilution coefficient and an inoculation volume correction factor to obtain the titer of the varicella virus inactivated vaccine, namely expressing the titer of the inactivated varicella virus inactivated vaccine by lg PFU/mL, wherein no plaque is formed in the titer result of the inactivated varicella virus inactivated vaccine.
Example 4 varicella virus inactivation and verification of inactivation
(1) Obtaining virus concentrated solution according to the steps (1) to (7) of the preparation method of the inactivated vaccine of the human using the poxvirus;
(2) detecting the protein content and the virus titer of the virus concentrated solution obtained in the step (1), adding an inactivating agent beta-propiolactone (BPL) into the virus concentrated solution with the protein content of 35-95 mg/mL and the virus titer of not less than 4.0lgPFU/mL according to the table 1-4 for inactivation and hydrolysis reaction:
TABLE 1
TABLE 2
TABLE 3
TABLE 4
(3) And (3) determining that the protein content is 35-95 mg/mL according to the result of the step (2), and when the virus titer is not less than 4lgPFU/mL, adding the inactivator in a ratio of 1: 2000-1: 8000, inactivating at the temperature of 3-5 ℃, inactivating for 12-36 h, hydrolyzing at the temperature of 34-37 ℃ for 1.5-3 h, inoculating the inactivated virus solution into a 24-well plate growing into monolayer cells under the conditions, adsorbing at the temperature of 34-37 ℃ for 1-2 h, supplementing 1mL of virus culture solution, culturing at the temperature of 34-37 ℃ for 70-96 h, collecting the virus solution, inoculating into the 24-well plate growing into monolayer cells, continuously conducting blind passage for 3 generations, collecting the virus solution, and measuring the virus titer by using a plaque method;
(4) the inactivated virus titer was 0 as determined in example 2, indicating that the inactivated vaccine was not infectious.
EXAMPLE 5 purification of inactivated vaccine against varicella Virus for human use
The inactivated virus solution is obtained according to the preparation methods (1) to (9) of the inactivated vaccine for human poxvirus. Purifying the inactivated virus liquid by using a Sepharose4FF chromatographic column, loading the virus inactivated liquid in batches according to the loading amount of the chromatographic column, and collecting a varicella virus protein peak under 280nm ultraviolet detection to obtain the inactivated virus purified liquid.
EXAMPLE 6 preparation of inactivated vaccine against varicella virus for lyophilized human
Obtaining an inactivated virus purified solution according to the preparation methods (1) to (10) of the inactivated vaccine of the human poxvirus, adding a protective agent, subpackaging each dose of 0.5mL, and freeze-drying to obtain a white loose body which is a clear liquid after redissolution.
Example 7
(1) Obtaining virus concentrated solution according to the preparation methods (1) to (7) of the inactivated vaccine of the human poxvirus;
(2) adding a proper amount of formaldehyde into the virus concentrated solution for inactivation, sampling at different time points to detect virus infectivity, and performing blind transmission for 3 generations, wherein the results are shown in the following table:
| inactivation time (h) | Infectivity |
| 24 | With formation of plaque |
| 48 | With formation of plaque |
| 72 | No formation of plaque |
| 96 | No formation of plaque |
(3) Adding formaldehyde and beta-propiolactone into the virus concentrated solution with the same volume for inactivation, immunizing BALB/c mice, taking blood from veins after 4 weeks, and detecting the VZV IgG level in the serum by an ELISA method and a FAMA method as follows:
| VZV IgG positive conversion rate | Formaldehyde (I) | Beta-propiolactone |
| ELISA method (%) | 62 | 89 |
| FAMA method (%) | 100％ | 100％ |
(4) The comparison of the two inactivators is given in the following table:
the above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims (9)
1. The preparation method of the varicella virus inactivated vaccine for human use is characterized by comprising the following steps:
(1) passaging MRC-5 cells for no more than 34 passages;
(2) when the cells are expanded to the fusion degree of more than 90%, inoculating the varicella virus into the cells for infection;
(3) adding the cells infected with the varicella virus into a minimum necessary culture medium containing 2-5% fetal calf serum, and culturing in a cell culture box at 34-37 ℃;
(4) observing the degree of cytopathic effect, pouring out the culture medium when the cytopathic effect reaches more than 50%, and washing the cells by using a phosphate buffer solution;
(5) adding 0.02-0.05% of EDTA solution to elute infected cells, centrifuging the cell suspension, removing supernatant, combining and mixing uniformly, sampling in a single bottle for aseptic detection, and freezing and storing at-65 to-80 ℃;
(6) carrying out ultrasonic crushing and centrifugation on a sample for detecting sterility, and collecting supernatant, namely virus stock solution;
(7) carrying out ultrafiltration concentration on the virus stock solution by adopting an ultrafiltration membrane package, wherein the ultrafiltration concentration on the virus stock solution by adopting the ultrafiltration membrane package is carried out by 30-60 times by adopting the ultrafiltration membrane package with the molecular weight cutoff of 100KD or 300 KD;
(8) detecting the protein content and virus titer of the virus liquid after ultrafiltration concentration;
(9) mixing beta-propiolactone and virus concentrated solution with the protein content of 35-95 mg/mL and the virus titer of not less than 4.0lgPFU/mL according to the volume ratio of 1: 8000, inactivating for 12-36 h at the temperature of 3-5 ℃, and hydrolyzing for 1.5-3 h at the temperature of 34-37 ℃ to obtain inactivated virus solution;
(10) purifying the inactivated virus liquid by adopting column chromatography to obtain an inactivated virus purified liquid;
(11) blind transferring the inactivated virus purification solution in MRC-5 cells for at least 3 generations, and detecting the virus titer, wherein no plaque is formed in the result;
(12) subpackaging and freeze-drying the inactivated virus purified solution to obtain the varicella virus inactivated vaccine.
2. The method for preparing the inactivated vaccine against varicella virus for human use according to claim 1, wherein: and (3) inoculating the virus into the cells to infect, wherein the multiplicity of infection is 0.01-0.1.
3. The method for preparing the inactivated vaccine against varicella virus for human use according to claim 1, wherein: and (5) centrifuging for 10-15 minutes at 3000-4000 rpm at 3-5 ℃.
4. The method for preparing the inactivated vaccine against varicella virus for human use according to claim 1, wherein: and (4) centrifuging at 7000-8000 rpm at 3-5 ℃ for 10-15 minutes under the centrifugation condition in the step (6).
5. The method for preparing the inactivated vaccine against varicella virus for human use according to claim 1, wherein: and (4) detecting the protein content in the step (8) by using a Lowry method.
6. The method for preparing the inactivated vaccine against varicella virus for human use according to claim 1, wherein: the inactivation condition of the step (9) is inactivation at 4 ℃ for 24h, and then hydrolysis at 37 ℃ for 2 h.
7. The method for preparing the inactivated vaccine against varicella virus for human use according to claim 1, wherein: and (3) the purification mode in the step (10) is to load the virus inactivation solution in batches by using a Sepharose4FF chromatographic column and collect a varicella virus protein peak under 280nm ultraviolet detection, namely the inactivated virus purification solution.
8. The method for preparing the inactivated vaccine against varicella virus for human use according to claim 1, wherein: the virus titer in steps (8) and (11) was measured using the plaque method.
9. An inactivated vaccine against varicella virus for human use, which is prepared according to any one of claims 1 to 8.
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