CN111450246B - Trivalent influenza virus subunit vaccine and preparation method thereof - Google Patents
Trivalent influenza virus subunit vaccine and preparation method thereof Download PDFInfo
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- C12N2760/16211—Influenzavirus B, i.e. influenza B virus
- C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The invention belongs to the technical field of biology, and particularly relates to a trivalent influenza virus subunit vaccine and a preparation method thereof. The trivalent influenza virus subunit vaccine contains three types of H1N1, H3N2 and B, and is prepared by virus inoculation, virus propagation culture, allantoic fluid harvesting, clarification, inactivation, ultrafiltration concentration, cracking, ultracentrifugation purification, blending, filtration sterilization, subpackaging and packaging, wherein the inactivation is as follows: firstly, adding a carboxymethyl dextran solution into a monovalent virus harvesting solution, and then adding formaldehyde for inactivation. The method reduces the content of free formaldehyde in the prepared trivalent influenza virus subunit vaccine, prolongs the storage life of the vaccine, and increases the generation of antibody level after the vaccine is immunized.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a trivalent influenza virus subunit vaccine and a preparation method thereof.
Background
Influenza is caused by influenza virus and can cause acute respiratory diseases which are commonly suffered by large-scale epidemic people and livestock. Influenza viruses are classified into three types, i.e., A, B and C. Among them, type a is most pathogenic, can infect animals and humans and cause epidemics even worldwide pandemics; the pathogenic force of B is weak, which can cause local epidemic. Influenza viruses often undergo antigenic drift and antigen transfer, evading the body's immune system defenses, which is also responsible for pandemics. Influenza viruses are highly contagious and are airborne by droplets. After a short incubation period, high fever and chills are caused, the body temperature can reach 40 ℃ within 1-2 days, and the body temperature is accompanied by symptoms such as systemic debilitation, headache, myalgia, pharyngalgia and the like, complications such as pneumonia, bronchitis, myocarditis, pericarditis and the like can be caused, so that a large number of deaths of high-risk people such as old people, weak people and the like are caused, and huge losses are brought to the society.
The existing vaccines in the market are influenza whole virus inactivated vaccines and influenza virus split vaccines, and the traditional split vaccine purification method comprises the following steps: and (3) carrying out ultrafiltration concentration, purification with centrifugation in a sucrose density area, ultrafiltration to remove sucrose, cracking, purification with centrifugation in a sucrose density area again, ultrafiltration to remove sucrose again, sterilization and filtration, and adding a salix pump as a preservative. The process is complex, the steps are complicated, a large amount of manpower and material resources are consumed, and the risk of microbial pollution is increased.
Meanwhile, the traditional influenza vaccine has insufficient purity and contains adjuvants and preservatives harmful to human bodies. Adjuvants, while improving the immune response of a vaccine, come at the expense of increased reactogenicity, and even more severe. For example, the antibody levels increased in the elderly after vaccination with MF59 adjuvant influenza vaccine, but the local reactions were more abundant, and in a control study in the elderly, the elderly vaccinated with adjuvant had 6-fold increase in pain, 2-fold increase in local redness, and systemic reactions such as weakness and muscle pain increased compared to those vaccinated without adjuvant. In addition, it is reported that the aluminium hydroxide-adsorbed diphtheria-pertussis-tetanus vaccine produced by the Danish Statens serum research institute causes severe adverse reactions of intractable induration pruritus in 546 cases of injection sites. 77% of adverse reaction cases were determined to be allergic to aluminum hydroxide by skin tests. Currently, the national food and drug administration has banned the use of aluminum hydroxide adjuvants for the production of rabies vaccines. Salix thiopica has been used as a preservative for many vaccines, but the toxic and side effects of salix thiopica, which cause contact dermatitis, allergic conjunctivitis, ototoxicity, etc., have begun to attract attention from researchers, and the use of salix thiopica in children's vaccines has been banned in iowa, california, the united kingdom, and other european countries.
CN107537030A discloses a trivalent influenza virus subunit vaccine and a preparation method thereof, wherein the influenza vaccine adopts a cracking agent and a new purification method to further purify virus protein after cracking to prepare the trivalent influenza virus subunit vaccine, wherein each agent contains influenza hemagglutinin of A1 type (H1N1), A3 type (H3N2) and B type with the content of more than 80 percent, and the trivalent influenza virus subunit vaccine does not contain an adjuvant, thimerosal and other preservatives. The invention also provides a preparation method of the influenza vaccine, which comprises the following steps: inoculating virus, proliferating and culturing virus, harvesting allantoic fluid, clarifying, ultrafiltering, concentrating, inactivating, cracking, ultracentrifuging, purifying by gel filtration chromatography (ultrafiltration), mixing, filtering, sterilizing, packaging, and the like. The influenza virus subunit vaccine provided by the invention can improve the safety of the influenza vaccine, eliminate adverse reactions caused by an adjuvant and eliminate toxic and side effects caused by thimerosal.
Currently, in the production of influenza vaccines, inactivation of the virus is performed using formaldehyde as an inactivating agent. Formaldehyde is a highly irritating carcinogen. It can act on both the amino-containing nucleotide bases (e.g. A, G, U) of the virus and the viral coat protein. When acting on the viral capsid protein, the protein is easily crosslinked or the viral particles aggregate, and cannot act on the nucleic acid in the capsid protein any more. Thus, the antigenicity of the pathogen proteins can be severely compromised and there may be survival of the pathogen. Therefore, in order to fully inactivate the pathogen and avoid the virus dispersion accident, measures such as increasing the concentration of formaldehyde and prolonging the inactivation time are adopted in the production process of the vaccine. As a result, the redundant free formaldehyde aldehyde group continuously acts on the antigen of the virus in the storage process of the vaccine, so that the antigen is gradually degraded under the action of the aldehyde group, the effective components in the vaccine continuously pass, the storage life of the vaccine is shortened, and the generation of the antibody level after the vaccine is immunized is reduced.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a trivalent influenza virus subunit vaccine, which has less content of free formaldehyde, prolongs the storage life of the vaccine, and increases the generation of antibody level after the vaccine is immunized.
The second purpose of the invention is to provide the preparation method of the trivalent influenza virus subunit vaccine, the carboxymethyl glucan solution is added firstly in the inactivation process, and then formaldehyde is added for inactivation.
In order to realize the first purpose of the invention, the invention adopts the following technical scheme:
a trivalent influenza virus subunit vaccine comprises three types of A1 (H1N1), A3 (H3N2) and B (B), and is prepared by virus inoculation, virus propagation culture, allantoic fluid harvest, clarification, inactivation, ultrafiltration concentration, splitting and ultracentrifugation purification, mixing, filtration sterilization, subpackaging and packaging, wherein the inactivation is as follows: firstly, adding a carboxymethyl dextran solution into a monovalent virus harvesting solution, and then adding formaldehyde for inactivation.
Currently, in the production of influenza vaccines, inactivation of the virus is performed using formaldehyde as an inactivating agent. Formaldehyde is a highly irritating carcinogen. It can act on both the amino-containing nucleotide bases (e.g. A, G, U) of the virus and the viral coat protein. When acting on the viral capsid protein, the protein is easily crosslinked or the viral particles aggregate, and cannot act on the nucleic acid in the capsid protein any more. Thus, the antigenicity of the pathogen proteins can be severely compromised and there may be survival of the pathogen. Therefore, in order to fully inactivate the pathogen and avoid the virus dispersion accident, measures such as increasing the concentration of formaldehyde and prolonging the inactivation time are adopted in the vaccine production process. As a result, the redundant free formaldehyde aldehyde group continuously acts on the antigen of the virus in the storage process of the vaccine, so that the antigen is gradually degraded under the action of the aldehyde group, the effective components in the vaccine continuously pass, the storage life of the vaccine is shortened, and the generation of the antibody level after the vaccine is immunized is reduced.
In the invention, a method of firstly adding carboxymethyl dextran solution into monovalent virus harvesting solution and then adding formaldehyde for inactivation is adopted in the inactivation process, so that the content of free formaldehyde in the vaccine is greatly reduced, the storage life of the vaccine is prolonged, and the generation of antibody level after the vaccine is immunized is increased.
Further, the substitution degree of the carboxymethyl glucan is 0.5-0.7.
In the invention, after carboxymethyl glucan is modified by carboxymethyl, a charged hydrophilic group is introduced, so that the water solubility is increased, the higher the substitution degree is, the higher the water solubility is, but the conformation is seriously damaged, the activity is reduced, the water solubility can be increased on the basis of not damaging the biological activity by proper substitution degree, and the immunological activity cannot be influenced by the substitution degree smaller than 0.75.
Experiments show that the substitution degree of the carboxymethyl glucan has certain influence on the content of free formaldehyde in the prepared vaccine. When the substitution degree of the carboxymethyl glucan is 0.5-0.7, the content of free formaldehyde in the prepared trivalent influenza virus subunit vaccine is obviously lower than that of the trivalent influenza virus subunit vaccine prepared when the substitution degree of the carboxymethyl glucan is not in the range of 0.5-0.7. Therefore, in the present invention, it is preferable that the degree of substitution of the carboxymethyl dextran is 0.5 to 0.7.
In order to achieve the second purpose of the invention, the invention adopts the following technical scheme:
the preparation method of the trivalent influenza virus subunit vaccine comprises the following steps of virus inoculation, virus propagation culture, allantoic fluid harvesting, clarification, inactivation, ultrafiltration concentration, lysis, ultracentrifugation purification, mixing, filtration sterilization, subpackaging and packaging, wherein the inactivation is as follows: firstly, adding a carboxymethyl dextran solution into a monovalent virus harvesting solution, and then adding formaldehyde for inactivation.
Further, the volume ratio of the addition amount of the carboxymethyl dextran solution to the monovalent virus harvesting solution is 1: 0.1-0.5.
Further, the substitution degree of the carboxymethyl glucan is 0.5-0.7.
Further, the concentration of the carboxymethyl dextran solution is 1-5g/100 mL.
Furthermore, the carboxymethyl glucan solution is prepared by adding carboxymethyl glucan into water for injection to be dissolved.
Further, the final concentration of the formaldehyde is 100-150mg/ml, and the inactivation time is 35-45 hours.
Further, the cracking agent adopted in the cracking is nonoxynol-9.
Further, the concentration of the cracking agent is 0.01-2%, wherein the concentration is the mass percentage concentration.
In the present invention, the inoculation of virus, virus propagation culture, allantoic fluid harvesting, clarification, inactivation, ultrafiltration concentration, lysis and ultracentrifugation purification, compounding, filtration sterilization, packaging and packaging are carried out according to methods commonly used in the art, such as the method in CN 107537030A.
Compared with the prior art, the invention has the following advantages:
the trivalent influenza virus subunit vaccine prepared by the method has low content of free formaldehyde, prolongs the storage life of the vaccine, and increases the generation of antibody level after the vaccine is immunized.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, the carboxymethyl dextran solution was prepared by the following method:
adding carboxymethyl dextran into water for injection to dissolve to obtain 1-5g/100mL carboxymethyl dextran water solution, wherein the substitution degree of carboxymethyl dextran is 0.5-0.7.
Example 1
A production method of influenza A1 (H1N1) subunit vaccine monovalent stock solution sequentially comprises the following steps:
(1) virus inoculation: inoculating influenza A1 (H1N1) virus working seeds into allantoic cavities of 10-day-old healthy chick embryos;
(2) virus propagation culture: transferring the inoculated chicken embryo into an incubator at 34 ℃ for culturing for 48 hours to proliferate influenza virus;
(3) allantoic fluid harvesting: screening live chick embryos, standing the chick embryos at the temperature of 2 ℃ for 16 hours, and then harvesting allantoic fluid;
(4) clarification: removing most impurities such as chick embryo red blood cells by a centrifugation method;
(5) inactivation: adding a carboxymethyl glucan solution with the concentration of 3g/100mL into the monovalent virus harvesting solution, wherein the volume ratio of the addition amount of the carboxymethyl glucan solution to the monovalent virus harvesting solution is 1: 0.3, the substitution degree of the carboxymethyl glucan is 0.6, then formaldehyde with the final concentration of 120mg/ml is added, and inactivation is carried out for 35 hours at the temperature of 2 ℃;
(6) and (3) ultrafiltration concentration: performing ultrafiltration concentration on the fully inactivated influenza virus allantois harvest liquid by using an ultrafiltration membrane with the cut-off molecular weight of 1000 kD;
(7) cracking and ultracentrifugation purification: adding 1.0% of cracking agent nonoxynol-9 into the ultrafiltration concentrated solution, mixing uniformly, loading, synchronously cracking for 4 hours in the process of sucrose density zone centrifugation to achieve the purpose of cracking while separating, and harvesting 10-20% of cracking solution according to the concentration of sucrose;
(8) and (3) gel filtration chromatography purification: and (3) further purifying the solution obtained by the sucrose density gradient centrifugation in the last step by a SephadexG-25 sephadex gel filtration chromatography system to obtain monovalent stock solution of the influenza A1 (H1N1) subunit vaccine.
Secondly, the method for producing the influenza A3 (H3N2) subunit vaccine monovalent stock solution sequentially comprises the following steps:
(1) virus inoculation: inoculating influenza A3 (H3N2) virus working seeds into allantoic cavities of 10-day-old healthy chick embryos;
(2) virus propagation culture: transferring the inoculated chicken embryo into an incubator at 34 ℃ for culturing for 48 hours to proliferate influenza virus;
(3) allantoic fluid harvesting: screening live chick embryos, standing the chick embryos at the temperature of 2 ℃ for 16 hours, and then harvesting allantoic fluid;
(4) clarification: removing most impurities such as chick embryo red blood cells by a centrifugation method;
(5) inactivation: adding a carboxymethyl glucan solution with the concentration of 3g/100mL into the monovalent virus harvesting solution, wherein the volume ratio of the addition amount of the carboxymethyl glucan solution to the monovalent virus harvesting solution is 1: 0.3, the substitution degree of the carboxymethyl glucan is 0.6, then formaldehyde with the final concentration of 120mg/ml is added, and inactivation is carried out for 45 hours at the temperature of 2 ℃;
(6) and (3) ultrafiltration concentration: performing ultrafiltration concentration on the fully inactivated influenza virus allantois harvest liquid by using an ultrafiltration membrane with the molecular weight cutoff of 300 kD;
(7) cracking and ultracentrifugation purification: adding 0.5% of cracking agent nonoxynol-9 into the ultrafiltration concentrated solution, mixing uniformly, loading, synchronously cracking for 10 hours in the process of sucrose density zone centrifugation to achieve the purpose of cracking while separating, and harvesting 15-30% of cracking solution according to the concentration of sucrose;
(8) and (3) gel filtration chromatography purification: and (3) further purifying the solution obtained by the sucrose density gradient centrifugation in the last step by a SephadexG-25 sephadex gel filtration chromatography system to obtain monovalent stock solution of the influenza A3 (H3N2) subunit vaccine.
The production method of the influenza B (B) virus subunit vaccine monovalent stock solution sequentially comprises the following steps:
(1) virus inoculation: inoculating influenza B (B) virus working seeds into allantoic cavities of 10-day-old healthy chick embryos;
(2) virus propagation culture: transferring the inoculated chicken embryo into an incubator at 34 ℃ for culturing for 72 hours to proliferate influenza virus;
(3) allantoic fluid harvesting: screening live chick embryos, standing the chick embryos at the temperature of 2 ℃ for 16 hours, and then harvesting allantoic fluid;
(4) clarification: removing most impurities such as chick embryo red blood cells by a centrifugation method;
(5) inactivation: adding a carboxymethyl glucan solution with the concentration of 3g/100mL into the monovalent virus harvesting solution, wherein the volume ratio of the addition amount of the carboxymethyl glucan solution to the monovalent virus harvesting solution is 1: 0.3, the substitution degree of the carboxymethyl glucan is 0.6, then formaldehyde with the final concentration of 120mg/ml is added, and inactivation is carried out for 40 hours at the temperature of 2 ℃;
(6) and (3) ultrafiltration concentration: performing ultrafiltration concentration on the fully inactivated influenza virus allantois harvest liquid by using an ultrafiltration membrane with the cut-off molecular weight of 1000 kD;
(7) cracking and ultracentrifugation purification: adding 1.5% of cracking agent nonoxynol-9 into the ultrafiltration concentrated solution, mixing uniformly, loading, synchronously cracking for 8 hours in the process of sucrose density zone centrifugation to achieve the purpose of cracking while separating, and harvesting 10-30% of cracking solution according to the concentration of sucrose;
(8) and (3) gel filtration chromatography purification: and (3) further purifying the solution obtained by the sucrose density gradient centrifugation in the last step by a SephadexG-25 sephadex gel filtration chromatography system to obtain the influenza B (B) virus subunit vaccine monovalent stock solution.
Fourthly, the vaccine finished product blending and preparation method sequentially comprises the following steps:
(1) mixing: influenza a 1(H1N1), a 3(H3N2), and B (B) monovalent stock solutions were mixed at a hemagglutinin concentration of 1: 1: 1, mixing, and controlling the concentration of hemagglutinin of each virus to be 40-48 mg/ml;
(2) and (3) filtering and sterilizing: filtering and sterilizing the virus liquid mixed in the last step by a 0.22mm filter membrane to obtain a semi-finished product of the influenza virus subunit vaccine;
(3) subpackaging: subpackaging the vaccine semi-finished product into prefilled vials, each vial having a dose of 0.5ml for 1 time, wherein the dose for human use is 0.5ml, and the dose of hemagglutinin containing each influenza virus strain is 10-20 mg;
(4) packaging: and after the lamp is qualified, labeling, boxing and the like are carried out according to the quality standard, and the trivalent influenza virus subunit vaccine is obtained.
Example 2
The procedure is as in example 1, except that:
a production method of influenza A1 (H1N1) subunit vaccine monovalent stock solution comprises the following steps:
the step (3) is as follows: screening live chick embryos, standing the chick embryos at 5 ℃ for 16 hours to obtain allantoic fluid;
the step (5) is as follows: adding a carboxymethyl glucan solution with the concentration of 1g/100mL into the monovalent virus harvesting solution, wherein the volume ratio of the addition amount of the carboxymethyl glucan solution to the monovalent virus harvesting solution is 1: 0.5, the substitution degree of the carboxymethyl glucan is 0.5, then formaldehyde with the final concentration of 100mg/ml is added, and inactivation is carried out for 35 hours at the temperature of 5 ℃;
in the step (7), the concentration of the cracking agent nonoxynol-9 is 0.01%.
Secondly, in the production method of the influenza A3 (H3N2) subunit vaccine monovalent stock solution:
the step (3) is as follows: screening live chick embryos, standing the chick embryos at 5 ℃ for 16 hours to obtain allantoic fluid;
the step (5) is as follows: adding a carboxymethyl glucan solution with the concentration of 3g/100mL into the monovalent virus harvesting solution, wherein the volume ratio of the addition amount of the carboxymethyl glucan solution to the monovalent virus harvesting solution is 1: 0.5, the substitution degree of the carboxymethyl glucan is 0.5, then formaldehyde with the final concentration of 100mg/ml is added, and inactivation is carried out for 45 hours at the temperature of 5 ℃;
thirdly, in the production method of the influenza B (B) type subunit vaccine monovalent stock solution:
the step (3) is as follows: screening live chick embryos, standing the chick embryos at 5 ℃ for 16 hours to obtain allantoic fluid;
the step (5) is as follows: adding a carboxymethyl glucan solution with the concentration of 3g/100mL into the monovalent virus harvesting solution, wherein the volume ratio of the addition amount of the carboxymethyl glucan solution to the monovalent virus harvesting solution is 1: 0.5, the substitution degree of the carboxymethyl glucan is 0.5, then formaldehyde with the final concentration of 100mg/ml is added, and inactivation is carried out for 40 hours at the temperature of 5 ℃;
in the step (7), the concentration of the cracking agent nonoxynol-9 is 0.01%.
Example 3
The procedure is as in example 1, except that:
a production method of influenza A1 (H1N1) subunit vaccine monovalent stock solution comprises the following steps:
the step (3) is as follows: screening live chick embryos, standing the chick embryos at 8 ℃ for 16 hours to obtain allantoic fluid;
the step (5) is as follows: adding a carboxymethyl glucan solution with the concentration of 5g/100mL into the monovalent virus harvesting solution, wherein the volume ratio of the addition amount of the carboxymethyl glucan solution to the monovalent virus harvesting solution is 1: 0.7, the substitution degree of the carboxymethyl glucan is 0.7, then formaldehyde with the final concentration of 150mg/ml is added, and inactivation is carried out for 35 hours at the temperature of 8 ℃;
in the step (7), the concentration of the cracking agent nonoxynol-9 is 2%.
Secondly, in the production method of the influenza A3 (H3N2) subunit vaccine monovalent stock solution:
the step (3) is as follows: screening live chick embryos, standing the chick embryos at 8 ℃ for 16 hours to obtain allantoic fluid;
the step (5) is as follows: adding the mixture into a monovalent virus harvest solution in a volume ratio of 1: 0.3 amount of carboxymethyl dextran solution with a concentration of 3g/100mL is added, wherein the substitution degree of carboxymethyl dextran is 0.7, then formaldehyde with a final concentration of 150mg/mL is added, and inactivation is carried out for 45 hours at 8 ℃;
in the step (7), the concentration of the cracking agent nonoxynol-9 is 2%.
Thirdly, in the production method of the influenza B (B) type subunit vaccine monovalent stock solution:
the step (3) is as follows: screening live chick embryos, standing the chick embryos at 8 ℃ for 16 hours to obtain allantoic fluid;
the step (5) is as follows: adding the mixture into a monovalent virus harvest solution in a volume ratio of 1: 0.3 amount of carboxymethyl dextran solution with a concentration of 3g/100mL is added, wherein the substitution degree of carboxymethyl dextran is 0.7, then formaldehyde with a final concentration of 150mg/mL is added, and inactivation is carried out for 40 hours at 8 ℃;
in the step (7), the concentration of the cracking agent nonoxynol-9 is 2%.
Comparative example
The specific steps are the same as example 1, except that the inactivation process in the production method of the influenza virus subunit vaccine monovalent stock solution is as follows:
the inactivation in the step (5) of the production method of the influenza A1 (H1N1) subunit vaccine monovalent stock solution is as follows: formaldehyde was added to the monovalent virus harvest to a final concentration of 180mg/ml and inactivated at 2 ℃ for 80 hours.
The inactivation in the step (5) of the production method of the influenza A3 (H3N2) subunit vaccine monovalent stock solution is as follows: adding formaldehyde with the final concentration of 180mg/ml into the monovalent virus harvest solution, and inactivating the monovalent virus harvest solution for 100 hours at the temperature of 2 ℃;
the inactivation in the step (5) of the production method of the influenza B (B) virus subunit vaccine monovalent stock solution is as follows: formaldehyde was added to the monovalent virus harvest to a final concentration of 180mg/ml and inactivated at 2 ℃ for 120 hours.
Test example 1 inactivation Process antigenicity test
Virus solutions inactivated by different methods (example 1 and comparative) were diluted with PBS to a hemagglutinin content of 30. mu.g/ml. 10 healthy clean-grade ISR mice of 16-18g are respectively inoculated, the male and the female are half respectively, each abdominal cavity is injected with 0.2ml, and meanwhile, 10 mice are respectively injected with PBS buffer solution as negative control. Blood was collected from the posterior orbit at 21d, serum was separated, nonspecific inhibin was removed by potassium periodate treatment, and antibody titer was measured by hemagglutination inhibition assay, with the results shown in tables 1, 2 and 3.
TABLE 1 type A1 (H1N1) inactivated group antibody titer table
TABLE 2 type A3 (H3N2) inactivation group antibody titer table
TABLE 3 titer of inactivated group B (B) antibody
As can be seen from the above table, the virus liquid prepared by the inactivation method of the invention has better immune effect than the formaldehyde inactivation group.
Test example 2 examination of free Formaldehyde content
The test example investigates the influence of different inactivation methods and the substitution degree of carboxymethyl glucan on the content of free formaldehyde in the obtained trivalent influenza virus subunit vaccine.
Test samples:
test group 1: a trivalent influenza virus subunit vaccine prepared using the method of example 1;
test group 2: a trivalent influenza virus subunit vaccine prepared using the method of example 2;
test group 3: a trivalent influenza virus subunit vaccine prepared using the method of example 3;
control group 1: trivalent influenza virus subunit vaccine prepared by the method of the comparative example;
control group 2: specifically, the procedure was as in example 1, except that the substitution degree of carboxymethyl dextran was 0.4;
control group 3: specifically, the procedure of example 1 was repeated, except that the degree of substitution of carboxymethyl dextran was 0.8.
The test method comprises the following steps: the determination was carried out according to the "determination of free formaldehyde content" method described in "development of purification Process of influenza splitting inactivated vaccine" [ Master academic thesis of university of major associates ].
The results are shown in Table 4:
TABLE 4 examination of free Formaldehyde content
| Sample (I) | Free Formaldehyde content (μ g/ml) |
| Test group 1 | 7.3 |
| Test group 2 | 7.5 |
| Test group 3 | 7.4 |
| Control group 1 | 22.7 |
| Control group 2 | 13.5 |
| Control group 3 | 13.3 |
From the test results, the content of free formaldehyde in the trivalent influenza virus subunit vaccines (test group 1, test group 2 and test group 3) prepared by the method is obviously lower than that of the trivalent influenza virus subunit vaccine (control group 1) prepared by the formaldehyde inactivation method, and the substitution degree of carboxymethyl glucan has certain influence on the content of free formaldehyde in the prepared vaccines. When the substitution degree of the carboxymethyl glucan is 0.5-0.7, the content of free formaldehyde in the prepared trivalent influenza virus subunit vaccines (test group 1, test group 2 and test group 3) is obviously lower than that of the trivalent influenza virus subunit vaccines (control group 2 and control group 3) when the substitution degree of the carboxymethyl glucan is not in the range of 0.5-0.7.
Test example 3 efficacy test
The trivalent influenza virus subunit vaccines prepared in the embodiment 1 and the comparative example are respectively stored at (6 +/-2) DEG C for 18 months, 25 SPF (specific pathogen free) chickens with the age of 4w are taken, 10 SPF chickens with the age of 4w are immunized subcutaneously on the neck of each vaccine, 0.3ml is taken, the embodiment 1 is taken as a test group, the comparative example is taken as a control group, and 5 non-immunized SPF chickens are additionally arranged as a blank control group. The SPF chicken for the test is raised in a negative pressure isolator, 0.5ml of heart blood is collected after 21d and 35d of inoculation respectively, the heart blood is placed in a sterilized small bottle and stands for about 4h at normal temperature, after the blood serum is discharged, HI antibody (log2) of the blood serum is detected by a hemagglutination inhibition test, and a positive blood serum control group and a negative blood serum control group are arranged.
The results are shown in tables 5 and 6:
TABLE 5 detection results of 21d antibody after immunization of each group of vaccines
TABLE 6 detection results of 35d antibody after immunization of each group of vaccines
As can be seen from the results in tables 5 and 6, the test group of example 1 using the inactivation method of the present invention had 3 titers of serum antibodies at 21 days higher than the control group, and the "standard" specified that the antibody level of the vaccine was a good product at 6log2, while the control group of the comparative example using formaldehyde inactivation had apparently failed; the antibody level in the test group was increased at day 35, while the control group maintained the original level substantially unchanged. In this experiment, the antibody data of positive and negative sera were normal, indicating that the experiment was true.
The trivalent influenza virus subunit vaccines prepared in other examples of the present invention were also tested as described above, and similar results were obtained.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A trivalent influenza virus subunit vaccine contains three types of H1N1, H3N2 and B, and is prepared by virus inoculation, virus propagation culture, allantoic fluid harvesting, clarification, inactivation, ultrafiltration concentration, cracking, ultracentrifugation purification, blending, filtration sterilization, subpackaging and packaging, and is characterized in that the inactivation is as follows: firstly, adding a carboxymethyl glucan solution into a monovalent virus harvesting solution, and then adding formaldehyde for inactivation; the substitution degree of the carboxymethyl glucan is 0.5-0.7.
2. A method for preparing the trivalent influenza virus subunit vaccine of claim 1, comprising the steps of virus inoculation, virus propagation culture, allantoic fluid harvest, clarification, inactivation, ultrafiltration concentration, lysis and ultracentrifugation purification, blending, filtration sterilization, subpackaging and packaging, wherein the inactivation is as follows: firstly, adding a carboxymethyl glucan solution into a monovalent virus harvesting solution, and then adding formaldehyde for inactivation; the substitution degree of the carboxymethyl glucan is 0.5-0.7.
3. The method according to claim 2, wherein the ratio of the amount of the carboxymethyl dextran solution added to the volume of the monovalent virus harvest solution is 1: 0.1-0.5.
4. The method according to claim 3, wherein the concentration of the carboxymethyl dextran solution is 1 to 5g/100 mL.
5. The method according to claim 4, wherein the carboxymethyl dextran solution is prepared by dissolving carboxymethyl dextran in water for injection.
6. The method as claimed in claim 5, wherein the final concentration of formaldehyde is 100-150mg/ml, and the inactivation time is 35-45 hours.
7. The process according to any one of claims 2 to 6, wherein the cracking agent used in the cracking is nonoxynol-9.
8. The method of claim 7, wherein the concentration of the lytic agent is 0.01-2%.
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Denomination of invention: A trivalent influenza virus Subunit vaccine and its preparation method Effective date of registration: 20230706 Granted publication date: 20210409 Pledgee: China Everbright Bank Limited by Share Ltd. Tianjin branch Pledgor: ZHONGYI ANKE BIOTECHNOLOGY Co.,Ltd. Registration number: Y2023120000056 |