WO2005037310A1

WO2005037310A1 - Hepatitis a, b-combined vaccine and its lyophilized preparation

Info

Publication number: WO2005037310A1
Application number: PCT/CN2004/000874
Authority: WO
Inventors: Pengfu Wang; Jingye Liu; Guifan Guan; Baosheng Xie; Xuan Luo; Lingjiu Liu; Wange Chen
Original assignee: Changchun Institute Of Biological Products
Priority date: 2003-07-31
Filing date: 2004-07-28
Publication date: 2005-04-28
Also published as: CN1513555A

Abstract

The present invention relates to a hepatitis A, B-combined vaccine and its lyophilized preparation and, particularly to a hepatitis A, B-combined vaccine comprising a properly attenuated live hepatitis A virus and a purified hepatitis B surface antigen produced by a recombinant Chinese Hamster Ovary (CHO) cell and/or recombinant yeast, as well as its lyophilized preparation. The protectant used in the said preparation contains trehalose, glutamic acid, ascorbic acid, urea, sorbitol, inositol and dextran. The inflammation associated with hepatitis A virus and with hepatitis B virus can be prevented simultaneously by use of the hepatitis A, B-combined vaccine of the present invention.

Description

-Hepatitis B combined vaccine and its lyophilized preparation

The present invention relates to a combined hepatitis B and hepatitis B vaccine and its freeze-dried preparation, in particular to a hepatitis B vaccine virus containing live appropriately attenuated hepatitis B virus and purified hepatitis B expressed by recombinant CH0 cells and / or recombinant yeast Hepatitis A-B combined vaccines for surface antigens, and lyophilized preparations thereof. The invention also relates to a method for preparing the lyophilized hepatitis A-B combined vaccine, and a protective agent for the lyophilized hepatitis A-B combined vaccine. Background of the invention

Hepatitis A is a global acute infectious disease caused by the hepatitis A virus (HAV), which is widespread in nature. Because of the prevalence and rapid spread of the hepatitis A epidemic, the prevalence of hepatitis A has become a serious public health problem. Especially in developing countries including China, due to the large population and backward socio-economic and public health conditions, large-scale outbreaks and local epidemics of hepatitis A often occur. Even in the highly developed United States, every year There are also about tens of thousands of patients with hepatitis associated with hepatitis A virus infection.

Hepatitis B is an infectious disease caused by the hepatitis B virus ^. Hepatitis B has a high morbidity and mortality rate in developing countries and generally has a poor prognosis. To date, there is no effective way to treat hepatitis B. In China, carriers of hepatitis B virus account for about 8-10 of the total population, and as many as 1 million people have chronic hepatitis.

Due to the serious damage and high prevalence of Hepatitis A and Hepatitis B, the incidence of these two types of hepatitis in many countries in the world has always accounted for the highest incidence of all viruses' 1. Hepatitis, 70% -80 ° / . about. Facing the severe epidemic situation of hepatitis B virus, China has taken the lead in successfully developing a live attenuated hepatitis B vaccine. In particular, in order to solve the problem of the source of raw materials for the production of hepatitis B vaccine and possible infection with other pathogenic factors, in recent years, the development of blood-borne hepatitis B vaccine On this basis, a genetically engineered hepatitis B vaccine based on hepatitis B surface antigen (HBsAg) was further developed.

Since the launch of the Children's Vaccine Program (CVI) in 1991 by international organizations such as the United Nations Children's Fund and the World Health Organization, many laboratories and companies in the world engaged in vaccine research and development have been working on new vaccines. These new vaccines should be multivalent, multivalent, thermostable, and can be administered orally or by aerosol. From the perspective of clinical application, by using a combined vaccine, two or more diseases can be effectively prevented with one immunization, so as to reduce the cost of vaccine production, improve the efficiency of vaccination, and reduce the adverse reactions caused by multiple injections. The main development direction of vaccine research.

In recent years, researchers have made many beneficial attempts in developing multivalent and multivalent vaccines. As far as hepatitis vaccines are concerned, for example, European Patent No. 0339 667 discloses an A-B containing inactivated hepatitis A virus antigen and hepatitis B virus surface antigen and adjuvant, which can simultaneously prevent hepatitis A and hepatitis B Hepatitis Vaccine. In addition, U.S. Patent No. 6,451,320 provides a combination vaccine composition suitable for adolescent vaccination. The combined vaccine composition includes a hepatitis B virus surface antigen and a herpes simplex virus antigen, and one or more viral antigens selected from the hepatitis B virus antigen (HAV) and the EB virus (EBV) antigen, and the HAV is The inactivation originated from the Re-175 virus strain. However, the hepatitis A virus antigens used in these existing combination vaccines are all inactivated HAV viruses.

To date, there have been no reports of the production of a combined hepatitis A-B vaccine (adjuvant) vaccine and its lyophilized preparation using live attenuated hepatitis A vaccine viruses. Summary of the invention

It is an object of the present invention to provide a 曱 -Hepatitis B combined vaccine composition comprising a live attenuated hepatitis A vaccine virus and a hepatitis B virus surface antigen (HBsAg).

It is another object of the present invention to provide a freeze-dried form of a cricket-hepatitis B combined vaccine composition, including live attenuated cricket-hepatitis vaccine virus, hepatitis B virus surface antigen (HBsAg) and protective agent.

Yet another object of the present invention is to provide a method for preparing a freeze-dried form of a combined hepatitis A-B vaccine composition.

Other objects of the invention are found in the description of the invention herein.

The present invention relates to a combined hepatitis A and hepatitis B vaccine, and more particularly to a freeze-dried combined hepatitis A-B vaccine composition containing live hepatitis A vaccine virus and hepatitis B surface antigen protein and a protective agent. The invention further relates to a method for preparing said combination vaccine composition.

According to the present invention, a combined rubidium-hepatitis B vaccine contains live attenuated hepatitis A vaccine virus and hepatitis B surface antigen protein.

The live attenuated hepatitis A vaccine virus used in the present invention may be a known live attenuated hepatitis A vaccine virus. For example, a suitable attenuated hepatitis A vaccine that can be obtained from the stool of a naturally infected hepatitis A patient and obtained through serial dilutions of terminal attenuation, "cloning," purification, or other artificial mutagenesis methods can be used As a seed virus, a cell matrix that is sensitive to the hepatitis A vaccine virus, such as an established human embryo lung diploid cell, is used as the cell matrix according to the method described in detail in Chinese Patent No. 921 14998. 0. Invented live attenuated hepatitis A vaccine virus stock.

The vaccine stock solution prepared from the live attenuated hepatitis A vaccine L-A-1 vaccine strain is preferably used.

The hepatitis B surface antigen protein used in the present invention may be a known purified hepatitis B virus surface antigen protein. For example, appropriate expression systems can be constructed and selected, such as the expression of hepatitis B virus surface antigen proteins in prokaryotic, eukaryotic, or yeast hosts. For example, the HBsAg gene can be isolated from HBsAg-positive and hepatitis B e-antigen-negative sera of blood donors and cloned into an appropriate plasmid vector such as pBR 322 or a derivative thereof, and then the appropriate recombinant vector is used to transform the appropriate Host cells, such as Chinese hamster ovary (CH0) cells and / or yeast, and culture the recombinant CH0 cells and / or yeast under conditions suitable for expressing HBsAg protein, and then harvest from the recombinant cell culture medium and / or yeast and The desired HBsAg protein was purified. The preparation of hepatitis B surface antigen can be prepared in accordance with the "Manufacturing and Assay of Recombinant (CH0 Cells) Hepatitis B Vaccine" in the "Chinese Biological Products Regulations" of the 2000 edition.

Alternatively, density gradient centrifugation or various ion exchange chromatography methods can be used to isolate and purify the blood-borne hepatitis B surface antigen protein from the serum of HBsAg-positive virus carriers.

According to a preferred embodiment of the combined vaccine composition of the present invention, the live attenuated hepatitis A vaccine virus is wild-type hepatitis A virus isolated from the stool of a naturally infected hepatitis A patient. Hepatitis A LA-1 strain purified by terminal dilution "clone" purified and passaged attenuated in appropriate host cells.

According to a preferred embodiment of the vaccine composition of the present invention, the virus titer of the live attenuated hepatitis B vaccine virus contained in the combination vaccine is not less than 6.5 LogCCID50 / ml.

According to a preferred embodiment of the vaccine composition of the present invention, wherein the hepatitis B virus surface antigen (HBsAg) is obtained from recombinant CH0 cells and / or recombinant yeast and purified.

A preferred embodiment of the vaccine composition according to the present invention is characterized in that the protein content of the hepatitis B virus surface antigen (HBsAg) in the combination vaccine is not less than 10 g / ml.

The preferred ratio range of the attenuated hepatitis B vaccine virus and hepatitis B surface antigen protein in the combined vaccine is: 6. 5-7. 0LogCCID50 / ml to 12-14 g / ml.

In order to facilitate the transportation, storage, and use of the combined vaccine product of the present invention, to reduce the so-called "cold chain" pressure in these processes, and to ensure the effect of vaccination, the inventors successfully developed a lyophilized protective agent or stabilizer. This lyophilized protective agent or stabilizer can completely guarantee that the liquid combination vaccine product will not be inactivated during the freeze-drying process and during the transportation, storage and use after lyophilization.

Therefore, in the present invention, a freeze-dried tadpole-hepatitis B combined vaccine contains a live attenuated tadpole hepatitis virus and hepatitis B surface antigen protein and a protective agent.

The protective agent used in the combination vaccine of the present invention contains trehalose, glutamic acid, ascorbic acid, urea, sorbitol, inositol, and dextran.

Dextran can be high, medium and low molecular weight dextran, such as dextran 70, 40, 20; Low molecular dextran, such as dextran 20 is preferred.

Before freeze-drying, their contents (W / V) in the combined vaccine composition are: trehalose 3-10%, glutamic acid 0.5-1, 0%, ascorbic acid 0.1-0.3%, Urea 0.5-2.0%, sorbitol 0.5-2.0 ° / », inositol 0.5-1.0%, dextran 1-6%.

According to a preferred embodiment of the vaccine composition according to the invention, the protective agent consists essentially of trehalose, glutamic acid, ascorbic acid, urea, sorbitol, inositol and dextran.

The 曱 -Hepatitis B combined vaccine of the present invention and its freeze-dried product may further contain one or more adjuvants for improving its immunogenicity without causing any side effects. These adjuvants are generally known to those skilled in the art, and include, but are not limited to, aluminum hydroxide or aluminum phosphate gel, 3D-MPL (a ThI cell response stimulant), and the like (see Pharmaceutical Biotechnology, Vol. 61 Vaccine Des ign- the subuni t and adjuvant approch, Powel l and Newman, Plenum Press).

The preferred immune adjuvant of the present invention is an aluminum hydroxide solution.

According to a preferred embodiment of the vaccine composition of the present invention, it may further contain an appropriate amount of an immunological adjuvant, for example, 0.8-1.2 mg / ml aluminum hydroxide solution.

According to a preferred embodiment of the vaccine composition of the present invention, wherein the adjuvant is provided by a 0.8 to 1.2 mg / ml aluminum hydroxide solution contained in a lyophilized 曱 hepatitis B combined vaccine dilution.

According to the present invention, a method for producing a freeze-dried radon-hepatitis B combined vaccine, the method includes:

(1) Separately provided and mixed at appropriate ratios containing attenuated hepatitis B vaccine virus stock solutions and protein stock solutions expressed by recombinant CH0 cells and / or recombinant yeast and purified hepatitis B virus surface antigen (HBsAg) To obtain a mixed suspension of hepatitis A virus and hepatitis B virus surface antigens;

(2) adding an appropriate amount of a protective agent to the mixed suspension obtained in step (1) and mixing them to obtain the required hepatitis A and hepatitis B combined vaccine composition suspension; (3) The combination vaccine suspension containing the protective agent obtained in step (2) is lyophilized. According to a preferred embodiment of the method of the present invention, wherein the components of the protective agent and their concentration (W / V) in the suspension of the combined vaccine composition are trehalose 3-10%, glutamic acid 0. 5-1. 0 %, Ascorbic acid 0.1-0.3 ° / », urea 0.5-2.0% sorbitol 0.5-2. 0¾, inositol 0.5-1. 0% and dextran 1-6%.

According to a preferred embodiment of the method of the present invention, wherein said method further comprises, before use, the freeze-dried combination vaccine is diluted with the aluminum hydroxide solution to obtain the lyophilized combination vaccine composition obtained in step (3), so that the live content is reduced. The virus titer of the hepatitis B vaccine virus is not less than 6.5 LogCCID ₅ o / ml, the protein content of the hepatitis B virus surface antigen (HBsAg) is not less than 10 μg / ml, and the concentration of aluminum hydroxide is 0.8-1.2 mg / ml.

According to a preferred embodiment of the method of the present invention, wherein the adjuvant is provided by a solution of aluminum hydroxide contained in a lyophilized hepatitis A-B vaccine dilution. The animal and monkey body experimental observation results show that the combined vaccine composition of the present invention not only has the same good safety as its monovalent vaccine, but also has better immunogenicity than its monovalent vaccine. On the other hand, since the combined vaccine composition of the present invention contains a freeze-dried protective agent that can effectively protect hepatitis B vaccine virus and hepatitis B indicating the biological activity of the antigen protein, it can be made into a freeze-dried combined vaccine Preparations, and thus eliminates the "cold chain" stress during vaccine production, transportation, storage and use, which can more effectively ensure the vaccine vaccination effect. In particular, the original live hepatitis A vaccine virus, Therefore, the production cost and market price of the combined vaccine composition are greatly reduced, thereby facilitating its promotion and universal use in developing countries including China.

In order to prepare the lyophilized protective agent of the combined vaccine of the present invention, for example, the following ingredients may be first dissolved in 37 ° C distilled water (1000ml) at the indicated concentrations: sodium glutamate 100. 0 g / L, ascorbic acid 20. 0 g / L, urea 80.0 g / L, sorbitol 80.0 g / L, and inositol 80.0 g / L, combined with 0.1N HCL adjusts the pH to 7.0. The protective agent component A was obtained after filtering and sterilizing through a 0.22 μm filter membrane. Then, 500. 0 g of trehalose was weighed and dissolved in water for injection at a temperature of about 37 ° C to make up to 1000 ml, and the protective agent component B was obtained after filtering and sterilizing through a 0.22 μm filter membrane. Finally, 300. 0 g of low-molecular-weight dextrose was dissolved in water for injection at a temperature of about 37 ° C to 1000 ml, and the protective agent component C was obtained after autoclaving at 116 ° C for 40 minutes. During use, an appropriate volume of the protective agent components A, B, and C can be added to the above-mentioned mixed suspension of the hepatitis A vaccine stock solution and the hepatitis B vaccine stock solution, so that each component of the protection agent reaches the concentration range as defined above (see Example 2).

However, in the freeze-dried combination vaccine composition of the present invention, the adjuvant is not added during the preparation of the vaccine, but is used as a solubilizer and diluent for the freeze-dried product, and is added just before the freeze-dried combination vaccine is used. The content of the added aluminum hydroxide adjuvant is generally limited. When added as a solubilizer and diluent for lyophilized products, it can make hepatitis A vaccine virus and hepatitis B surface antigen protein immunogens in the combined vaccine Sex has increased. Generally speaking, the content of adjuvant aluminum hydroxide in the lyophilized tritium-hepatitis B combined vaccine ranges from about 0.8 to 1.2 mg / ml.

The combined vaccine for hepatitis B and hepatitis B of the present invention can be prepared by a simple mechanical mixing method. For example, firstly, according to an appropriate volume ratio (such as approximately 20: 1 (V / V)), the pre-prepared viral titer of the hepatitis A vaccine stock solution with a log concentration of greater than 7.0 Log CCID ₅₀ / ml and the pre-prepared protein concentration is The hepatitis B virus surface antigen stock solution greater than 100 g / ml is mixed, and then an appropriate amount of a pre-made lyophilized protective agent is added and gently mixed.

Then, the 199 comprehensive culture solution used as a vaccine diluent is diluted to the required content or concentration of each component in the combined vaccine to obtain a combined vaccine composition in the form of a suspension.

Finally, the above-mentioned mixed suspension can be freeze-dried using a conventional freeze-drying device. The freeze-drying conditions used are:-30 ° C to-55 Ό Pre-freeze at low temperature for 3-7 hours, and then at-30 Vacuum dry at ° C to 35 ° C for 8 to 16 hours. During lyophilization, the eutectic temperature of the vaccine lyophilized mixture was below -38 ° C.

Compared with before lyophilization, the lyophilized hepatitis B vaccine virus titer decreased slightly after lyophilization, but the decrease was not greater than about 0.5 LogCCID50 / ml. The lyophilized vaccine product thus obtained can be used at room temperature. It can be stored for a long period of time to 6 months without affecting the viral titer of the hepatitis A vaccine virus and the relative efficacy of the hepatitis B virus surface antigen (using the hepatitis B vaccine expressed by recombinant CH0 cells as a reference vaccine).

Before clinical application, the lyophilized combination vaccine obtained as above can be dissolved and diluted by using an aluminum hydroxide solution with a content of 0.8-1.2oig / ml to prepare the combination vaccine of the present invention in the form of a suspension. The aluminum hydroxide aqueous solution can be used not only as a solubilizing agent and diluent for a lyophilized combination vaccine, but also to provide an adjuvant with an immune enhancing effect.

A large number of experimental studies and vaccination experiments on a variety of animals including rhesus monkeys and red-faced monkeys have confirmed that the combined vaccine of the present invention, as a new pharmaceutical composition, can be effectively used to prevent hepatitis A and hepatitis B It will not interfere with each other due to the mixing of the two viral antigens, nor will it cause any immune response suppression or other adverse reactions. The results of safety experiments showed that all primates (rhesus monkeys or red-faced monkeys) inoculated with the combined vaccine of the present invention did not show an increase in serum aminotransferase (ALT) from 0-12 weeks after vaccination. Liver tissues of animals The pathological examination showed that no abnormal pathological changes of liver tissue were seen in all the tested animals (see Example 3).

Experimental data also show that the combined vaccine of the present invention appears to provide better immunogenicity than its monovalent vaccine compared to the live attenuated hepatitis B vaccine and / or hepatitis B vaccine. In addition, because the combination vaccine composition of the present invention contains effective protective or stabilizing ingredients, the hepatitis A vaccine virus titer and hepatitis B vaccine are during the freeze-drying process of the vaccine or during long-term storage and transportation after freeze-drying. The relative effectiveness did not decrease significantly.

The test results of the two components of the pre-prepared combination vaccine showed that the titer of hepatitis V vaccine was not less than 6.5 LogCCID50 / ml, and the content of HBsAg antigen protein was not less than 10 μg / ml.

The results of the combined vaccine immunogenicity test showed that when the rhesus or red-faced monkey was vaccinated with the combined vaccine for 4 weeks, the positive rate of anti-HAV antibody in the animal serum reached 80-100%. At the same time, the positive rate of anti-HBsAg antibody in animal serum was about 20%; the positive rate of anti-HBsAg antibody at 8 weeks was 40-80%; and the positive rate of anti-HBsAg antibody after ² weeks of inoculation Can reach 80-100%, and Further comparison experiments show that compared with the monovalent vaccine, the primate (rhesus or red-faced monkey) vaccinated with the lyophilized hepatitis A-B hepatitis combined vaccine of the present invention has the same good safety and security as the monovalent vaccine. Seems better immunogenic (see Example 4-①).

Cellular immunological experiments confirmed that after inoculating BALB / C pure line mice with the combined vaccine composition of the present invention, they were tested for lymphokine surface markers, natural killer (NK) cell activity, and lymphokine-activated killer (LAK) cell activity. The results of detection, detection of macrophage phagocytosis, and detection of some cytokines (such as: IFN-γ, IL- ² , IL-18) showed that the animals produced significant cell-mediated immune responses. In addition, further comparison experiments showed that Compared with the monovalent hepatitis A vaccine and hepatitis B vaccine, the combined hepatitis A-B vaccine of the present invention can produce a better cellular immune-mediated response (see Example 4 -②).

Further experimental results on storage stability of the combined vaccine indicate that the lyophilized combination vaccine of the present invention can be stored at 2-8 ° C and room temperature (25 ° C) for more than 18 and 5 months, respectively. The hepatitis A vaccine virus titer and The relative efficacy of the hepatitis B vaccine is stable (see Example 5).

The following examples are intended to further illustrate, but not limit the invention. Those skilled in the art can understand that, without departing from the spirit and principles of the present invention, any parallel changes and modifications to the present invention will fall within the scope of the claims of the present invention. Example 1: Preparation of hepatitis B vaccine virus stock solution

First, human embryo lung diploid cells were expanded and cultured in a basic medium (MEM) (pH 7. 2-7. 6) supplemented with 10-15% calf serum. After the cells formed a dense monolayer, the growth medium was discarded, and the cells were repeatedly washed with fresh Ear le's solution, and then the seed virus solution prepared according to the method described in Example 1 of Chinese Patent No. 92114998. 0 was inoculated. After 34 ° C-36 ° C adsorption for 3-4 hours, supplement the cell growth maintenance solution (lactalbumin hydrolysate with vitamin C) to the culture and incubate at 34 ° C-36 ° C for about 4 weeks. Change the fluid once a week. After the cultivation, the maintenance solution and the remaining calf serum were discarded, and the 199 comprehensive culture medium containing low-concentration salt (for example, 1-1 OmM NaCl) and no phenol red was directly added and continued to culture (34 ° C-36 ° C) To 4 days. Cells were then collected and centrifuged to collect cells after repeated freeze-thaw and sonication. The supernatant was collected to obtain the hepatitis B vaccine virus stock solution. Example 2: Preparation of a freeze-dried hepatitis A-B combined vaccine

A 2.0 L stock vaccine virus (L-A-1. Strain with a virus titer of 8.0 LogCCID50 / ml) and 72 ml of recombinant type B expressed from recombinant CH0 cells and / or recombinant yeast were used. Hepatitis virus surface antigen protein stock solution (protein content: 1334 g / ml) was mixed, and then a protective agent stock solution prepared as follows: a protective agent component A550 ml, a protective agent component B 700 ml, and a protective agent component C 450 ml.

Then, an appropriate amount of the 199 comprehensive culture liquid as the virus vaccine matrix liquid was added to the thus obtained mixed suspension, so that the total volume of the liquid combination vaccine reached 8000 ml, and the titer of the hepatitis 疫苗 vaccine virus was not less than 6, 5 Log CCID50 / ml, hepatitis B virus surface antigen protein content is not less than 10μ _§ / ιη1. The result of such dilution also led to the concentration (W / V) of each component of the protective agent added to the vaccine suspension reaching trehalose 3-10%, glutamic acid 0.5-1. 0%, ascorbic acid 0.1- 0.3%, urea 0.5-2.0%, sorbitol 0.5-2. 0 ° /. , Inositol 0. 5-1. 0% and dextran 1-6%.

0 g In order to prepare a lyophilized protective agent stock solution, the following ingredients can be first dissolved in distilled water (1000ml) at the indicated concentrations: sodium glutamate 100. 0g / L, ascorbic acid 20. 0 g / L, urea 80. 0 g 0。 L, sorbitol 80.0 g / L and inositol 80. 0 g / L, and adjusted the pH value to 7.0 with 0.1N HCL. The protective agent component A was obtained after filtering and sterilizing through a 0.22 μm filter membrane. Then weighed 5.00g of trehalose and dissolved in water for injection at a temperature of about 37 ° C to make up to 1000ml, and filtered through a 0.22 μm filter to sterilize to obtain the protective agent component B. Finally, 30.0 g of dextran 20 was dissolved in water for injection at a temperature of about 37 ° C to 1000 ml, and the protective agent component C was obtained after autoclaving at 116 ° C for 40 minutes.

Dispense the combined vaccine suspension at a volume of 1 ml per ampoule (or vial), and place the ampoule in a closed vacuum freeze dryer (FS150-SS20C, Hull Co., USA), as follows Conditional freeze-drying: First, pre-freeze the vaccine suspension at about -40 ° C for 4 hours, and then gradually raise the temperature to 32 ° C and vacuum-dry for about 15 hours, so as to obtain the required freeze-dried pupal-Hepatitis B vaccine. According to the requirements of the World Health Organization (WHO) and the State Drug Administration (SDA) for the preparation and inspection of joint vaccines and freeze-dried preparations and quality standards, a comprehensive inspection of the combined vaccine provided by the present invention has been achieved. Or it exceeds the requirements of the manufacturing inspection regulations stipulated by the World Health Organization (WHO) and the State Drug Administration (SDA). The titer of hepatitis A vaccine virus is not less than 6.5 LogCCID50 / ml, and the protein content of hepatitis B surface antigen (HBsAg) is not less than 1 (g / ml.) Example 3: Lyophilized hepatitis A-B Safety trials of combined vaccines

Thirty-five healthy red-faced monkeys or rhesus monkeys (average weight 1. 5-4. 5 Kg) with negative anti-HAV antibodies and anti-HBV antibodies and normal ALT were selected and randomly divided into 7 groups, 5 in each group animal. On the day of the experiment, each animal of the 5 experimental groups was injected with different batches of the lyophilized combination vaccine of the present invention by injection of 1ml each (hepatitis A virus titer of 6. 5-7. 0 LogCCID50 / ml, hepatitis B The virus indicates that the content of antigen protein is 10-12 g / ml, and its relative potency is 2.45-3.06.) The animals of the two control groups received the same dose of attenuated live hepatitis A virus vaccine (lyophilized preparation) and recombinant Hepatitis B vaccine expressed in CH0 cells.

The venous blood of the immunized animals was collected at 0, 4, 8 and 12 weeks after vaccination, and the serum was separated and the serum alanine aminotransferase (SGPT) or (ALT) was detected by conventional biochemical detection and enzyme-linked immunosorbent assay, respectively. Levels, and the levels of anti-HAV and anti-HBsAg antibodies, and the relative efficacy of the hepatitis B vaccine (the ratio of the ED50 of the test vaccine to the ED50 of the reference vaccine). The anti-HAV detection kit is used to detect the anti-HAV antibody by an enzyme-linked immunosorbent assay (ELISA); the anti-HSsAg reagent is used to detect the anti-HBsAg antibody by the radioimmunoassay (RIA). Simultaneously with the blood collection, the liver tissues of the test animals were collected by liver puncture and liver pathological examination was performed. The results are shown in Listing 1 below. Table 1 曱 -Hepatitis B combined vaccination monkey body safety test vaccine 曱 Liver virus titer Hepatitis B serum SGPT abnormalities (weeks) Hepatic tissue pathological abnormalities (weeks) Lot No. LogCCID ₅ . / ml Relative potency ^ ~~ 2 ~~ 4 ~~ 8 ~~ 12 ~ 0 4 8 12

980401 6. 50 2. 79 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

980 402 6. 67 2. 58 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

980403 6. 67 2. 88 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

980501 6. 67 3. 06 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

980502 7. 00 2. 45 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

HA980501 6. 67 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Grind 80401 2. 55 0/5 0/5 0/5 0/5 0 / 5 0/5 0/5 0/5 0/5

HA: lyophilized live attenuated hepatitis A virus vaccine; HB: recombinant CHO vaccine expressed in CH0 cells. As can be seen from the results shown in Table 1, after monkeys were vaccinated with the lyophilized cricket-hepatitis B combined vaccine and control group No abnormal elevation of serum liver enzymes occurred. Liver biopsy also revealed no abnormal pathological changes in liver tissue in all test animals. It has been shown that the freeze-dried 曱 hepatitis B combined vaccine has the same good safety as its monovalent vaccine. Example 4: Lyophilized hepatitis A-B vaccine combination immunogenicity test

① Vaccine-induced humoral immune response

Thirty-five healthy rhesus monkeys (average body weight 1.5-4.5 Kg) with negative HAV antibody and HBV antibody and normal ALT were selected. They were randomly divided into 柒 groups with 5 animals in each group. On the day of the experiment, each animal of the 5 experimental groups received different batches of the lyophilized hepatitis A-B vaccine of the present invention through the intravenous route, each 1ml (曱 liver virus titer 6. 5-7. 0 LogCCID ₅₀ / ml, hepatitis B virus showed that the antigen protein content was 10-12 g / ml, and its relative potency was 2.45-3. 06), and the other two control animals received the same dose of attenuated atrium Live liver virus vaccine (lyophilized preparation) or hepatitis B vaccine expressed by recombinant CH0 cells.

Venous blood was collected from immunized animals at 0, 4, 8 and 12 weeks after vaccination, and serum was isolated Animals were tested for serum anti-HAV and anti-HBsAg antibody levels, and HAV-IgM antibody titers (as indicators of primary infection after vaccination) using conventional enzyme-linked immunosorbent assays. The results are shown in Listings 2, 3, 4, and 5 below.

Immunogenicity test of monkeys vaccinated with freeze-dried crickets and hepatitis B vaccine (1)

980401 6. 50 2. 79 5 0/5 3/5 5/5 5/5 0/5 4/5 3/5 0/5

980402 6. 67 2. 58 5 0/5 5/5 5/5 5/5 0/5 5/5 3/5 0/5

980403 6. 67 2. 88 5 0/5 4/5 4/5 5/5 0/5 5/5 2/5 0/5

980501 6. 67 3. 06 5 0/5 2/5 5/5 5/5 0/5 4/5 3/5 1/5

980502 7. 00 2. 45 5 0/5 3/5 4/5 5/5 0/5 3/5 2/5 1/5

HA980501 6. 67 5 0/5 3/5 5/5 5/5 0/5 4/5 2/5 0/5

HA: lyophilized live attenuated hepatitis A virus vaccine

Monkey freeze-dried hepatitis A-B combined vaccination monkey body immunogenicity test (2) vaccine titer hepatitis virus titer hepatitis B test anti-HBS positive (W) batch number LogCCIDso / ml relative number of animals 0 4 8 12 16

980401 6. 50 2. 79 5 0/5 0/5 2/5 5/5 5/5

980402 6. 67 2. 58 5 0/5 1/5 3/5 4/5 4/5

980403 6. 67 2. 88 5 0/5 1/5 4/5 5/5 5/5

980501 6. 67 3. 06 5 0/5 1/5 3/5 5/5 5/5

980502 7. 00 2. 45 5 0/5 1/5 3/5 4/5 4/5

HB980401 2. 55 5 0/5 1/5 3/5 4/5 4/5 Hepatitis B vaccine expressed by recombinant CH0 cells Detection of Hepatitis A Antibody Levels in Monkeys After Freeze-dried Hepatitis B Combined Vaccination

― HAV titer HB antibody level (m IU / ml)

Sample number LogCCID ₅ . / ml relative potency animal (4W) (8W) (12W) (16W)

321 1409 11008 69136 12769

322 389 6422 80589 46978

980401 6. 50 2. 79 323 1215 4984 70055 74418

324 1586 6962 15958 10224

325 1451 6336 14725 44275

(X ± SD) 1089. 13 + 1. 79 6891. 30 + 1. 34 39 128. 26 ± 2. 36 28913. 57:

436 1389 1447 71535 12438

437 472 1489 69572 41287

980402 6. 67 2. 58 438 1441 1375 80109 49078

439 1511 1491 15123 57325

440 1312 1583 12263 10115

(X ± SD) 1133. 71 + 1. 64 1475. 45 ± 1. 05 37477. 78 + 2. 53 27098. 93:

326 1309 1479 15416 11098

327 1538 1592 13817 12495

HA980501 6. 67 328 415 1319 15958 49907

329 1375 1563 6751 58732

330 1319 1601 7059 43285

(X soil SD) 1086. 67 ± 1. 72 1506. 96 ± 1. 08 11012. 73 ± 1. 54 28123. 6 +

HA980501 is a live monovalent freeze-dried hepatitis A attenuated live vaccine Detection of anti-HBs levels in monkeys vaccinated with freeze-dried hepatitis A-B vaccine

HAV titer Hepatitis B and Hepatitis B anti-HBS ¾¾ degree (mlU / ml) (week) Sample No. LogCCID ₅ . / ml relative potency

Animal No. 8 12 16

321 0 652. 45 286. 68

322 341. 73 3 586. 68 4931. 91

980401 6. 50 2. 79 323 67. 63 785. 54 3346. 65

324 158. 45 2 038. 51 1307. 63

325 0 1 156. 66 2037. 75

(X soil SD) 1340. 2 3 ± 2. 02 1660. 09 + 3. 01 o o

436 163. 56 3143. Bu 86 2193. 86

437 0 845. 65 1198. 73

980402 6. 67 2. 58 438 186. 70 2104. 45 3586. 68

439 61. 85 584. 36 797. 45

440 0 0 0

(X soil SD) 1344. 05 ± 2. 19 1656. 08 ± 1.94

331 98. 75 3296. 71 1758. 70

332 65. 56

HB980401 2. 55 333 152. 41 2345. 84 3346. 15

334 0 714. 58 652. 68

335 0 0 0

(X soil SD) 134 0. 44 ± 2. 36 1657. 55 ± 1. 98

HB90401 is a hepatitis B vaccine expressed by recombinant CHO cells From the results shown in Tables 2, 3, 4 and 5, it can be seen that the primate (rhesus or red-faced monkey) was vaccinated with different batches of the 曱 -Hepatitis B combined vaccine of the present invention at the 4th week. Animal serum anti-HAV antibody positive conversion rate was 80%-100%, and reached 100% after 8 weeks. The positive rate of anti-HBs antibody in animal serum was 20% 4 weeks after the combined vaccine; 40-80% at 8 weeks, and 80-100% at 12 weeks. There was no significant difference in the antibody levels of the combined vaccine compared with its monovalent liver vaccine and hepatitis B vaccine (P> 0.05). These results indicate that the combination vaccine of the present invention has the same good immunogenicity as its monovalent hepatitis A and B vaccines. In addition, after the animals were vaccinated with the combined vaccine, the serum anti-hepatitis virus IgM antibodies, which are indications of primary hepatitis A infection, also turned positive at 2-4 weeks after vaccination, and gradually disappeared after 8 weeks (Table 2) .

② Vaccine-induced cellular immune response

A Test animal selection

It was selected from 40 pure BALB / C female mice of 6-8 weeks old (provided by the Animal Center of Changchun Institute of Health) and randomly divided into four groups of 10 animals each: a blank control group, a liver vaccine group, and a hepatitis B vaccine group , A

-Hepatitis B combined vaccine group. Animals in each experimental group were injected intraperitoneally with 0.5 ml of the corresponding vaccine, while animals in the blank control group were injected with the same volume of diluted aluminum hydroxide (concentration: 1.0 mg / ml).

B lymphocyte surface marker detection

The animals were sacrificed 10 days after inoculation, and the spleens of each group of animals were aseptically removed and single cell suspensions were prepared. The resulting cell suspension was then dispensed into test tubes at a volume of 1 ml per tube. Anti-CD3, anti-CD4 and anti-CD8 monoclonal antibodies (0.1ml each) combined with fluorescein isothiocyanate (FITC) were added to each tube. Then use a flow cytometer (Becton Dickinson, USA) for flow cytometry. The results are shown in Table 6 below, where the values are expressed as the mean soil standard deviation of each group of animals. Detection of T lymphocyte surface markers in immunized animals

CD 3+ (%) CD4 + (%) CD8 + (%) CD4 + / CD8 + (%)

①Control group 35. 44 ± 6. 47 16. 19 ± 3. 62 8. 90 ± 0. 03 1. 81 ± 0. 04

② Hepatitis A vaccine group 47. 46 + 5. 60 25. 86 ± 2. 85 9. 24 ± 0. 75 2. 80 ± 0. 01

③ Hepatitis B vaccine group 47. 23 ± 4. 25 26. 91 ± 0. 41 9. 56 ± 0. 21 2. 81 soil 0.03

④ Combination vaccine group 50. 66 ± 7. 77 32. 74 soil 6. 85 9. 72 ± 0. 43 3. 41 ± 0. 15

From the results shown in Table 6, it can be seen that the overall CD3 + of the animals in each test group was significantly increased compared with the control group (P <0.05). The CD4 + in the experimental groups was also significantly higher than that in the control group, and the CD4 + in the combined vaccine group was higher than that in the monovalent vaccine group (P <0.05). Furthermore, it can be seen that the CD4 + / CD8 + values of the animals in the combined vaccine group were significantly higher than those in the monovalent vaccine group and the blank control group (P <0.05). These results indicate that the degree of activation of immune cells in vivo after animals are vaccinated with the combination vaccine of the present invention is significantly higher than that of animals vaccinated with a monovalent hepatitis A or hepatitis B vaccine.

C Natural killer (NK) cell activity test

The animals were sacrificed 10 days after inoculation, and the spleens of the group animals were aseptically removed and single cell suspensions were prepared. The resulting cell suspension was then dispensed into test tubes at a volume of 1 ml per tube. Anti-mouse Nl.1 monoclonal antibody (0.1 ml each) combined with PE (stearylethanolamine) was added to each tube. Flow cytometry (Becton Dickin son, USA) was then used to perform flow cytometry. The results are shown in Table 7 below, where the values are expressed as the mean ± standard deviation of each group of animals.

Detection of D lymphokine-activated killer (LAK) cell activity Interleukin 2 (IL-2) was added to the suspension of spleen lymphocytes of the immunized mice at a final concentration of 15 μl / ml, and the required concentration was obtained after incubation at 37 ° C and 5% C0 ₂ for 72 hours. Lymphokine activated killer (LAK) cells. In the experiment, H ³ -TdR-labeled K562 cells were used as target cells, and the two cells were mixed in a ratio of 100: 1 effector cell: target cell. The mixed cell suspension was incubated at 37 ° C and 5% CO ₂ for 8 hours. Then, the cells were sufficiently washed and collected by centrifugation, and the radioactivity (cpm value) of the cells in each group was measured using a gamma counter. The results are shown in Table 7 below, where all values are expressed as the mean ± standard deviation of each group of animals.

E peritoneal macrophage swallowing p complex function assay

Before the experiment, all immunized mice were injected intraperitoneally with a 2% glycogen aqueous solution. After 24 hours, 5% chicken red blood cell suspension was injected intraperitoneally with 1 ml each. Animals were sacrificed after 30 minutes, and peritoneal macrophages were collected and smeared. The resulting cell smear was then incubated at 37 ° F for 30 minutes. After the cells were sufficiently washed with phosphate buffered saline (PBS) and air-dried, the cells were stained with Giemsa, and then the number of peritoneal macrophages of mice that had phagocytosed chicken red blood cells was observed and counted under a light microscope to calculate the percentage of phagocytosis. The results are shown in Table 7 below, where all values are expressed as the mean ± SD of the animals in each group. NK activity, LAK activity, and M φ phagocytic activity in immunized animals

N activity (%) LAK activity (W M φ phagocytic activity

①Control group 4. 46 ± 0.51 33. 5 + 5. 60 28. 58 ± 3. 06

② Liver vaccine group 11. 80 ± 0.71 64. 10 ± 6. 38 50. 62 ± 2. 67

③ Hepatitis B vaccine group 8. 79 soil 1. 21 63. 98 ± 4. 71 48. 20 ± 1. 38

④ Combined vaccine group 12. 20 ± 1. 69 66. 60 + 7. 42 56. 64 ± 19. 13

From the results shown in Table 7, it can be seen that the NK cells, LAK cells, and M φ phagocytic cells of animals were immunized with the 曱 -Hepatitis B combined vaccine and its monovalent 曱 Hepatitis B and Hepatitis B vaccine. Both had significant improvement in sexuality, and the difference was very significant compared with the control group (p <0.05). Compared with the animals vaccinated with the monovalent vaccine, the activity of peritoneal macrophages of the animals vaccinated with the combined hepatitis A-B vaccine was significantly improved (P <0.05). These results indicate that the phagocytosis of macrophages and the antigen presentation thereof induced by animals inoculated with the combined vaccine of the present invention are significantly higher than those of animals vaccinated with simple liver and hepatitis B vaccines.

Quantitative detection of F cytokines.

RT-PCR (reverse transcription polymerase chain reaction) method was used to quantitatively detect the DNA encoding cytokines IFN-γ, IL-2 and IL-18. At the same time, RNA was extracted from the spleen cells of immunized mice, and then mRNA was reverse transcribed into cDNA using bandit LV-RT (mouse leukemia virus reverse transcriptase), and amplified by competitive PCR using appropriate cytokine primers. Cytokine-specific cDNA. Finally, the obtained amplified products were quantitatively analyzed by agarose gel electrophoresis. In the experiment, β-acting was used as a control sample. The results are shown in the following list 8-10. Quantitative Comparison of Vaccine-stimulated IFN-γ Production

Control group Hepatitis A vaccine group Hepatitis B vaccine group Lianhe vaccine group Control sample 70. 3 52. 47 65. 34 45. 16 Experimental sample 29. 7 47. 53 35. 53 54. 84

The results shown in Table 8 indicate that the ability of the hepatitis A vaccine group and the hepatitis A-B combined vaccine group to stimulate the body to produce IFN-V is only recent (P> 0.05), but significantly higher than the control group and the hepatitis B vaccine group (P <0. 05). Quantitative comparison of vaccine-stimulated IL-2 production in the body Control group 曱 Liver vaccine group HBV vaccine combination and vaccine group control sample 62. 27 59. 1 60. 28 43. 04 Experimental sample 37. 73 40. 99 39. 22 56. 95 The results shown in Table 9 show that: -The ability of Hepatitis B vaccine and vaccine group to stimulate the body to produce IL-2 is significantly better than that of hepatitis A vaccine group, hepatitis B vaccine group and control group (P <0.05). Since the main role of IL-2 is to induce the proliferation and differentiation of T lymphocytes, it can be considered that the 曱 -Hepatitis B vaccine of the present invention is significantly better than the monovalent vaccines of 曱 and Hepatitis B in terms of the ability to induce T lymphocyte proliferation and differentiation P <0. 05). Quantitative Comparison of Vaccine Stimulating IL-18 Production

Control group 曱 Liver vaccine group HBV vaccine group Hehe vaccine group Control sample 100 72. 33 100 52.1 Experimental sample 0 27. 67 0 47. 9

The results shown in Table 10 show that the ability of the hepatitis A-B vaccine group to stimulate the body to produce IL-18 is significantly better than that of the liver vaccine group, the hepatitis B vaccine group, and the control group (P <0.05). Since the main role of IL-18 lies in the secretion of activated macrophages and significantly enhanced NK cell activity, it can be considered that the 曱 -Hepatitis B vaccine of the present invention is significantly superior to the monovalent 曱 liver in enhancing NK cell activity. Or hepatitis B vaccine (P <0. 05). Example 5: Thermal stability test of lyophilized tadpole-hepatitis B combined vaccine The combination vaccine is stored at different temperatures for different times, and then samples are taken for testing of the titer of the liver vaccine virus, the efficacy of the hepatitis B vaccine, and the residual moisture content to evaluate the thermal stability of the freeze-dried hepatitis A-B vaccine of the present invention. .

The lyophilized cricket-hepatitis B combined vaccine of the present invention was stored in the dark at a temperature of 2-8 ° C, and samples were taken at 9, 12, 15, 21, and 24 months for hepatitis A vaccine virus titer and hepatitis B virus. Vaccine efficacy, residual moisture content testing. The hepatitis B vaccine expressed by recombinant CH0 cells was used as the reference vaccine. The results are shown in Listings 11 and 12 below. Table 11 Viral titer and relative potency of hepatitis A-B combined vaccine after storage at 2--8 ° C for different times

Vaccine Hepatitis A virus titer (LogCCID ₅ „/ ml) (month) Relative efficacy of hepatitis B

Lot number 0 9 12 15 18 21 24 0 9 12 15 18 24

980401 6. 50 6. 50 6. 67 6. 50 6. 67 6. 50 6. 00 2. 79 2. 73 2. 81 2. 64 2. 57 2. 54

980402 6. 67 6. 50 6. 67 6. 50 6. 50 6. 67 6. 33 2. 58 2. 49 2. 51 2. 41 2. 34 2. 32

980403 6. 67 6. 67 6. 50 6. 50 6. 67 6. 67 6. 50 2. 88 2. 86 2. 81 2. 77 2. 79 2. 78

980501 6. 67 6. 67 6. 67 6. 67 6. 50 6. 50 6. 33 3. 06 3. 08 2. 98 2. 87 2. 81 2. 82

980502 7. 00 7. 00 7. 00 7. 00 6. 67 6. 67 6. 50 2. 45 2. 47 2. 36 2. 41 2. 31 2. 39

Table 12 曱 -Hepatitis B combined vaccine 2--8 ° C Determination of residual water content at different time storage Residual water content (%) Ϊ) ~~

Vaccine batch number 0 9 12 15 18 21 24

980401 2. 23 2. 30 2. 29 2. 37 2. 41 2. 44 2. 90

980402 2. 15 2. 25 2. 27 2. 31 2. 39 2. 40 2. 43

980403 2. 30 2. 30 2. 29 2. 33 2. 32 2. 89 2. 39

980501 2. 28 2. 31 2. 30 2. 34 2. 31 2. 79 2. 95

980502 2. 26 2. 28 2. 28 2. 31 2. 29 2. 42 W

The lyophilized hepatitis A-B vaccine of the present invention was stored for 6 months at room temperature in the dark, and samples were taken at 0, 2, 3, 4, 5, and June to measure the titer of the liver vaccine virus, the efficacy of the hepatitis B vaccine, and Detection of residual moisture content. The hepatitis B vaccine expressed by recombinant CH0 cells was used as a reference vaccine. The results are shown in Tables 1 3 and 15 below.

Tibia-hepatitis B combined vaccine titration of tadpole hepatitis virus and relative efficacy of HBV stored at room temperature

Vaccine and liver virus titer (LogCCID ₅ ./ml) (month) Relative efficacy of hepatitis B (month)

Lot number 0 2 3 4 5 6 0 2 3 4 5 6

980401 6. 50 6. 67 6. 50 6. 50 6. 50 6. 33 2. 79 2. 68 2. 71 2. 69 2. 61 2. 59

980402 6. 67 6. 50 6. 67 6. 50 6. 50 6. 00 2. 58 2. 60 2. 54 2. 50 2. 41 2. 38

980403 6. 67 6. 67 6. 67 6. 50 6. 50 6. 00 2. 88 2. 84 2. 71 2. 75 2. 87 2. 70

980501 6. 67 6. 67 6. 67 6. 50 6. 50 6. 33 3. 06 3. 08 3. 10 2. 98 2. 89 2. 78

980502 7. 00 7. 00 7. 00 7. 00 6. 67 6. 50 2. 45 2. 47 2. 39 2. 33 2. 21 2. 19 The lyophilized cricket-hepatitis B combined vaccine of the present invention Store at 37 ° C. Samples are taken at weeks 0, 1, 2, 3, 4 and 5 for testing of hepatitis A vaccine virus titer, hepatitis B vaccine efficacy, and residual moisture content. The hepatitis B vaccine expressed by recombinant CH0 cells was used as a reference vaccine. The results are shown in Listings 14 and 15 below. Table 14 Hepatitis A-B combined vaccine titer and relative potency of hepatitis B virus stored at 37 ° C

Vaccine Hepatitis virus titer (LogCCID50 / ml) (week) Hepatitis B efficacy (ED50) (week) Lot number 0 1 2 3 4 5 0 1 2 3 4

980401 6. 50 6. 50 6. 33 6. 50 6. 00 6. 00 2. 79 2. 76 2. 71 2. 60 ~ 2? 68 ~ 2

980402 6. 67 6. 50 6. 50 6. 67 6. 00 6. 00 2. 58 2. 51 2. 53 2. 47 2. 41 2

980403 6. 67 6. 67 6. 50 6. 50 6. 33 5. 67 2. 88 2. 84 2. 77 2. 69 2. 58 2

980501 6. 67 6. 67 6. 33 6. 67 6. 33 6. 00 3. 06 3. 02 3. 06 2. 98 2. 95 2

980502 7. 00 7. 00 6. 50 6. 67 6. 50 6. 00 2. 45 2. 48 2. 41 2. 39 2. 30 2

Table 15 Residual water of hepatitis A-B combined vaccine after storage at room temperature and 37 ° C for different times Partial content determination Initial vaccine room temperature residual moisture (¾) (month) 37 ° C residual moisture (° /.) (Week) Lot number moisture (%) 2 4 5 6 1 2 3 4

980401 2. 23 2. 25 2. 30 2. 34 2. 89 2. 24 2. 26 2. 30 2. 37

980402 2. 15 2. 19 2. 41 2. 50 3. 10 2. 20 2. 27 2. 29 2. 36

980403 2. 30 2. 29 2. 34 2. 41 2. 78 2. 30 2. 31 2. 35 2. 39

980501 2. 28 2. 30 2. 31 2. 39 2. 40 2. 33 2. 33 2. 40 2. 41

980502 2. 26 2. 27 2. 29 2. 38 2. 39 2. 29 2. 30 2. 38 2. 40

From the data shown in Tables 6 to 15 above, it can be seen that compared with the monovalent vaccines of Hepatitis A or Hepatitis B, the freeze-dried 曱 -Hepatitis B combined vaccine of the present invention is at room temperature, 2 ° C -8 ° C, 37 ° C. After being stored for different periods of time, the titer of the hepatitis B vaccine virus and the relative efficacy of the hepatitis B vaccine did not decrease significantly, and even the efficacy of the hepatitis B vaccine was higher than the monovalent vaccine of the hepatitis B vaccine. Also. Although the lyophilized hepatitis A-B combined vaccine of the present invention is stored at different temperatures for different times, its residual moisture content has increased, but it does not affect the overall lyophilized cricket-Hepatitis B combined vaccine of the present invention. Quality requirements.

Claims

Claim

1. A combined hepatitis A and B vaccine comprising live attenuated hepatitis B vaccine virus and hepatitis B virus surface antigen protein.

2. The rhenium-hepatitis B combined vaccine according to claim 1, further comprising an immune adjuvant.

3. The combined rubidium-hepatitis B vaccine according to claim 2, wherein the immune adjuvant is aluminum hydroxide.

8-1. 2 mg / ml。 4. The rubidium-hepatitis B combined vaccine according to claim 3, wherein the content of aluminum hydroxide is 0.8 to 1.2 mg / ml.

5. The combined gallium-hepatitis B vaccine according to claim 1, further comprising a virus vaccine base fluid.

6. The combined gallium-hepatitis B vaccine according to claim 1, wherein said live attenuated gallium hepatitis vaccine virus is a live attenuated gallium vaccine L-A-1 vaccine strain.

7. The tadpole-hepatitis B combined vaccine according to claim 1, wherein the hepatitis B virus surface antigen (HBsAg) protein is obtained by recombinant CH0 cells and / or recombinant yeast and purified.

8. The combined hepatitis A-B vaccine according to claim 7, wherein the hepatitis B virus surface antigen (HBsAg) protein is obtained by expressing and purifying recombinant CH0 cells.

9. LogCCID _5。 9. The 曱 -Hepatitis B combined vaccine according to claim 1, characterized in that said live attenuated 曱 hepatitis vaccine virus titer is not lower than 6.5 LogCCID ₅ . / ml.

10. The combined hepatitis A-B vaccine according to claim 1, characterized in that the content of said hepatitis B virus surface antigen protein is not less than 10 μg / ml.

11. The combined rubidium-hepatitis B vaccine according to claim 2, wherein the virus vaccine-based shield solution is 199 comprehensive culture solution.

12. A lyophilized combination of hepatitis A and B vaccine, comprising live attenuated hepatitis B vaccine virus, hepatitis B virus surface antigen protein and a protective agent.

13. The lyophilized radon-hepatitis B combined vaccine according to claim 12, wherein the protective agent comprises trehalose, glutamic acid, ascorbic acid, urea, sorbitol, inositol, and low-molecular dextran.

14. The lyophilized radon-hepatitis B combined vaccine according to claim 12, wherein, before lyophilization, the concentration (W / V) of the protective agent in the combined vaccine is trehalose 3-10%, and glutamic acid 0. 5-1. 0%, ascorbic acid 0.1-1.0.3, urea 0.5-2.0%, sorbitol 0.5-2. 0%, inositol 0.5-1. 0% and low molecular weight Dextran 1-6%.

15. The lyophilized radon-hepatitis B combined vaccine according to claim 12, further comprising an immune adjuvant.

16. The lyophilized hepatitis A-B combined vaccine according to claim 12, further comprising a virus vaccine matrix fluid.

17. The lyophilized hepatitis A-B combined vaccine according to claim 12, wherein said live attenuated hepatitis A vaccine virus is a live attenuated hepatitis A vaccine L-A-1 vaccine strain.

18. The lyophilized hepatitis A-B combined vaccine according to claim 12, wherein the hepatitis B virus surface antigen (HBsAg) protein is expressed by recombinant CH0 cells and / or recombinant yeast and obtained after purification.

19. The lyophilized hepatitis A-B combined vaccine according to claim 12, wherein the hepatitis B virus surface antigen (HBsAg) protein is expressed by recombinant CH0 cells and obtained after purification.

20. The lyophilized radon-hepatitis B combined vaccine according to claim 12, characterized in that said live attenuated hepatitis A vaccine virus titer is not lower than 6.5 LogCCID ₅ . / ml.

21. The lyophilized hepatitis A-B combined vaccine according to claim 12, characterized in that the content of said hepatitis B virus surface antigen protein is not less than 10 g / ml.

22. The lyophilized hepatitis A-B combined vaccine according to claim 16, wherein the virus vaccine base fluid is 199 comprehensive culture fluid.

23. A freeze-dried cricket-hepatitis B combined vaccine according to claim 12, wherein the adjuvant is a solution containing 0.8-1.2 mg / ml aluminum hydroxide as a dilution of the freeze-dried hepatitis A-B combined vaccine which provided.

24. A method for preparing a lyophilized radon-hepatitis B combined vaccine, the method comprising:

(1) Provide and mix attenuated hepatitis B vaccine virus stock and Recombinant CHO cells or recombinant yeast express and purified the original hepatitis B virus surface antigen protein stock solution to obtain a mixed solution of hepatitis B vaccine virus and hepatitis B virus surface antigen.

(2) Add an appropriate amount of a protective agent system to the mixed solution obtained in step (1) and mix them to obtain the desired combination of hepatitis A and hepatitis B vaccine composition suspension.

(3) freeze-drying the combined vaccine suspension containing the protective agent obtained in step (2) to obtain a lyophilized 曱 -Hepatitis B combined vaccine.

25. The method according to claim 24, wherein said freeze-drying step of step (3) comprises first pre-freezing said combination vaccine composition at a low temperature of about -30 to -55 ° C for 3-7 hours, Then gradually increase the temperature to 35 ° C and dry under vacuum for 8 to 16 hours.