CN1513555A - Combined hepatitis A-B vaccine and its freeze-dried preparation - Google Patents

Abstract

The present invention relates to hepatitis A and hepatitis B combined vaccine and its freeze-dried preparation, especially relates to the hepatitis B vaccine virus containing live suitably attenuated and expressed and purified by recombinant CHO cells and/or recombinant yeast Surface antigen combined hepatitis A-B vaccine, and its freeze-dried preparation. In freeze-dried formulations, the protective agents used include trehalose, glutamic acid, ascorbic acid, urea, sorbitol, inositol and dextran. The combination vaccine of hepatitis A and hepatitis B of the present invention can prevent hepatitis A and hepatitis B at the same time.

Description

Combined hepatitis A-B vaccine and its freeze-dried preparation

technical field

The present invention relates to hepatitis A and hepatitis B combined vaccine and its freeze-dried preparation, especially relates to the hepatitis B vaccine virus containing live suitably attenuated and expressed and purified by recombinant CHO cells and/or recombinant yeast Surface antigen combined hepatitis A-B vaccine, and freeze-dried formulations thereof. The present invention also relates to a method for preparing the freeze-dried hepatitis A-B combined vaccine and a protective agent for the freeze-dried combined hepatitis A-B vaccine.

Background of the invention

Hepatitis A is a global acute infectious disease caused by Hepatitis A virus (HAV), which exists widely in nature. The prevalence of hepatitis A has become a serious public health problem due to its prevalence and rapid spread. Especially in developing countries including China, due to the large population, relatively backward socioeconomic 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 about tens of thousands of cases of hepatitis patients related to hepatitis A virus infection.

Hepatitis B is an infectious disease caused by the hepatitis B virus. Hepatitis B has high morbidity and mortality rates in developing countries and generally has a poor prognosis. So far, there is no effective method for the treatment of hepatitis B. In China, hepatitis B virus carriers account for about 8-10% of the total population, and there are more than 10 million chronic hepatitis patients.

Due to the serious harm and high prevalence of hepatitis A and hepatitis B, the incidence of these two types of hepatitis in many countries in the world always accounts for the first place in the incidence of all viral hepatitis, which is about 70%-80%. Facing the severe epidemic situation of hepatitis A-B, China has taken the lead in successfully developing a live attenuated hepatitis A vaccine. In particular, in order to solve the source of raw materials for hepatitis B vaccine production and possible infection by other pathogenic factors, in recent years, on the basis of developing blood-borne hepatitis B vaccines, further developed a vaccine based on hepatitis B surface antigen (HBsAg) ) genetically engineered hepatitis B vaccine.

Since the "Children's Vaccine Initiative" (CVI) was initiated and implemented by international organizations such as UNICEF and the World Health Organization in 1991, many laboratories and companies engaged in vaccine research and development in the world have been working on the development of new vaccines. These new vaccines should be polyvalent, heat-stable, and administered orally or by aerosol. From the perspective of clinical application, through the use of combined vaccines, one immunization can effectively prevent two or more diseases, so as to reduce vaccine production costs, improve vaccination efficiency, and reduce adverse reactions caused by multiple injections. The main development direction of vaccine research.

In recent years, researchers have made many beneficial attempts in the development of multivalent multivalent vaccines. As far as hepatitis vaccine is concerned, for example, European Patent No. 0339 667 discloses a kind of A-B vaccine containing inactivated hepatitis A virus antigen, hepatitis B virus surface antigen and adjuvant, which can prevent hepatitis A and hepatitis B simultaneously. Hepatitis combination vaccine. In addition, US Patent No. 6,451,320 provides a combined vaccine composition suitable for vaccination of adolescents. The combined vaccine composition comprises hepatitis B virus surface antigen and herpes simplex virus antigen, and one or more virus antigens selected from hepatitis A virus antigen (HAV), Epstein-Barr virus (EBV) antigen, etc., and wherein HAV is The inactivated one was derived from the HM-175 strain. However, the hepatitis A virus antigens used in these existing combined vaccines are all inactivated HAV viruses.

So far, there has been no report on the use of live attenuated hepatitis A vaccine virus to produce combined (adjuvant) vaccine and freeze-dried preparation of hepatitis A-B.

Contents of the invention

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

Another object of the present invention is to provide a combined hepatitis A-B vaccine composition in freeze-dried form, comprising live attenuated hepatitis A vaccine virus, hepatitis B virus surface antigen (HBsAg) and a protective agent.

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

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

The present invention relates to hepatitis A and hepatitis B combined vaccine, in particular to a freeze-dried form of hepatitis A-hepatitis B combined vaccine composition containing live hepatitis A vaccine virus and hepatitis B surface antigen protein and protective agent. The present invention further relates to a method for producing said combination vaccine composition.

According to the present invention, a combined hepatitis A-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, suitable attenuated hepatitis A vaccine strains isolated from the feces of naturally infected hepatitis A patients and obtained through serial attenuation, passage, terminal dilution, "cloning" purification or other artificial mutagenesis methods can be used As the seed virus, a cell matrix sensitive to hepatitis A vaccine virus, such as established human embryonic lung diploid cells, is used as the cell matrix, and the living virus used in the present invention is prepared according to the method described in detail in Chinese Patent No. 92114998.0. Attenuated hepatitis A vaccine virus stock solution.

Preferably, the vaccine stock solution prepared by using the live attenuated hepatitis A vaccine L-A-1 vaccine strain is used.

The hepatitis B surface antigen protein used in the present invention may be known purified hepatitis B virus surface antigen protein. For example, constructing and selecting an appropriate expression system can be used, such as expressing the HBsAg protein 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, cloned into an appropriate plasmid vector such as pBR322 or its derivatives, and then transformed into an appropriate host with the resulting recombinant vector cells, such as Chinese hamster ovary (CHO) cells and/or yeast, and culture recombinant CHO cells and/or yeast under conditions suitable for expression of HBsAg protein, then harvest and purify from recombinant cell culture fluid and/or yeast Get the desired HBsAg protein.

The HBsAg can be prepared according to the "Recombinant (CHO Cell) Hepatitis B Vaccine Manufacture and Verification Regulations" in the 2000 edition of "China Biological Products Regulations".

Alternatively, density gradient centrifugation or various ion exchange chromatography methods can also be used to separate and purify the blood-derived 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, wherein said live attenuated hepatitis A vaccine virus is the wild-type hepatitis A virus isolated from the feces of patients with hepatitis A who are naturally infected, through Final dilution "clones" the purified and attenuated Hepatitis A L-A-1 strain obtained by passage in appropriate host cells.

According to a preferred embodiment of the vaccine composition of the present invention, it is characterized in that the viral titer of the live attenuated hepatitis A vaccine virus contained in the combined vaccine is not less than 6.5 LogCCID50/ml.

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

According to a preferred embodiment of the vaccine composition of the present invention, it is characterized in that the protein content of hepatitis B virus surface antigen (HBsAg) in the combined vaccine is not less than 10 μg/ml.

The preferred ratio range of attenuated hepatitis A vaccine virus and hepatitis B surface antigen protein in the combined vaccine is: 6.5-7.0 LogCCID50/ml to 12-14 μg/ml.

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

Thus, according to the present invention, a combined hepatitis A-B vaccine in freeze-dried form comprises live attenuated hepatitis A vaccine virus and hepatitis B surface antigen protein and a protective agent.

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

Dextran can be high, medium or low molecular weight dextran, such as dextran 70, 40, 20; preferably low molecular dextran, such as dextran 20.

Before freeze-drying, their contents (W/V) in the combined vaccine composition are respectively: 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 of the present invention, wherein the protective agent consists essentially of trehalose, glutamic acid, ascorbic acid, urea, sorbitol, inositol and dextran.

The combined hepatitis A-B vaccine of the present invention and its freeze-dried product may also contain one or more adjuvants for improving its immunogenicity without causing any side effects. These adjuvants are generally well known to those skilled in the art, including but not limited to aluminum hydroxide or aluminum phosphate gel, 3D-MPL (a Th1 cell response stimulator) etc. (see Pharmaceutical Biotechnology, Vol.61 Vaccine Design -the subunit and adjuvantapproch, Powell and Newman, Plenurn Press).

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

According to a preferred embodiment of the vaccine composition of the present invention, it may also contain an appropriate amount of immune 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, the adjuvant is provided by a 0.8-1.2 mg/ml aluminum hydroxide solution contained in the lyophilized combined hepatitis A and B vaccine dilution.

According to the present invention, a kind of production method of freeze-dried hepatitis A-B combined vaccine, the method comprises:

(1) The protein stock solution containing the attenuated hepatitis A vaccine virus stock solution and the purified hepatitis B virus surface antigen (HBsAg) expressed by recombinant CHO cells and/or recombinant yeast, respectively provided and mixed in an appropriate proportion , to obtain a mixed suspension of hepatitis A virus and hepatitis B virus surface antigen;

(2) Add an appropriate amount of protective agent to the mixed suspension obtained in step (1) and mix it uniformly to obtain the required suspension of the combined vaccine composition of hepatitis A and hepatitis B;

(3) freeze-drying the combined vaccine suspension containing the protective agent obtained in step (2).

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 combined vaccine composition suspension 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%, and dextran 1-6%.

According to a preferred embodiment of the method of the present invention, said method further includes, before using the lyophilized combined vaccine, diluting the lyophilized combined vaccine composition obtained in step (3) with an aluminum hydroxide solution, so that the live attenuated The virus titer of toxic hepatitis A vaccine virus is not less than 6.5LogCCID ₅₀ /ml, the protein content of hepatitis B virus surface antigen (HBsAg) is not less than 10μg/ml, and the concentration of aluminum hydroxide is 0.8-1.2mg/ml ml.

According to a preferred embodiment of the method of the present invention, wherein the adjuvant is provided by the aluminum hydroxide solution contained in the lyophilized combined hepatitis A and B vaccine dilution.

The combination vaccine composition of the present invention can be effectively used to prevent hepatitis A and hepatitis B. The observation results of various animal and monkey experiments 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 combination vaccine composition of the present invention contains the biologically active lyoprotectant that can effectively protect the hepatitis A vaccine virus and the hepatitis B antigenic protein, it can be made into a freeze-dried combination vaccine Preparations, and thus avoid the "cold chain" pressure in the process of vaccine production, transportation, storage and use, so as to more effectively guarantee the vaccination effect of the vaccine. In particular, since the live hepatitis A vaccine virus with good safety and immunogenicity has been fully proven in the combined vaccine composition of the present invention, the production cost and market price of the combined vaccine composition are greatly reduced, thereby It is conducive to its promotion and general use in developing countries including China.

In order to prepare the combined vaccine lyoprotectant of the present invention, for example, the following ingredients can be dissolved in 37°C distilled water (1000ml) at first according to the indicated concentration: sodium glutamate 100.0g/L, ascorbic acid 20.0g/L, urea 80.0g /L, sorbitol 80.0g/L and inositol 80.0g/L, and adjust the pH value to 7.0 with 0.1NHCL. Protective agent component A was obtained after filtration and sterilization with a 0.22 μm filter membrane. Then weigh 500.0 g of trehalose and dissolve it in water for injection at a temperature of about 37 ° C to make up to 1000 ml, filter and sterilize through a 0.22 μm filter membrane to obtain the protective agent component B. Finally, 300.0 g of low-molecular-weight dextran was dissolved in water for injection at a temperature of about 37° C. to make up to 1000 ml, and sterilized by high-pressure steam at 116° C. for 40 minutes to obtain the protective agent component C. When in use, an appropriate volume of protectant components A, B and C can be added to the mixed suspension of the above-mentioned hepatitis A vaccine stock solution and hepatitis B vaccine stock solution, so that each component of the protectant reaches the concentration range defined above (see Example 2).

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

The combination vaccine composition of hepatitis A and hepatitis B of the present invention can be prepared by simple mechanical mixing method. For example, first, according to an appropriate volume ratio (such as about 20:1 (V/V)), the pre-prepared hepatitis A vaccine stock solution with a virus titer greater than 7.0 Log CCID ₅₀ /ml and the pre-prepared protein concentration greater than 100 μg /ml of hepatitis B virus surface antigen stock solution, and then add an appropriate amount of pre-prepared lyoprotectant and mix gently.

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

Finally, the aliquoted mixed suspension can be freeze-dried using a conventional freeze-drying device, wherein the freeze-drying conditions used are: pre-freeze at -30°C to -55°C for 3-7 hours, and then freeze at -30°C Vacuum dry at 35°C for 8 to 16 hours. During the freeze-drying process, the co-thawing temperature of the vaccine freeze-dried mixture is lower than -38°C.

Compared with before freeze-drying, although the virus titer of hepatitis A vaccine decreased slightly after freeze-drying, the drop rate was not much about 0.5LogCCID50/ml. The freeze-dried vaccine product obtained in this way can be preserved to 6 months at room temperature for a long time without affecting the viral titer of its hepatitis A vaccine virus and the relative efficacy of hepatitis B virus surface antigen (using the hepatitis B vaccine expressed by recombinant CHO cells) as a reference vaccine).

Before clinical application, the lyophilized combined vaccine obtained above can be dissolved and diluted with an aluminum hydroxide solution with a content of 0.8-1.2 mg/ml, and the combined vaccine of the present invention can be made into a preparation in the form of a suspension. The aluminum hydroxide aqueous solution can not only be used as the dissolving agent and diluent of the freeze-dried combined vaccine, but also can provide it with an adjuvant with immune enhancing effect.

A large number of experimental studies and various animal inoculation experiments 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 , will not interfere with each other due to the mixing of the two virus antigens, nor will it cause any immune response suppression or other adverse reactions. The safety test results show that all primates (rhesus monkeys or red-faced monkeys) inoculated with the combined vaccine of the present invention do not have elevated serum transaminases (ALT) in 0-12 weeks after inoculation. Liver tissue of animals The pathological examination showed that all the tested animals had no abnormal pathological changes of liver tissue (see Example 3).

Experimental data also show that the combined vaccines of the present invention appear to provide better immunogenicity than their monovalent counterparts compared to live attenuated hepatitis A vaccines and/or hepatitis B vaccines alone. In addition, since the combined vaccine composition of the present invention contains effective protector or stabilizer components, the hepatitis A vaccine virus titer and hepatitis B vaccine There was no significant decrease in relative potency.

The test results of the components in the two pre-prepared combined vaccines showed that the hepatitis A vaccine virus titer was not lower than 6.5 LogCCID50/ml, and the HBsAg antigenic protein content was not lower than 10 μg/ml.

The results of the immunogenicity experiment of the combined vaccine showed that when rhesus monkeys or red-faced monkeys were inoculated with the combined vaccine for 4 weeks, the positive conversion rate of anti-HAV antibodies in the animal serum reached 80-100%. Observe simultaneously, the positive conversion rate of anti-HBsAg antibody in animal serum is about 20%; The positive conversion rate of anti-HBsAg antibody at 8 weeks is 40-80%; Reach 80-100%, in addition, further comparative experiment shows, compared with inoculation monovalent vaccine, the primate (rheses monkey or red-faced monkey) that inoculates freeze-dried Hepatitis B combined vaccine of the present invention, has The monovalent vaccine also has good safety and seems to have better immunogenicity (see Example 4-①).

Cellular immunology experiments confirmed that after inoculating the combined vaccine composition of the present invention to BALB/C pure line mice, the lymphokine surface marker detection, natural killer (NK) cell activity detection, 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 a significant cell-mediated immune response. In addition, further comparative experiments showed that , compared with inoculation of monovalent hepatitis A vaccine and hepatitis B vaccine, the combination vaccine of hepatitis A and hepatitis B of the present invention can produce better cellular immune-mediated response (see Example 4-②).

Further combined vaccine storage stability test results show that the freeze-dried combined 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, and the hepatitis A vaccine virus titer and B Hepatitis vaccines are relatively potent (see Example 5).

The following examples are intended to further illustrate, but not limit, the invention. Those skilled in the art can understand that any parallel changes and modifications to the present invention will fall within the scope of the claims of the present invention without departing from the spirit and principles of the present invention.

Embodiment 1: preparation of hepatitis A vaccine virus stock solution

Firstly, human embryonic lung diploid cells are amplified and cultured using minimal medium (MEM) (pH 7.2-7.6) added with 10-15% calf serum. After the cells formed a dense monolayer, the growth medium was discarded, and after the cells were washed repeatedly with fresh Earle's solution, the seed virus solution prepared according to the method described in Example 1 of Chinese Patent No. 92114998.0 was inoculated. After adsorption at 34°C-36°C for 3-4 hours, add cell growth maintenance solution (lactoalbumin hydrolyzate with vitamin C) to the culture and culture at 34°C-36°C for about 4 weeks, changing the medium every week once. After culturing, discard the maintenance solution and residual calf serum, directly add 199 comprehensive culture solution containing low concentration of salt (such as 1-10mM NaCl) and does not contain phenol red, and continue culturing (34°C-36°C) for 2 to 4 days . Then the cells are collected, and the supernatant is collected by centrifugation after repeated freezing and thawing and sonicating the cells to obtain the stock solution of the hepatitis A vaccine virus.

Embodiment 2: the preparation of lyophilized hepatitis A-hepatitis B combined vaccine

The stoste of 2.0L hepatitis A vaccine virus (L-A-1. strain, virus titer is 8.0LogCCID50/ml) and 72ml are expressed by recombinant CHO cell and/or recombinant yeast and purified hepatitis B virus surface antigen protein stoste ( The protein content is 1334 μg/ml), and then add the protective agent stock solution prepared as follows: 550ml of protective agent component A, 700ml of protective agent component B and 450ml of protective agent component C.

Then, add an appropriate amount of 199 comprehensive culture solution as the virus vaccine matrix solution in the mixed suspension obtained in this way, make the total volume of liquid combination vaccine reach 8000ml, make wherein hepatitis A vaccine virus titer not less than 6.5Log CCID50/ ml, and the content of hepatitis B virus surface antigen protein is not less than 10 μg/ml. As a result of such dilution, the concentration (W/V) of each component of the protective agent added in the vaccine suspension also reaches 3-10% trehalose, 0.5-1.0% glutamic acid, 0.1-0.3% ascorbic acid, and 0.5-0.5% urea. 2.0%, sorbitol 0.5-2.0%, inositol 0.5-1.0%, and dextran 1-6%.

In order to prepare the stock solution of lyoprotectant, the following ingredients can be dissolved in distilled water (1000ml) at the indicated concentration first: sodium glutamate 100.0g/L, ascorbic acid 20.0g/L, urea 80.0g/L, sorbitol 80.0g /L and inositol 80.0g/L, and adjust the pH value to 7.0 with 0.1N HCL. Protective agent component A was obtained after filtration and sterilization with a 0.22 μm filter membrane. Then weigh 500.0g of trehalose and dissolve it in water for injection at a temperature of about 37°C to make up to 1000ml, filter and sterilize through a 0.22μm filter membrane to obtain the protective agent component B. Finally, 300.0 g of dextran 20 was dissolved in water for injection at a temperature of about 37° C. to make up to 1000 ml, and sterilized by high-pressure steam at 116° C. for 40 minutes to obtain the protective agent component C.

According to the volume of each ampoule (or vial) of 1ml, the above combined vaccine suspension is packed, and then the ampoule is placed in a closed vacuum freeze dryer (FS150-SS20C type, Hull Co., USA), according to the following conditions Freeze-drying: first pre-freeze the vaccine suspension at about -40°C for 4 hours, then gradually warm up to 32°C and dry in vacuum for about 15 hours, so as to obtain the desired freeze-dried combined hepatitis A-B vaccine.

According to the World Health Organization (WHO) and the State Drug Administration (SDA) relevant combination vaccine and freeze-dried preparation inspection regulations and quality standard requirements, the combination vaccine provided by the present invention is fully tested, and each quality index of its result all reaches Or exceed the requirements of the inspection procedures stipulated by the World Health Organization (WHO) and the State Drug Administration (SDA). Among them, the hepatitis A vaccine virus titer is not lower than 6.5LogCCID50/ml, and the hepatitis B surface antigen (HBsAg) protein content is not lower than 10 μg/ml.

Embodiment 3: Safety experiment of freeze-dried hepatitis A-hepatitis B combined vaccine

Select 35 healthy red-faced monkeys or rhesus monkeys (average body weight 1.5-4.5Kg) with negative anti-HAV antibody and anti-HBV antibody and normal ALT index, and randomly divide them into 7 groups with 5 animals in each group. On the day when the experiment started, each animal of 5 experimental groups injected different batches of freeze-dried combined vaccine of the present invention by intravenous route. The content is 10-12 μ g/ml, and its relative efficacy is 2.45-3.06), and the animals in the two control groups receive the same dose of attenuated hepatitis A live virus vaccine (lyophilized preparation) and the hepatitis B vaccine expressed by recombinant CHO cells respectively.

The venous blood of the immunized animals was collected at 0, 4, 8 and 12 weeks after vaccination, the serum was separated, and the serum alanine aminotransferase (SGPT) or (ALT) of the animals was detected by conventional biochemical detection method and enzyme-linked immunosorbent assay, respectively. Levels, as well as anti-HAV and anti-HBsAg antibody levels and the relative efficacy of hepatitis B vaccine (the ratio of the ED50 of the tested vaccine to the ED50 of the reference vaccine). The anti-HAV detection kit was used to detect anti-HAV antibody by enzyme-linked immunosorbent assay (ELISA); the anti-HSsAg reagent was used to detect anti-HBsAg antibody by radioimmunoassay (RIA). Simultaneously with blood collection, living liver tissues of the tested animals were collected by liver puncture and histopathological examination was performed. The results are shown in Table 1 below.

Table 1 Safety test in monkeys inoculated with combined hepatitis A-B vaccine

Vaccine Hepatitis A virus titer Hepatitis B Abnormal serum SGPT (week) Abnormal liver histopathology (week)

Lot Number LogCCID ₅₀ /ml Relative Potency 0 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

980402 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

HB980401 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: hepatitis B vaccine expressed in recombinant CHO cells

It can be seen from the results shown in Table 1 that after the lyophilized hepatitis A-B combined vaccine and the control group vaccine were inoculated into monkeys, no abnormal elevation of serum liver enzymes occurred in the animals. Pathological examination of liver biopsy also showed that no abnormal pathological changes of liver tissue were found in all experimental animals. It shows that the freeze-dried combined hepatitis A and B vaccine has the same good safety as its monovalent vaccine.

Embodiment 4: Freeze-dried hepatitis A-hepatitis B combined vaccine immunogenicity experiment

① Vaccine-induced humoral immune response

Select 35 healthy rhesus monkeys (average body weight 1.5-4.5Kg) with negative HAV antibody and HBV antibody and normal ALT index, and randomly divide them into seven groups with 5 animals in each group. On the day when the experiment started, each animal in the 5 experimental groups received each 1ml of different batches of freeze-dried hepatitis A-hepatitis B combined vaccine of the present invention through intravenous route (hepatitis A virus titer is _{6.5-7.0LogCCID} Hepatitis B virus shows that the antigenic protein content is 10-12 μ g/ml, and its relative effectiveness is 2.45-3.06), and the animals of the remaining 2 control groups then receive the same dose of attenuated hepatitis A live virus vaccine (lyophilized preparation) or recombinant Hepatitis B vaccine expressed in CHO cells.

The venous blood of the immunized animals was collected at 0, 4, 8 and 12 weeks after vaccination, the serum was separated, and the serum anti-HAV and anti-HBsAg antibody levels of the animals, as well as the HAV-IgM antibody titer were detected by conventional enzyme-linked immunosorbent assay (as an indication of primary infection following vaccination). The results are shown in Tables 2, 3, 4 and 5 below.

         Table 2 Immunogenicity test in monkeys vaccinated with freeze-dried combined hepatitis A-B vaccine (1)

Vaccine Hepatitis A Virus Titer Hepatitis B Test Anti-HAV Positive (W) HAV-IgM Positive (W)

Batch No. LogCCID ₅₀ /ml Relative potency Number of animals 0 2 4 8 0 2 4 8

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 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

              Table 3 Immunogenicity test in monkeys inoculated with freeze-dried combined hepatitis A-B vaccine (2)

Vaccine Hepatitis A Virus Titer Hepatitis B Test Anti-HBS Positive (W)

Lot Number LogCCID ₅₀ /ml Relative Potency 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 5 0/5 1/5 3/5 4/5 4/5

HB: Hepatitis B vaccine expressed in recombinant CHO cells

Table 4 Detection of hepatitis A antibody levels in monkeys inoculated with freeze-dried combined hepatitis A and B vaccine

- HAV titer Hepatitis B Antibody level (mIU/m1)

Sample No. LogCCID ₅₀ /ml Relative Potency Animal No. (4W) (8W) (12W) (16W)

|

322 389 6422 80589 46978

980401 6.50 2.79 323 1215 4984 70055 74418

|

                                                              ,

(X±SD) 1089.13±1.79 6891.30±1.34 39128.26±2.36 28913.57±2.40

...

|

980402 6.67 2.58 438 1441 1375 80109 49078

...

                                                                       ,

(X±SD) 1133.71±1.64 1475.45±1.05 37477.78±2.53 27098.93±2.26

                                                                      ,

...

HA980501 6.67 328 415 1319 15958 49907

...

|

(X±SD) 1086.67±1.72 1506.96±1.08 11012.73±1.54 28123.6±2.23

HA980501 is a monovalent freeze-dried live attenuated hepatitis A vaccine

           Table 5 Detection of anti-HBs levels in monkeys after inoculation with freeze-dried combined hepatitis A-B vaccine

                                                                   

Sample No. LogCCID ₅₀ /ml Relative Potency

Animal No. 8 12 16

321 0 652.45 286.68

|

980401 6.50 2.79 323 67.63 785.54 3346.65

...

                                                                   ,

(X±SD) 1340.23±2.02 1660.09±3.01

|

437 0 845.65 1198.73

980402 6.67 2.58 438 186.70 2104.45 3586.68

...

440 0 0 0

(X±SD) 1344.05±2.19 1656.08±1.94

331 98.75 3296.71 1758.70

|

HB980401 2.55 333 152.41 2345.84 3346.15

                                                                                                                                                                              ,

335 0 0 0

(X±SD) 1340.44±2.36 1657.55±1.98

HB90401 is a hepatitis B vaccine expressed by recombinant CHO cells

As can be seen from the results shown in Tables 2, 3, 4 and 5, after primates (rhesus or red-faced monkeys) were inoculated with different batches of the present invention's A-B combined vaccine, at the 4th week The positive conversion rate of anti-hepatitis A virus antibody in animal serum is 80%-100%, and all reach 100% after 8 weeks. The positive conversion rate of anti-HBs antibody in animal serum was 20% after 4 weeks of inoculation with combined vaccine; it was 40-80% at 8 weeks, and reached 80-100% at 12 weeks. Compared with the monovalent hepatitis A vaccine and hepatitis B vaccine, the antibody level of the combined vaccine had no significant difference (P>0.05). These results show that the combination vaccine of the present invention has the same good immunogenicity as its monovalent hepatitis A and hepatitis B vaccines. In addition, after the animals were vaccinated with the combined vaccine, the serum anti-hepatitis A virus IgM antibodies as the indication of primary hepatitis A infection also turned positive 2-4 weeks after inoculation, and gradually disappeared after the 8th week (Table 2) .

② Vaccine-induced cellular immune response

A test animal selection

Selected from 40 female BALB/C pure-line mice aged 6-8 weeks (provided by the Animal Center of Changchun Health Research Institute), they were randomly divided into four groups with 10 mice in each group: a blank control group, a hepatitis A vaccine group, a hepatitis B vaccine group, Combined hepatitis A-B vaccine group. Animals in each experimental group were injected intraperitoneally with 0.5 ml of the corresponding vaccine, and animals in the blank control group were injected with an equal volume of diluent of aluminum hydroxide (concentration: 1.0 mg/ml) in the same way.

B lymphocyte surface marker detection

The animals were sacrificed 10 days after inoculation, and the spleens of the animals in each group were aseptically removed and single-cell suspensions were prepared respectively. The resulting cell suspension was then dispensed into test tubes in volumes of 1 ml per tube. Anti-CD3, anti-CD4 and anti-CD8 monoclonal antibodies (each 0.1 ml) conjugated with fluorescein isothiocyanate (FITC) were added to each tube. Then flow cytometry (Becton Dickinson, USA) was used for flow cytometry detection. The results are shown in Table 6 below, where the values are expressed as the mean ± standard deviation of the animals in each group.

                                                                              

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

①Control group 35.44±6.47 16.19±3.62 8.90±0.03 1.81±0.04

②Hepatitis A vaccine group 47.46±5.60 25.86±2.85 9.24±0.75 2.80±0.01

③Hepatitis B vaccine group 47.23±4.25 26.91±0.41 9.56±0.21 2.81±0.03

④ Combined vaccine group 50.66±7.77 32.74±6.85 9.72±0.43 3.41±0.15

From the results shown in Table 6, it can be seen that the CD3+ of the animals in each test group was significantly higher than that in the control group (P<0.05). The CD4+ of the animals in each test group was also significantly higher than that of the control group, and the CD4+ of the animals in the combined vaccine group was higher than that in the monovalent vaccine group (P<0.05). Moreover, it can also be seen that the CD4+/CD8+ value of the combined vaccine group was significantly higher than that of the monovalent vaccine group and the blank control group (P<0.05). These results show that after animals are vaccinated with the combined vaccine of the present invention, the degree of activation of immune cells in the body is significantly higher than that of animals vaccinated with monovalent hepatitis A or hepatitis B vaccine.

C natural killer (NK) cell activity detection

The animals were sacrificed 10 days after inoculation, and the spleens of the animals in each group were aseptically removed and single-cell suspensions were prepared respectively. The resulting cell suspension was then dispensed into test tubes in volumes of 1 ml per tube. PE (phosphatidylethanolamine)-conjugated anti-mouse NK1.1 monoclonal antibody (0.1 ml each) was added to each tube. Then flow cytometry (Becton Dickinson, USA) was used for flow cytometry detection. The results are shown in Table 7 below, where the values are expressed as mean ± standard deviation of animals in each group.

D lymphokine-activated killer (LAK) cell activity assay

Add interleukin-2 (IL-2) at a final concentration of 15 μl/ml to the above splenic lymphocyte suspension of immunized mice, and incubate at 37°C and 5% _CO2 for 72 hours to obtain the desired activation of lymphokines killer (LAK) cells. In the experiment, H ³ -TdR-labeled K562 cells were used as target cells, and the two kinds of cells were mixed according to the effector cell:target cell ratio of 100:1. The mixed cell suspension was incubated at 37 °C and 5% _CO2 for 8 h. Then, the cells were fully washed and collected by centrifugation, and the radioactivity (cpm value) of the animal cells in each group was measured using a gamma counter. The results are shown in Table 7 below, where all values are expressed as mean ± standard deviation of animals in each group.

Phagocytosis of E peritoneal macrophages

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

Table 7 NK activity, LAK activity and Mφ phagocyte activity of immunized animals

Group NK activity (%) LAK activity (%) Mφ phagocyte activity

①Control group 4.46±0.51 33.5±5.60 28.58±3.06

②Hepatitis A vaccine group 11.80±0.71 64.10±6.38 50.62±2.67

③Hepatitis B vaccine group 8.79±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

As can be seen from the results shown in Table 7, the activities of NK cells, LAK cells and Mφ phagocytes were significantly improved after the animals were immunized with the combined hepatitis A-B vaccine and its monovalent hepatitis A and hepatitis B vaccines. Compared with the control group, the difference was very significant (p<0.05). Among them, compared with animals vaccinated with monovalent vaccine, the activity of peritoneal macrophages in animals vaccinated with combined hepatitis A-B vaccine was significantly increased (P<0.05). These results show that the phagocytosis of macrophages and the antigen presentation caused by the combined vaccine of the present invention are significantly higher than those of animals vaccinated with simple hepatitis A 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. Simultaneously extract RNA from splenocytes of immunized mice, then use MMLV-RT (mouse leukemia virus reverse transcriptase) to reverse transcribe mRNA into cDNA, and use appropriate cytokine primers to amplify cytokines by competitive PCR specific cDNA. Finally, the amplified products were quantitatively analyzed by agarose gel electrophoresis. β-acting was used as a control sample in the experiments. The results are shown in Tables 8-10 below.

Table 8 Quantitative comparison of vaccine-stimulated body to produce IFN-γ Control group Hepatitis A vaccine group Hepatitis B vaccine group Combined vaccine group Control sample 70.3 52.47 65.34 45.16 Experimental sample 29.7 47.53 35.5 3 54.84

The results shown in Table 8 show that the ability of the hepatitis A vaccine group and the combined hepatitis A-B vaccine group to stimulate the body to produce IFN-γ is similar (P>0.05), but significantly higher than that of the control group and the hepatitis B vaccine group (P<0.05).

Table 9 Quantitative comparison of vaccine-stimulated body to produce IL-2 Control group Hepatitis A vaccine group Hepatitis B vaccine group Combined 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 showed that the ability of the combined hepatitis A-B vaccine group to stimulate the body to produce IL-2 was significantly better than that of the hepatitis A vaccine group, hepatitis B vaccine group and control group (P<0.05). Because the main effect of IL-2 is to induce T lymphocyte proliferation and differentiation, it can be considered that the combination vaccine of A-hepatitis B of the present invention is obviously better than hepatitis A and hepatitis B monovalent vaccine in inducing T lymphocyte proliferation and differentiation ability (P< 0.05).

Table 10 Quantitative comparison of vaccine-stimulated body to produce IL-18 Control group Hepatitis A vaccine group Hepatitis B vaccine group Combined vaccine group Control sample 100 72.33 100 52.1 Experimental sample 0 27.67 0 47.9

The results shown in Table 10 showed that the ability of the combined hepatitis A-B vaccine group to stimulate the body to produce IL-18 was significantly better than that of the hepatitis A vaccine group, hepatitis B vaccine group and control group (P<0.05). Since the main function of IL-18 is that activated macrophages secrete and significantly enhance NK cell activity, it can be considered that the combined hepatitis A-B vaccine of the present invention is significantly better than monovalent hepatitis A or hepatitis B vaccine in enhancing NK cell activity. Hepatitis B vaccine (P<0.05).

Embodiment 5: freeze-dried hepatitis A-hepatitis B combined vaccine thermal stability experiment

The combined vaccine is stored at different temperatures for different times, and then samples are taken to detect the virus titer of the hepatitis A vaccine, the efficacy of the hepatitis B vaccine, and the residual moisture content, so as to evaluate the thermal stability of the freeze-dried combined vaccine of hepatitis A and B of the present invention.

The freeze-dried combined hepatitis A-hepatitis B vaccine of the present invention is stored in the dark at a temperature of 2-8°C, during which samples are taken at 9, 12, 15, 21, and 24 months for hepatitis A vaccine virus titer, hepatitis B vaccine Potency, residual moisture content testing. The hepatitis B vaccine expressed by recombinant CHO cells was used as the reference vaccine. The results are shown in Tables 11 and 12 below.

Table 11 Determination of virus titer and hepatitis B relative potency after storage of different time at 2-8°C for hepatitis A-hepatitis B combined vaccine

Vaccine Hepatitis A virus titer (LogCCID ₅₀ /ml) (months) Hepatitis B relative potency (months)

Batch No. 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 Determination of residual moisture content of combined hepatitis A-B vaccine stored at 2-8°C for different periods of time

Residual moisture content (%) (month)

Vaccine lot 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

The freeze-dried combined hepatitis A-hepatitis B vaccine of the present invention was stored in the dark at room temperature for 6 months, and samples were taken at 0, 2, 3, 4, 5 and 6 months to carry out hepatitis A vaccine virus titer, hepatitis B vaccine efficacy and residual Moisture content detection. The hepatitis B vaccine expressed by recombinant CHO cells was used as the reference vaccine. The results are shown in Tables 13 and 15 below.

Table 13 Determination of Hepatitis A Virus Titer and Hepatitis B Relative Effectiveness in Room Temperature Storage of A-Hepatitis B Combined Vaccine

Vaccine Hepatitis A virus titer (LogCCID ₅₀ /ml) (months) Hepatitis B relative potency (months)

Batch No. 0 2 3 4 5 6 0 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 freeze-dried hepatitis A-hepatitis B combined vaccine of the present invention is stored at 37°C, and samples are taken at 0, 1, 2, 3, 4 and 5 weeks to detect the hepatitis A vaccine virus titer, hepatitis B vaccine efficacy and residual water content. The hepatitis B vaccine expressed by recombinant CHO cells was used as the reference vaccine. The results are shown in Tables 14 and 15 below.

Table 14 Determination of Hepatitis A Virus Titer and Hepatitis B Relative Efficacy of Hepatitis A-Hepatitis B Combined Vaccine Stored at 37°C

Vaccine Hepatitis A virus titer (LogCCID50/ml) (week) Hepatitis B efficacy (ED50) (week)

Batch No. 0 1 2 3 3 4 5 5 0 1 2 3 4 5

980401 6.50 6.50 6.33 6.50 6.00 6.00 2.79 2.76 2.71 2.60 2.68 2.59

980402 6.67 6.50 6.50 6.67 6.00 6.00 2.58 2.51 2.53 2.47 2.41 2.34

980403 6.67 6.67 6.50 6.50 6.33 5.67 2.88 2.84 2.77 2.69 2.58 2.50

980501 6.67 6.67 6.33 6.67 6.33 6.00 3.06 3.02 3.06 2.98 2.95 2.89

980502 7.00 7.00 6.50 6.67 6.50 6.00 2.45 2.48 2.41 2.39 2.30 2.21

Table 15 Determination of residual moisture content of combined hepatitis A-B vaccine stored at room temperature and 37°C for different periods of time

Vaccine Preliminary Test Residual moisture at room temperature (%) (month) Residual moisture at 37°C (%) (week)

Batch No. Moisture (%) 2 2 4 5 6 1 2 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

As can be seen from the data shown in the above Table 6-15, compared with hepatitis A or hepatitis B monovalent vaccine, the freeze-dried hepatitis A-hepatitis B combined vaccine of the present invention is stored differently at room temperature, 2°C-8°C, and 37°C. After a period of time, the viral titer of the hepatitis A vaccine and the relative efficacy of the hepatitis B vaccine did not decrease significantly, and the efficacy of the hepatitis B vaccine was even higher than that of the monovalent hepatitis B vaccine. in addition. After the freeze-dried hepatitis A-hepatitis B combined vaccine of the present invention is stored at different temperatures for different times, although its residual moisture content increases to some extent, it does not affect the overall quality of the freeze-dried hepatitis A-hepatitis B combined vaccine of the present invention. Quality requirements.

Claims (12)

1. A combined hepatitis A and hepatitis B vaccine, comprising live attenuated hepatitis A vaccine virus and hepatitis B virus surface antigen protein. 2. A freeze-dried hepatitis A and hepatitis B combined vaccine, comprising live attenuated hepatitis A vaccine virus, hepatitis B virus surface antigen protein and protective agent. 3. The hepatitis A-B combined vaccine according to claim 1 or 2, which also contains an appropriate amount of immune adjuvant. 4. according to claim 1 or 2 Hepatitis A combined vaccine, wherein said live attenuated hepatitis A vaccine virus is hepatitis A attenuated live vaccine L-A-1 vaccine strain or other attenuated live vaccine strain . 5. The combined hepatitis A-B vaccine according to claim 1 or 2, wherein the hepatitis B virus surface antigen (HBsAg) protein is expressed and purified by recombinant CHO cells and/or recombinant yeast. 6. The combined hepatitis A-B vaccine according to claim 1 or 2, characterized in that the viral titer of said live attenuated hepatitis A vaccine is not lower than 6.5 LogCCID ₅₀ /ml. 7. The combined hepatitis A-B vaccine according to claim 1 or 2, characterized in that the content of said hepatitis B virus surface antigen protein is not less than 10 μg/ml. 8. The combined hepatitis A-B vaccine according to claim 2, wherein the protective agent comprises trehalose, glutamic acid, ascorbic acid, urea, sorbitol, inositol and dextran. 9. according to claim 2 Hepatitis A combined vaccine, wherein the composition of protective agent and their concentration (W/V) in combined vaccine composition suspension 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%, and dextran 1-6%. 10. The hepatitis A-B combined vaccine according to claim 2, wherein the adjuvant is provided by a solution containing 0.8-1.2 mg/ml aluminum hydroxide as a diluent of the lyophilized hepatitis A-B combined vaccine. 11. A method for preparing freeze-dried combined hepatitis A-B vaccine, the method comprising: (1) respectively provide and mix the attenuated hepatitis A vaccine virus stock solution and the purified hepatitis B virus surface antigen protein stock solution expressed by recombinant CHO cells or recombinant yeast in appropriate proportions to obtain hepatitis A vaccine virus and B A mixture of hepatitis virus surface antigens. (2) Add an appropriate amount of protective agent to the mixture obtained in step (1) and mix it uniformly to obtain the desired suspension of the combined hepatitis A and hepatitis B vaccine composition. (3) freeze-drying the combined vaccine suspension containing the protective agent obtained in step (2) to obtain the freeze-dried hepatitis A-B combined vaccine. 12. The method according to claim 11, wherein said freeze-drying step in step (3) comprises at first said combined vaccine composition at about -30 to -55°C under low temperature conditions, pre-freezing for 3-7 hours, and then Gradually raise the temperature to 35°C and dry in vacuum for 8 to 16 hours.