WO2000029018A1

WO2000029018A1 - Combined vaccine against both hav and measle virus and method of producing it

Info

Publication number: WO2000029018A1
Application number: PCT/CN1999/000162
Authority: WO
Inventors: Pengfu Wang; Jingye Liu; Guangpu Li; Yifan Nan; Meihua Wang; Wei Wang; Jianjun Guo; Baosheng Xie; Zongming Song; Cuibo Qu; Lisha Liu; Jinfeng Huang; Zongju Wan
Original assignee: Changchun Institute Of Biological Products Ministry Of Public Health
Priority date: 1998-11-12
Filing date: 1999-10-13
Publication date: 2000-05-25

Abstract

The invention discloses a kind of combined vaccine against both HAV and measle virus and a lyophilization protective agent. It also discloses a process of producing the combined vaccine in the same host cell medium, of preparing the said lyophilized combined vaccine using the said protective agent and a method of producing the lyophilization protective agent.

Description

Hepatitis A-measles combined vaccine and production method thereof

The present invention relates to a combined hepatitis-measles-measles vaccine (HM) and its lyophilized protective agent. The present invention further relates to a method for preparing said hepatitis A-measles combined vaccine (HM) and its lyophilized protective agent. technical background

Since the launch of the Children's Vaccine Initiative (CVI) 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 committed to Development of new vaccines. These new vaccines should be multivalent, thermally stable, and can be administered in the form of oral or aerosol. From the perspective of clinical application, people hope that the combined vaccine can be effective after a single immunization To prevent two or more diseases in order to reduce vaccination costs, improve immunization efficiency, and reduce the workload of health care doctors and epidemic prevention departments, thereby improving the current low immunization rate of infants and young children. In recent years, a new type of combination has been approved The vaccine is a combination vaccine of whole cell diphtheria (combination of diphtheria, pertussis and tetanus toxoid) and Haemophilus influenzae B. In addition, given the feasibility of using the same syringe to administer a mixed vaccine product, related principles It has also been used to inoculate different parts of the human body at the same time. The so-called "mix and match" procedure for vaccine preparations. However, to date, there have been no reports concerning the combined preparation of combined live vaccines (HM) with live attenuated liver vaccines and other attenuated vaccines, such as measles vaccines, especially in the same Reports on the preparation of a combined vaccine by successive inoculation of two or more viruses in the matrix of a sensitive host cell.

An object of the present invention is to provide a combined vaccine for hepatitis A measles (HM) The vaccine contains hepatitis A live attenuated vaccine virus and live attenuated attenuated vaccine virus that do not interfere with each other and are effective in preventing two viral diseases such as hepatitis A and measles, with or without live vaccine virus protection Agent.

According to the combined hepatitis V measles vaccine (HM) of the present invention, the effective virus titer of the live attenuated hepatitis V vaccine is》 10 ⁶⁰ CCID ₅₀ / ml, and the effective virus titer of the live attenuated measles vaccine is> 10 ^{3 5} CCID ₅₀ / mL

Wherein, the combined hepatitis A measles vaccine (HM) provided by the present invention is in a freeze-dried form.

According to the present invention, the hepatitis 曱 -measles combined vaccine (HM), wherein the live vaccine virus protection agent is a human serum albumin consisting of a weight / volume ratio (w / v%) of 0 to 2%, 0.5 to 1 % Gelatin, 5 ~ 10% trehalose, 0.75 ~ 1.5% sodium glutamate, 0.05 ~ 0.55% ascorbic acid, 0.5 ~ 2.8% urea, 5 ~ 10% sorbitol and / or mannitol and 0.5 ~ 1 % Inositol and other aqueous solutions.

Another object of the present invention is to provide a method for producing and preparing hepatitis 曱 -measles combined vaccine (HM), which method comprises reducing hepatitis 曱 hepatitis virus with a virus titer of> 10 ^{7 0} CCID ₅₀ / ml obtained by conventional methods. The live virus vaccine stock solution and the virus titer of> 10 ⁴⁵ CCID ₅₀ / ml of the live attenuated attenuated vaccine of measles are mixed in an appropriate ratio. In the combined vaccine stock solution obtained, the virus titer of the live attenuated vaccine for hepatitis A virus Not less than 10 ⁰ CCID _5Q / ml, and the virus titer of the measles live attenuated vaccine stock solution is not less than 10 ⁴ CCID ₅ . / ml.

Yet another object of the present invention is to provide a method for producing a hepatitis B measles-measles combined vaccine (HM) by using the same host cell matrix, the method comprising the following steps:

1) Provide the attenuated live attenuated hepatitis A vaccine virus strains and measles live attenuated vaccine virus strains by conventional methods;

2) Inoculate human embryo lung diploid cells with the live attenuated hepatitis B virus vaccine virus provided in step 1) and culture said cells under appropriate conditions to proliferate the virus. When the hepatitis A virus is infected When the positive rate of the cells reaches more than 75%, re-inoculate the measles live attenuated vaccine virus strain provided in step 1) and continue to culture it;

3) When the positive rate of hepatitis B virus-infected cells reaches more than 90%, and more than 90% of the diploid stromal cells show a typical cytopathic disease (CPE) caused by measles virus, the cells are harvested, and according to those skilled in the art Known methods such as: freeze-thaw, ultrasound, centrifugation, purification and other steps to obtain the required hepatitis 曱 -measles combined vaccine stock solution.

According to a preferred embodiment of this object of the present invention, the infection rate (positive rate) and viral titer LogCCID _{5 of the} diploid cell matrix of said hepatitis B virus infection. / ml, was detected by standard immunofluorescence (IF) and ELISA methods.

According to another preferred embodiment of this object of the present invention, the method further comprises the step of: synthesizing the hepatitis A measles combined vaccine stock solution obtained in step 3) with a live vaccine provided by the present invention in a ratio of about 1: 1. Virus protection agents are mixed and lyophilized in a conventional manner.

The live vaccine virus protection agent is basically composed of human serum albumin with a weight / volume ratio (w / v%) of 0 ~ 2%, 0.5 ~ 1% gelatin, 5 ~ 10% trehalose, 0.75 A suspension aqueous solution composed of ~ 1.5% sodium glutamate, 0.05 ~ 0.55% ascorbic acid, 0.5 ~ 2.8% urea, 5 ~ 10% sorbitol and / or mannitol, and 0.5 ~ 1% inositol.

It is still another object of the present invention to provide a method for preparing a lyophilized preparation of the combined hepatitis A measles vaccine (HM) as defined above, the method comprising:

1) Provide a live attenuated live vaccine for hepatitis A vaccine and a live attenuated live vaccine for measles with effective virus titers, respectively, and mix them into a suspension of the live vaccine for hepatitis A-measles combined vaccine;

2) In the combined vaccine stock solution described in step 1), the live vaccine virus protection agent as defined above is added at a ratio of about 1: 1 (v / v) and uniformly mixed;

3) The combination vaccine composition obtained in step 2) is freeze-dried.

According to a preferred embodiment of this object of the present invention, wherein said step 3) comprises first pre-freezing said combination vaccine composition at a temperature of about -20 ~ -50 ° C for 3 ~ 6 hours, Then in a suitable freeze-drying device, the temperature of the product is lower than its eutectic melting point, and the temperature is gradually increased from -35 ° C to 35 ° C, and the product is dried under vacuum for 8-20 hours.

According to another preferred embodiment of this object of the present invention, the effective titer of hepatitis A virus in the lyophilized hepatitis A measles combined vaccine is> 10 ^6. ° CCID ₅ . / ml, effective titer of measles virus is> 10 ^{3 5} CCID ₅₀ / mL

The present invention provides a hepatitis A-measles combined vaccine preparation (HM) composed of live hepatitis A attenuated live vaccine virus stocks and measles live attenuated live vaccine virus stocks, which have effective virus titers. The combined vaccine formulation (HM) may or may not contain a live vaccine virus protectant.

Measles is a severe infectious disease that is caused by the measles virus and is transmitted through the respiratory tract, which seriously endangers human health. Usually, it can obtain lasting or even lifetime protection after the first immunization and booster immunization. The primary immunization is usually carried out at the age of 8 months, and the booster immunization is given again one year later or before school age, so that the vaccinated person can obtain lasting or even lifetime immunity. With the implementation of the Childhood Immunization Program (EPI), measles will become the second infectious disease to be completely eliminated globally after polio. At present, live attenuated measles vaccines commonly used in China are prepared and provided based on attenuated strains of Measles -47 or Shanghai -191, and are mostly subcultured using chicken embryo primary cells. However, our research shows that the use of human embryo lung diploid cell monolayers or cell suspensions as the stromal cells for the measles virus passage and proliferation will facilitate the virus's proliferation and the preservation of the virus's antigenicity, as well as more effectively avoid other foreign sources. Factor pollution and so on.

On the other hand, as is well known, hepatitis A is another acute infectious disease with a high global incidence caused by hepatitis A virus, which is widely present in nature. In recent years, the inventors have used their own attenuated hepatitis B virus strain LA-1 as a seed virus, and used human embryo lung diploid cells as stromal cells to achieve the industrialized large-scale production of a live attenuated hepatitis A vaccine. A lot of research and production practice experience. So far, more than 80 million susceptible people have been vaccinated with the vaccine, which not only has excellent safety in use, but also has a protection rate of one immunization. Above 95%, the protection rate of the second immunization reached 100%.

We have found in long-term research and production practice that human embryo lung diploid cells are not only a good host for hepatitis A vaccine virus, but also a good host for measles vaccine virus. In particular, we found that the measles vaccine virus proliferated faster in human embryo lung diploid host cells. Specifically, we ca n’t find out in advance that after inoculating human embryo lung diploid cell matrix firstly with the live attenuated liver attenuated live vaccine vaccine species, culturing the cells in an appropriate temperature and medium for about 2 to 3 weeks, When the hepatitis B vaccine virus has increased to a sufficiently high virus titer (for example> 10 ^{5 5} CCID _5Q / ml), the measles vaccine virus is inoculated and cultured. After about one week, the two live vaccine viruses reach corresponding high proliferation at the same time. Level, a combined vaccine stock solution containing two attenuated live vaccine virus liquids can be obtained at the same time. This discovery constitutes the basis for the proliferation of two live attenuated vaccine viruses in the same host cell (for example, human embryo lung diploid cells), and thus the preparation of a combined hepatitis-Measles vaccine.

Of course, the hepatitis A live attenuated vaccine stock solution and the measles live attenuated vaccine stock solution containing effective virus titers can also be prepared separately, and the two live vaccine stock solutions are mixed in an appropriate ratio to obtain the liver-measles-measles in suspension form. Combined vaccine stock solution. The effective virus titer mentioned here is generally the nail liver vaccine virus titer is> 10 ^{7 0} CCID ₅₀ / ml, and the measles vaccine virus titer is> 10 ^{4 5} CCID ₅ . / ml.

Therefore, the present invention further provides a method for producing a combined hepatitis B measles vaccine (HM) using the same host stromal cells, the method comprising:

1) To provide attenuated live attenuated hepatitis B vaccine virus virus strains and measles live vaccine virus strains by conventional methods;

2) Inoculate human embryo lung diploid cells with the live attenuated hepatitis B virus vaccine virus provided in step 1) and culture the cells under appropriate conditions to proliferate the virus. When the positive rate reaches more than 75%, re-inoculate the measles live attenuated vaccine virus strain provided in step 1) and continue to cultivate it;

3) When the positive rate of hepatitis B virus-infected cells reaches more than 90%, and more than 90% of the diploid stromal cells show the typical cytopathic (CPE) caused by measles virus, harvest the fine Cells and virus-free maintenance solution containing no newborn bovine serum, and according to methods known to those skilled in the art, such as: freeze-thaw, ultrasound, centrifugation, purification and other steps, to obtain the required hepatitis A measles combined vaccine stock solution. On the other hand, in order to prepare the combined hepatitis A-measles vaccine (HM) of the present invention, first, a live attenuated hepatitis A vaccine virus stock solution and a live-attenuated attenuated vaccine virus stock solution can be prepared according to known methods. For example, a live hepatitis B attenuated live attenuated vaccine virus virus stock solution can be prepared according to the method described in Chinese Patent No. 92114998.0. At the same time, in accordance with the current Chinese biological product regulations, a long-47 or Shanghai-191 measles virus strain was used to prepare a live attenuated live vaccine virus stock.

Hepatitis I virus can proliferate in many tissue culture cells, such as human embryo lung diploid cells, but the proliferation rate is relatively slow and does not cause morphologically obvious cytopathic changes. On the contrary, measles virus can not only proliferate in many primary or passage cells, but also has a relatively short period of proliferation, a high rate of virus proliferation in the same time, and can produce typical fusion giant cell-like lesions, especially our Experiments have further shown that human embryo lung diploid cells are sensitive host cells for both the aforementioned hepatitis B vaccine virus strains and measles vaccine virus strains. Therefore, we have selected human embryo lung diploid cells as the cultured proliferative liver vaccine virus and measles vaccine. A preferred common host cell matrix of the virus is used to prepare the combined hepatitis A measles vaccine (HM) of the present invention.

To achieve this, human embryo lung diploid cells are first expanded and passaged to a sufficient cell volume and cell density according to conventional methods, for example, growing into a dense cell monolayer in a culture container. Inoculate the cell matrix with live attenuated liver attenuated live vaccine vaccine at a multiplicity of infection of 0.02-10 (mo 丄), culture at approximately 32-35 ° C, and use known immunofluorescence (IF) to directly monitor the virus Infection rate. After about 2 to 3 weeks, when more than 75% of the virus-infected cells are immunofluorescent, replace the MEM maintenance solution containing 2 ~ 8% newborn bovine serum, and inoculate the measles attenuated at 0.01-10. Live vaccine virus.

After inoculation, human embryo lung diploid cells that have been successively infected with hepatitis B virus and measles virus are cultured at 33 ~ 35 ° C, and the maintenance solution is replaced every 3 to 5 days according to the degree of cell growth and maintenance. Observe cell morphology changes daily. When 90 ~ 100% of infected cells appear measles When the typical cytopathic effect (CPE) caused by viral infection, and the positive rate of hepatic virus infection reaches more than 90%, the cell culture is moved to a temperature of 2 ~ 8 ° C for cold release for 1-3 days, and then low temperature

Store the cells frozen (below -20 ° C).

Alternatively, as another alternative operation method, when the positive rate of diploid cells infected with hepatitis B virus reaches 75% or more, discard the maintenance solution, and wash with a balanced salt solution (PBS or Earle's solution) Rinse cells to remove residual newborn bovine serum. Then replaced with 0.01 ~ 0.25%

(w / v) Eagle's MEM of human serum albumin or other suitable medium (for example, 199 comprehensive medium), and inoculated with a multiplicity of infection (m.o.i.) of 0.01 to 10 to inoculate the live measles attenuated vaccine virus. The resulting culture was cultured at 33 ~ 35 ° C and samples were taken daily to observe the typical CPE caused by measles virus infection. When the CPE reaches 90 ~ 100%, and when the positive rate of the cells infected by the liver vaccine virus also reaches more than 90%, the cell culture is cold-released at 2 ~ 8 ° C for 1 ~ 3 days, and then collected and cooled (Below -20 ° C) Keep it frozen.

After three freeze-thaw and ultrasonic treatments, the cells prepared according to the method described above were disrupted, the cell debris was removed by centrifugation, and the supernatant was collected to obtain a stock solution of hepatitis A-measles combined vaccine in the form of a suspension.

Of course, after preparing a live attenuated hepatitis B vaccine stock solution and a measles live attenuated vaccine stock solution with effective virus titers, the two attenuated activities containing the required virus titer may be mixed in an appropriate volume ratio. The vaccine stock solution is prepared as a suspension of the hoe liver-measles combined vaccine stock solution. The effective virus titer mentioned here is generally the nail liver vaccine virus titer is> 10 ⁷⁰ CCID ₅₀ / ml, the measles vaccine virus titer is> 10 ^{4 5} CCID ₅₀ / mL

The present invention further provides a method for preparing a lyophilized preparation of the combined hepatitis A measles vaccine as defined above, which method comprises:

1) Provide a live attenuated hepatitis B vaccine and a live attenuated vaccine for measles with effective virus titers, respectively, and mix them to form a suspension of the live vaccine for hepatitis-measles combined vaccine;

2) In the combined vaccine stock solution described in step 1), the live vaccine virus protection agent as defined above is added at a ratio of about 1: 1 (v / v) and mixed uniformly; 3) freeze-drying the combined vaccine composition obtained in step 2).

The invention further provides a freeze-dried liver-measles combined vaccine and a method for producing a freeze-dried protective agent thereof. The thus-prepared suspension-shaped liver-measles combined vaccine is prepared into a lyophilized protective agent Vaccine preparations, further changing that suspended liquid vaccines must be transported, stored, and used under low-temperature refrigeration conditions (that is, through the so-called "cold chain"), enabling the large-scale promotion and use of live attenuated vaccines, especially in less developed regions and tropical regions And restricted use in subtropical regions.

In order to prepare a freeze-dried liver-measles combined vaccine (HM) in the form of a suspension prepared as described above into a lyophilized preparation, the prepared suspension of the liver-measles-measles combined vaccine stock solution and a lyophilized attenuated live vaccine can be prepared. The protective agent is mixed in a ratio of about 1: 1 (v / v). After aliquoting under aseptic conditions, the resulting mixture is placed in an appropriate freeze-drying device for freeze-drying treatment to obtain a freeze-dried form of hepatitis A-measles live attenuated vaccine composition of the present invention.

According to a preferred embodiment of the present invention, the freeze-drying process includes firstly suspending the hepatitis A-measles live attenuated vaccine stock solution in the form of a suspension at about -30 ° C to -50 ° C, preferably at about- Pre-freeze at 40 ° C for 3 ~ 6 hours, and then vacuum-dry at -35 ° C to 35 ° C for 8 ~ 20 hours, to obtain frozen hepatitis C measles-measles combined vaccine (HM).

According to another preferred embodiment of the present invention, the lyophilized protective agent is substantially composed of human serum albumin having a weight / volume ratio (w / v%) of 0 to 2%, gelatin of 0.5 to 1%, 5 ~ 10% trehalose, 0.75 ~ 1.5% sodium glutamate, 0.05 ~ 0.55% ascorbic acid, 0.5 ~ 2.8% urea, 5 ~ 10% sorbitol and / or mannitol and 0.5 ~ 1% inositol And other composition of aqueous solution.

In the live vaccine virus protection agent of the present invention as described above, human serum albumin and gelatin mainly provide protein and colloid scaffolding functions, and provide space support and partial nutrition protection for live or lyophilized live viruses; Trehalose has the function of stabilizing the structure of cells and proteins and improving the ability to resist high temperature damage. Studies have shown that certain bioactive materials such as antibodies, enzymes, viruses, etc. that have been freeze-dried in the presence of trehalose can regain their vitality after rehydration (Roser B. JFood Sci & Technol. 2 (7): 166-169, 1991; Roser B., Biopharm. 4 (8): 47-53, 1991; Roser B., Colace C., New Scientist, 138: 24-28, 1993) . At present, the use of trehalose to lyophilize polio vaccine has been successful. Basic amino acid salts such as sodium glutamate, urea, sorbitol and / or mannitol and inositol, and ascorbic acid in the lyophilized protective agent of the present invention mainly play a role in adjusting the pH, stabilizing the hydration state, or osmotic pressure during dehydration. And antioxidant effects.

However, it should be particularly pointed out that our experiments show that when used for some non-enveloped viruses, such as hepatitis A virus, measles virus and polio virus, the lyophilized protection agent may not contain humans. Serum albumin is self-contained, but still has a good freeze-drying effect. Therefore, when preparing the hepatitis A-measles combined vaccine preparation of the present invention, the lyophilized protective agent used may not include the more expensive human serum albumin.

The live vaccine virus protection agent of the present invention can be prepared in a suitable container according to a conventional reagent preparation method, but before mixing trehalose, gelatin, sorbitol and / or mannitol, and inositol, these substances should be firstly incubated at 37 ° C. Pre-heat for 24 to 48 hours under conditions, and mix the protective agent and the lyophilized vaccine stock solution at a ratio of about 1: 1 (v / v) uniformly within 0.5 to 2 hours before the vaccine is dispensed. A conventional freeze-drying device can be used to pre-freeze the aliquoted mixture at -30 ~ -50 ° C for 3 ~ 6 hours, and then vacuum-dry at -35 ° C to 35 ° C for 8 ~ 20 hours. Within this temperature range, the frozen mixture will gradually sublimate and be dried. During the freeze-drying process, the eutectic temperature of the vaccine frozen mixture is lower than -30 ° C.

In order to confirm the protective effect of the live vaccine virus protective agent of the present invention on its live vaccine virus in its freeze-drying process and its effect on the storage stability of the live vaccine virus after freeze-drying, we combined the liver-measles-measles combination of the present invention The vaccine is taken as an example, and the effect of the freeze-dried protective agent of the present invention on the titer of the virus before and after lyophilization, and the effect of the lyophilized protective agent on the storage stability of the vaccine were performed. A series of comparative experiments. The test results show that the live vaccine virus protection agent of the present invention not only has excellent protective effect on virus activity during the freeze-drying process, but also can significantly improve the freeze-dried hepatitis-measles combined vaccine (HM) at room temperature. Storage stability under elevated conditions and temperature conditions.

The experimental results show that the hepatitis 曱 -measles combined vaccine (HM) of the present invention has excellent specificity. Using the freeze-dried cricket hepatitis-measles combined vaccine (HM) prepared according to the method of the present invention, the in vitro cricket liver virus identification test and the measles virus identification test were performed respectively. The results show that the lyophilized combined vaccine sample of the present invention is marked with a certain label Amounts of hepatitis A virus and measles virus can be completely neutralized after acting on their corresponding heterologous high titer specific immune serum.

Our in vivo tests using rhesus monkeys or red-faced monkeys as test subjects have further shown that the freeze-dried liver-measles combined vaccine (HM) of the present invention has excellent safety and high use in primates. Immunogenicity. The test results showed that during the 8-week observation period, the healthy rhesus monkeys or red-faced monkeys were injected with an appropriate amount of the lyophilized hepatitis A-measles combined vaccine (HM) of the present invention, and no paralysis and any other nervous system occurred in the animals. Symptoms and histopathological examination of the central nervous system showed no abnormal changes. In addition, continuous liver histopathological examinations and serum alanine aminotransferase (SGPT) measurements performed before and after intravenous administration of the combination vaccine of the present invention to test animals did not reveal any significant changes. At the same time, it was found that the serum anti-HAV IgM and anti-measles virus IgM antibodies appeared in the second week after the animals were vaccinated, but gradually disappeared by the eighth week. On the other hand, specific anti-HAV and anti-measles virus hemagglutination inhibitory (HI) antibodies showed 50% animal antibody positive conversion at the second week, and the positive conversion of both antibodies reached 100% at the fourth week. In addition, the titers of liver dysentery and measles virus neutralizing antibodies in the serum 8 weeks after the animals were vaccinated with the combined vaccine were tested, and the results showed that the serum dilutions were greater than 1: 4.

Finally, in order to prove the applicability and tolerability of the human hepatitis B measles combined vaccine inoculated in humans, the inventors further vaccinated healthy infants aged 8-12 months with live attenuated hepatitis B vaccine (LA -1) and lyophilized live attenuated measles vaccine (Chang-47) and clinical observations of live attenuated live attenuated hepatitis B vaccine and live attenuated live attenuated measles vaccine. It was found that all 275 subjects did not show any local redness and swelling allergic reactions during the 72-hour observation period, and did not see any related systemic adverse reactions during the 72-day observation period. Statistics The results of medical treatment showed that there was no significant difference in the positive rates of anti- 病毒 hepatitis virus antibodies and anti-measles virus antibodies between the two groups after vaccination with any one of the vaccines and the combined use of the two vaccines; There was no significant difference in the geometric mean titers (GMT) of the two antibodies produced by the researchers.

Therefore, it can be speculated on this basis that it is possible to make two or even multiple vaccine preparations of hepatitis V vaccine and measles vaccine that do not interfere with each other, and gradually incorporate them into the children's planned immunization. For example, especially for infants and young children in areas with high incidence of hepatitis A, an effective preventive dose of the combined hepatitis A-measles vaccine of the present invention can be injected at the age of 10-13 months to complete the initial prevention of hepatitis A and measles at the same time. Immunization, or injecting a combined vaccine for hepatitis A and measles in school-aged children to complete the vaccination of hepatitis A vaccine and booster immunity for measles vaccine at the same time, so as to achieve the purpose of early prevention of two diseases. Embodiments of the present invention Example 1 Preparation of Hepatitis A-Measles Combined Vaccine

First, human embryo lung diploid cells were cultured using Eagle's minimal medium (MEM) according to a conventional method. After about 5 days, when the cells proliferate to form a dense monolayer, inoculate Hepatitis A virus LA-1 as described in Chinese Patent No. ZL 92114998.0 at about 4.5 (moi), supplement the maintenance solution (plus 2 to 5 % Newborn bovine serum in MEM medium), continue to culture the cells, and continue to culture for 3 weeks, change the fresh medium once a week, and monitor the proliferation level of the 曱 liver vaccine virus by indirect immunofluorescence (IF). After 3 weeks, when about 75% of the infected cells were positive for immunofluorescence, the maintenance solution was replaced again, and the measles virus long-47 strain was inoculated at about 4.5 m.o.i.

Then the human embryo lung diploid cells that have been successively infected with the hepatitis B vaccine virus and the measles vaccine virus continue to be cultured at a temperature of 32 ~ 35 ° C, and the culture medium is replaced every 4-7 days, and the cells are taken daily The morphological changes of the samples were observed under a high magnification microscope. When about 50% of the cells come out When a typical cytopathic disease caused by measles virus is present, the remaining newborn bovine serum is washed away with a balanced salt solution such as Earle's solution. Then the cells were re-cultured in Eagle's MEM culture medium (vaccine solution) without newborn bovine serum, and continued to be cultured until the incidence of cytopathic disease reached 90 ~ 100%, and the positive rate of 曱 hepatitis virus infected cells reached about 95%. At that time, the culture was terminated and the cell culture was cold-released at 2-8 ° C for about 72 hours, and then the cell culture was frozen and stored at -20 ° C.

Cryopreserved cells were repeatedly frozen and thawed three times, and the cells were sonicated for 10-15 minutes to disrupt the cells. After centrifugation (2000 rpm, 4 ° C, 15 minutes) to remove cell debris, the supernatant was collected and purified to obtain suspended liquid mash. Liver-measles combined vaccine stock solution.

Example 2 Preparation of a lyophilized protective agent for hepatitis 曱 -measles combined vaccine

First, a lyophilized protective agent for the preparation of a lyophilized liver-measles combined vaccine was prepared as follows:

Dissolve 0.8 g of medical gelatin in 30 ml of distilled water, heat and boil to dissolve it, sterilize by autoclaving at about 116 ° C for 40 minutes, and then cool to room temperature to about 30 ° C. To the gelatin solution was added 1.0 g of human serum albumin obtained through sterilization using a 0.22 μηι filter membrane or filter column, and the mixture was thoroughly stirred and set aside, and then 8.0 g of trehalose and 1.0 g of sodium glutamate were weighed separately. 0.1 g of ascorbic acid, 2.5 g of urea, 8.0 g of sorbitol, and 0.6 g of inositol, and then add them to 50 ml of distilled water in sequence. After shaking and stirring, the mixture is preheated at 37 ° C for 24 hours. The next day, the mixture was cooled to room temperature (about 25 ° C) and mixed with the above-mentioned gelatin-human serum albumin solution, and then sterilized water for injection was added to 100 ml, and the pH was adjusted to 7.0 with 0.1N HC1. After filtering and sterilizing again, the required vaccine lyoprotectant was obtained.

This lyophilized protective agent was mixed with the suspension liquid hepatitis A-measles combined vaccine stock solution prepared according to the method described in Example 1 in a volume ratio of 1: 1, and aseptically dispensed in a vial (1.2 ml / bottle) and placed in a Bacterial sealed vacuum freeze dryer (FS150-SS20C, Full Co., USA) for lyophilization: first pre-freeze at about -40 ° C for 4 hours, and then gradually increase the temperature to 32 ° C for 15 hours and vacuum dry, Thus, a freeze-dried hepatitis A-measles combined vaccine preparation (HM) was obtained. Example 3 Thermal Stability of a Freeze-dried Liver-Measles Combined Vaccine

In this embodiment, the lyophilized hepatitis A-measles combination vaccine of the present invention is measured before and after lyophilization, and after storage at 2-8 ° C, room temperature (25 ° C) and 37 ° C for different times. The virus titer confirmed that the freeze-dried liver-measles combined vaccine (HM) of the present invention has good thermal stability.

Five batches of combined vaccine samples (HM1-5) produced continuously were taken before and after lyophilization, and were stored at 2-8 ° C, 25 ° C, and 37 ° C for different periods of time, and the combined vaccines were tested separately. Titers of cricket liver virus and measles virus.

Because measles virus infects human embryo lung diploid cells to produce a cytopathic effect (CPE), the measles virus should be neutralized first when measuring the titer of hepatitis virus. To this end, a 10-fold serial dilution of the lyophilized combination vaccine sample to be tested, take 0.1ml of a 10 · ² dilution sample and add 0.9ml of anti-measles virus serum diluted by the instruction manual (supervised by China Pharmaceutical and Biological Products Testing Institute), mix It was then placed at 2-8 ° C overnight to neutralize the measles virus. The next day a sample of the vaccine to be performed during and after the 10-fold dilution series, and by ^10-3-- ⁸ virus dilution was inoculated to a suitable culture has human diploid monolayer cells. Cells were harvested after 4 weeks of culturing according to the method described above. After routine disruption of cells, centipede liver antigens were collected by centrifugation, and then titers of pancreas virus were measured by ELISA, and LogCCID _{5 was} calculated by Reed-Muench method. / ml value.

In order to measure the measles virus titer in the freeze-dried liver-measles combined vaccine (HM) of the present invention, first, a 10-fold serial dilution of the combined vaccine sample and the measles virus reference product (provided by China Pharmaceutical and Biological Products) was used. ^10-26 dilution of virus suspension (1ml / vial) FL or Vero cells were seeded and cells were cultured at 34 ° C 1 week and then the above-described conventional method for detecting measles virus titer.

The results of this example are shown in Tables 1 and 2 below. Comparison of lyophilized liver-measles combined vaccine (HM) virus titer before and after lyophilization CCID50 / ml before lyophilization CCID50 / ml after lyophilization Sample batch number

曱 Liver Anesthesia 曱 Liver Measles

HM-1 7.0 * 5.38 * 6.50 * 5.13 *

HM-2 6.50 5.38 6.33 5.50

HM-3 6.50 6.13 6.00 5.75

HM-4 6.67 5.50 6.33 5.38

HM-5 6.50 5.00 6.33 4.63 indicates that the data given is the virus titer per unit volume (Log CCID ₅₀ / ml). Table 2 Storage stability of freeze-dried liver-measles combined vaccine (HM) at different temperatures

* The data in the table is the virus titer per unit volume (Log CCID _{5 /} ml). From the results shown in Tables 1 and 2, it can be seen that in the presence of the lyophilized vaccine virus protectant, the virus titer of the lyophilized liver-measles combined vaccine (HM) of the present invention is freeze-dried than lyophilized. There was a decrease, but the values of the titers of the two viruses in all five batches were <0.51 ^ ¾ CCID ₅ . / ml. In addition, the frozen chitosan liver-measles combined vaccine (HM) of the present invention is stored at 2-8 ° C for 12 months, The titers of both vaccine viruses were stable when stored at room temperature (25 ° C) for 3 months and at 37 ° C for 7 days. Therefore, it is shown that the lyophilized vaccine virus protection agent provided by the present invention has a good protection effect on the combined vaccine in its lyophilization process and the lyophilized vaccine virus. Example 4 Animal Experiments Demonstrating the Safety and Immunogenicity of the Freeze-Dried Liver-Measles Combined Vaccine (HM)

The rhesus monkey or red-faced monkey, a sensitive animal, was vaccinated with the freeze-dried liver-measles-measles combined vaccine of the present invention, and observed its clinical manifestations and examined the pathological changes of liver and brain tissues, as well as anti-HAV and measles blood in serum. Changes in titers of coagulation inhibiting antibodies (or neutralizing antibodies) are used to evaluate the safety and immunogenicity of combination vaccines.

In each group, 5 healthy rhesus monkeys or red monkeys with negative serum anti-HAV and anti-measles virus antibodies weighing 1.5-4.5 kg were vaccinated with 1.0 ml of freeze-dried liver-measles-measles combined vaccine sample by intravenous route, and each mouse The intracranial thalamus sites on both sides of the animals were punctured with 0.5 ml of vaccine samples. After the inoculation, the animals were observed for paralysis and other nervous system-related abnormalities, and the observation was continued for 21 days. Liver histopathology of liver tissue puncture was performed at 0, 4 and 8 weeks after vaccination; blood was collected at 0, 2, 3, 4, 6 and 8 weeks after vaccination to detect serum alanine aminotransferase (SGPT), and anti-diarrhea Liver virus and anti-measles virus antibodies. At the same time, monovalent vaccines were set as controls. After the last liver tissue puncture and blood collection, the animals were sacrificed under anesthesia to dissect the brain tissue of the animal, and a specimen was taken for pathological examination of the brain tissue.

In order to measure the neutralizing antibody titers in the serum of the animals after immunization, two known hepatitis A virus and measles vaccine viruses were diluted to 100 CCID ₅₀ / ml virus titers, respectively. The serum collected before and after vaccination of the animals with the combined vaccine of the present invention is heated at 56 ° C for 30 minutes to inactivate the serum, and the serum is diluted at different dilutions and mixed with an equal volume of the above-mentioned virus dilution to neutralize the corresponding virus. The obtained neutralization reaction products were then used to inoculate human embryo lung diploid cells and FL cells, respectively, and the results were judged by conventional methods known to those skilled in the art 7 days and 28 days after the cells were inoculated.

The results of this example are shown in Tables 3 and 4 below. Table 3 shows the inoculation of HM- Serum SGPT levels (measured by modified Reich's method,> 25 units / ml are abnormally elevated SGPT), and serum anti-HAV antibodies in three batches of liver-measles combined vaccine And anti-measles virus hemagglutination inhibition (HI) antibody positive rate (data are given as the number of antibody-positive animals in 5 animals).

Table 3 Serum transaminase levels and anti-hepatic liver and anti-measles antibody positive rates at 0 to 4 weeks after monkey body vaccination with the combined vaccine The sheep SGPT (u / ml) increased anti-HAV antibodies anti-measling virus HI antibody lot number 0 2 4 0 2 4 0 2 4

HM-1 0/5 0/5 0/5 0/5 0/5 · 3/5 * 5/5 * 0/5 * 4/5 * 5/5 '

H -3 0/5 0/5 0/5 0/5 0/5 5/5 5/5 0/5 4/5 5/5

HM-5 0/5 0/5 0/5 0/5 0/5. 3/5 4/5 0/5 5/5 5/5

H vaccine 0/5 0/5 0/5 0/5 0/5 3/5 5/5

M vaccine 0/5 0/5 0/5 0/5 0/5 4/5 5/5

* The data given are the number of animals that showed positive antibody response in the corresponding weeks after 5 animals received the combination vaccine. Determination of serum neutralizing antibody titers in monkeys before and after vaccination

* HM is the English abbreviation for Hepatitis-Measles Vaccine

** For virus control

From the results shown in Tables 3 and 4, it can be seen that within 4 weeks of inoculation of the combined liver-measles-measles vaccine of the present invention, the serum aminotransferases of all test animals were normal, and the pathology of living liver tissue was normal. The results of the medical examination showed that no abnormal pathological changes of liver tissue were found in all the tested animals. Clinical observation and pathological examination of the brain did not show any abnormalities in the peripheral and central nervous system. The experimental results showed that the anti-hepatitis virus neutralizing antibody titer of the animals was> 1: 4 and the measles neutralizing antibody titer was> 1: 4 after 4 weeks of inoculation with the freeze-dried liver-measles combined vaccine. In addition, changes in serum 曱 liver and measles Ig M antibodies that were used as indications for primary infection after the combined vaccination were also detected. The results showed that serum Ig M was positive at 2 weeks after vaccination, but by the 8th week Ig M gradually disappeared (data not shown). Example 5 Human observations to prove the safety and immunogenicity of the combined liver-measles-measles vaccine In order to further confirm the human clinical applicability of the combined hepatitis-measles-measles vaccine provided by the present invention, this example further observes the simultaneous vaccination of humans with liver-measles Clinical efficacy of two live attenuated vaccines against measles.

A total of 275 healthy infants from August to December were randomly divided into three groups: the monovalent live attenuated liver vaccine (H), the monovalent live attenuated vaccine vaccine (M) and the live attenuated liver and measles Simultaneous vaccination group (HM). The virus titers of the live attenuated liver vaccine (LA-1) and live attenuated measles vaccine (Chang-47) (both produced by the Ministry of Health and the Chun Institute of Biological Products) have virus titers of 6.5 Log CCID ₅₀ / ml and 4.0 Log CCID ₅ . / ml. As usual, 1.0 ml of 曱 liver vaccine and / or 0.2 ml of measles vaccine were injected subcutaneously into the deltoid muscle of the upper arm of each person. The local reaction at the injection site and the systemic reaction (temperature change) within 42 days were regularly observed within 72 hours after the inoculation, and the number of clinical reactions was recorded as weak, medium and strong. Serum SGPT was detected at 4 and 8 weeks after immunization (using Shimadzu microfluidic cell spectrophotometer for a normal value of 120 units). Using an ELISA kit

(Abbott Kit ELISA, American Abbott Corp.) was used to detect anti-hepatitis A virus antibodies in serum, and a domestic ELISA kit was used to detect anti-measles HI antibodies.

The results showed that 2 out of 103 people who had received live attenuated liver and measles attenuated vaccines

(1.94%) Weak systemic reactions occurred, and one person (0.97%) had moderate systemic reactions (weak and moderate systemic reactions were both 0.70% in the control group H), and none of the other subjects had any localized reactions. And systemic clinical response.

The results of antibody titer analysis showed that after 8 weeks of monovalent hepatitis A vaccine or combined immunization with liver disease and measles vaccine, the anti-hepatitis antibody positive rate in the HM group was 92.27%, and the H group was 93.66%. There was no significant difference between the two groups ( X ² = 0.19, p> 0.05 geometric mean titer of antibody in two groups of subjects

(GMT) were 5.005 ± 2.538 and 4.886 ± 2.610, respectively, and there was no significant difference between the two groups.

(t = 0.196, ρ> 0.05).

In addition, after 8 weeks of monovalent measles vaccine or combined vaccination with liver and measles vaccine, the positive rate of anti-measles virus HI antibody in the ΗM group was 98.05%, and the M group was 96.66%. There was no significant difference in the antibody positive rate between the two groups ( X ² = 0.204, p> 0.05) „The geometric mean titers (GMT) of the antibodies in the two groups of subjects were 16.22 ± 2.29 and 13.93 ± 2.59, and there was no significant difference between the two groups.

(t-0.86, p> 0.05 industrial applicability

In summary, it can be concluded that the freeze-dried hepatitis-measles combined vaccine (HM) provided by the present invention is not significantly different from the monovalent vaccine group in terms of safety and immunogenicity, and is used in clinical applications. It is feasible in both industrial and industrial production. For children, especially infants and young children in areas with high incidence of hepatitis A, a vaccinated freeze-dried hepatitis A measles vaccine provided by the present invention can be injected once in an effective dose at the age of 10-12 months. (HM), in order to complete the combined immunization of hepatitis B vaccine and measles vaccine at the same time, to achieve the purpose of early prevention of two diseases, and also to inoculate the freeze-dried hepatitis B measles vaccine of the present invention in preschool or school-age children, In order to complete the primary / or booster vaccine of hepatitis B vaccine and the booster immunity of measles vaccine at the same time, the purpose of ultimate control or elimination of both diseases is achieved.

Claims

Claim

1. A combined vaccine for hepatitis A and measles, characterized in that the combination vaccine contains virus titers of vaccines that are effective for preventing two types of viral diseases, hepatitis A and measles, and does not contain live vaccines. Virus protection agent.

2. The combined hepatitis A-measles vaccine according to claim 1, wherein the effective virus titer of the live attenuated hepatitis A vaccine is> 10 ⁶⁰ CCID ₅₀ / ml, and the effective virus titer of the live attenuated measles vaccine > 10 ^{3 5} CCID ₅₀ / mL

3. The combined hepatitis 肝 -measles vaccine according to claim 1, wherein said combination vaccine can also be in a freeze-dried form.

4. The combined hepatitis 肝 -measles vaccine according to any one of claims 1 to 3, wherein said live vaccine virus lyoprotectant is a human whose weight / volume ratio (w / v) is 0 to 2%. Serum albumin, 0.5 ~ 1% gelatin, 5 ~ 10% trehalose, 0.75 ~ 1.5% sodium glutamate, 0.05 ~ 0.55% ascorbic acid, 0.5 ~ 2.8% urea, 5 ~ 10% sorbitol and An aqueous solution consisting of mannitol and 0.5 to 1% inositol.

5, a process for preparing hepatitis Yue combination vaccine of measles, which will include a conventional vaccine virus titers were obtained proliferative "10 ^{7 0} CCID ₅₀ / ml of the live attenuated hepatitis A vaccine Yue stock virus titer 10 ^{4 5} CCID ₅₀ / ml live measles attenuated vaccine stock solution was mixed in an appropriate ratio.

6. A method for preparing a combined hepatitis A-measles vaccine, which is characterized in that the method uses the same host cell matrix to prepare a combined hepatitis A-measles vaccine (HM), including the following steps:

1) Provide hepatitis A live attenuated live vaccine virus strains and measles live attenuated live vaccine virus strains prepared according to conventional methods;

2) Inoculate human embryo lung diploid cells with the hepatitis B attenuated live attenuated vaccine virus virus provided in step 1) and culture said cells to proliferate the virus. When the positive rate of hepatitis A vaccine virus-infected cells reaches When it is above 75%, re-inoculate the measles live attenuated vaccine virus strain provided in step 1) and continue to cultivate it; 3) When the positive rate of hepatitis B vaccine virus-infected cells reaches more than 90%, and more than 90% of the diploid stromal cells show typical cytopathic effects caused by measles virus, the cells are harvested, and subjected to freeze-thaw, ultrasound, centrifugation, Purification step to obtain the required hepatitis 曱 -measles combined vaccine stock solution.

7. The method according to claim 6, wherein the positive rate and viral titer Log CCID ₅₀ / ml of human diploid cell matrix infected by the hepatitis B vaccine virus is using a standard immunofluorescence method (IF) And ELISA method.

8. The method according to claim 5 or 6, further comprising mixing the obtained hepatitis A measles combined vaccine virus stock solution with a lyophilized live vaccine virus protection agent at a ratio of about 1: 1, and freezing in accordance with a conventional method Thousands of dryness.

9. The method according to claim 8, wherein the live vaccine virus protecting agent is basically composed of human serum albumin having a weight / volume ratio (w / v) of 0 to 2%, gelatin of 0.5 to 1%, 5 ~ 10% trehalose, 0.75 ~ 1.5% sodium glutamate, 0.05 ~ 0.55% ascorbic acid, 0.5 ~ 2.8% urea, 5 ~ 10% sorbitol and / or mannitol and 0.5 ~ 1% inositol Of an aqueous solution.

10. A method for preparing a lyophilized preparation of the combined hepatitis A measles vaccine obtained according to any one of claims 5 to 9, the method comprising:

2) In the combined vaccine stock solution described in step 1), add the frozen thousand live vaccine virus protection agent as defined above and mix it uniformly at a ratio of about 1: 1 (v / v);

3) The vaccine composition obtained in step 2) is freeze-dried.

11. The method according to claim 10, wherein step 3) comprises first pre-freezing said vaccine composition at a temperature of about -20 ~ -50 ° C for 3 to 6 hours, and then at -35 ~ 35 ° C Dry under vacuum for 8 to 20 hours.