TW201642901A - Tranquilizer composition for vaccine and preparation method thereof, and classical swine fever vaccine containing the same - Google Patents

Abstract

This invention provides a tranquilizer composition for vaccine, which is based on the total volume of the tranquilizer composition (vol%) and contains 0.2 vol% to 10 vol% of chitosan, 1 vol% to 10 vol% of lactose, and 80 vol% to 99.8 vol% of water. This invention further provides a preparation method of vaccine, including evenly mixing the aforementioned tranquilizer composition with the virus solution in a volume ratio from 10:1 to 1:10 to form the mixture; freezing the mixture at a temperature from -20 to -80 DEG C for 12 to 48 hours; and, dehydrating the frozen mixture for 12 to 48 hours to obtain the vaccine. This invention further provides a classical swine fever vaccine, which contains the attenuated swine fever virus and the aforementioned tranquilizer composition. This invention may achieve the efficacies of stably maintaining the vaccine potency of high dosage, long preservation period, and excellent biocompatibility.

Description

Stabilizer composition for vaccine, preparation method thereof and swine fever vaccine containing the same

The present invention relates to a stabilizer composition for a vaccine, and more particularly to a stabilizer composition for a vaccine containing chitosan and lactose. The invention further relates to a process for the preparation of a stabilizer composition for a vaccine, in particular to a process for the preparation of a stabilizer composition comprising chitosan and lactose. The invention further relates to a swine fever vaccine, in particular to a swine fever vaccine comprising a stabilizer composition of chitosan and lactose.

Hog cholera (classical swine fever; CSF), is a highly contagious disease caused by the CSFV (classical swine fever virus) caused by severe pigs have fatal (Rau et al, 2006;. Holinka et al,. 2009). Hog may cause fever, anorexia, diarrhea, death, etc., and may have neurological symptoms. Sows may abort or give birth to dead pigs. Hog Cholera is one of 16 legal infectious diseases listed in the World Organisation for Animal Health (Marconi et al ., 2006; Tews et al ., 2009). The swine fever virus is flaviviridae and pestivirus (Martha et al ., 1994), which is highly contagious and may cause mortality of 20% to 90% (Rossi et al ., 2004). Piglets have been in existence for 150 years, but the damage of this disease is still very high. The outbreaks have been caused in China and in Europe and other parts of the world, causing significant economic losses in the pig industry (Liu et al ., 2009). In order to stop the epidemic, it is necessary to reduce the export volume of animals in the affected areas, especially in pig-raising areas. In order to achieve this, there are two methods currently available, one is direct culling and the other is emergency vaccination (Jantien et al ., 2008). In the Netherlands, pig mites broke out between 1997 and 1998, and the culling policy was implemented, causing major economic losses in the Netherlands (Belak et al ., 2008). However, the subsequent bird flu experience in 2001 and 2003 changed the public's perception that animals should be fully vaccinated for immune protection (Holinka et al ., 2009). In order to achieve eradication and control of swine fever, safe and effective attenuated swine fever vaccine is the best choice. Today the availability of classical swine fever virus vaccine are mainly two, namely the traditional live vaccine and subunit vaccine (Bouma et al., 1999) .

The most important quality of live vaccines is the preservation of antigenic activity. Therefore, the use of vaccine stabilizers is mainly to protect the vaccine antigen components from the external environment for a long time, such as temperature and pH changes can still maintain a certain effect (Schmidt and Lennette, 1976; Kissmann et al ., 2008 ). Gelatin is the most commonly used stabilizer for vaccines, and its constituents are mainly derived from animal collagen (Nemoto et al ., 1999). Gelatin is generally considered to be low immunogenic and hypoallergenic for humans and protects viral antigens at high temperatures (Chun et al ., 2004). However, because the source of gelatin may be contaminated with strained Prion protein (Scrapie prion protein; PrPsc, the cause of mad cow disease) and the new mutant Creutzfeldt-Jakob disease, non-animal source stabilizers There is a need for its development (Nemoto et al ., 1999). Carbohydrates, especially disaccharides, are another widely used stabilizer and have proven to be the most effective substance for protecting and stabilizing vaccine antigens in freeze-dried vaccines (Crowe and Carpenter, 1993a; Crowe et al . , 1993b). The possible reasons for the disaccharide-stabilizing antigenic substance are: 1. The disaccharide can produce a hydrogen bond with the antigenic substance similar to the water displacement reaction; 2. The disaccharide can form a high-viscosity sugar glass, embedding the antigenic substance therein to produce high Activation energy provides future chemical reaction energy requirements (Crowe and Carpenter, 1993a; Crowe et al ., 1993b; Leslie et al ., 1995; Allison, 1999). Commonly used disaccharides are sucrose and trehalose, which are used in liposomes, proteins, and viral vaccines because they are inactive (Leslie et al ., 1995). In addition, there are glucose, lactose, and maltose, which are reducing sugars that can be reduced by the Maillard reaction, causing damage to the antigen (Li, 1996).

In Japan, polyvinylpyrrolidone (PVP) and lactose are widely used as stabilizers for the preservation of swine fever vaccines, and their storage and solubility have good effects (Yang, et al . 1973). Although PVP is a compound approved by the US FDA, in recent years more and more reports have indicated that individuals have an allergic reaction to PVP, whether it is subcutaneous absorption (Yoshida et al ., 2008; Adachi et al ., 2003) or oral (Rönnau). Et al ., 2000). Sarkar (2003) et al. also explored lactatedbumin hydrolysate-sucrose (LS), Weybridge medium (WBM), buffered gelatin-sorbitol (BUGS) and trehalose. The trehalose dihydrate (TD) was added to the active attenuated vaccine (Peste des petits ruminants, PPR) for lyophilization, and the preservation efficacy of the small ruminant vaccine was tested under different temperature and time conditions. It was found that LS and TD were superior to the other two in the preservation stability of the small ruminant vaccine.

The current type of vaccine is liquid and freeze-dried. The use of freeze-drying forms is primarily to avoid degradation of the vaccine antigenic material, while dehydration and the addition of a suitable stabilizer to stabilize the virus (Worrall et al ., 2001). In use, the liquid vaccine has the disadvantages of being difficult to transport, short in efficacy, unstable in force price (number of viable cells), and the freeze-drying type has the advantages of light weight and convenient transportation. A freeze-dried vaccine must possess the following characteristics: 1. long-term stability; 2. fast resolving rate; 3. formation of fine cakes; 4. maintaining the original characteristics of the antigen; 5. first-time sheets when remelting Solution (Bedu-Addo, 2004). Freeze drying mainly includes processes such as freezing and drying. In the freezing process, the liquid is mainly formed into ice crystals, and the remaining substances form a thick substance, which no longer produces crystals (Bedu-Addo, 2004). Improper manipulation of this process can damage proteins and viruses due to the presence of large amounts of ice water, which can be destroyed by differences in pressure during freezing (Pikal-Cleland et al ., 2000; Searles et al ., 2001). The freezing process is the structure and dry state that determines the formation of dry cakes in the future (Searles et al ., 2001). Drying can be divided into primary and secondary drying. Traditional freeze-drying has only one drying process, but there is a secondary drying process in order to enhance the heat resistance of the vaccine product. Litamoia et al. (2005) added chitosan to freeze-dried viruses to achieve heat-tolerant effects. This relatively natural, non-toxic, chitosan that enhances and maintains its immune response may have potential as a stabilizer (Jaganathan et al ., 2005).

The swine fever vaccine can effectively reduce the incidence of highly contagious and high mortality in pigs caused by acute viral diseases of swine fever, thereby reducing major economic losses. However, the cost of vaccines is often burdened by livestock farmers because of the large number of swine fever vaccines. Therefore, the development of single-bottle high-dose swine fever vaccines is expected to reduce the cost of livestock farmers. The use of tranquilizers allows the swine fever vaccine to maintain the stability of the virus in a range of temperature and humidity environments, so that the antigen of the virus retains its activity and exerts its immune function. Traditional swine fever vaccine stabilizers contain chemical ingredients and are dosed from 10 to 20 doses per bottle (Log 10 ⁴ TCID ₅₀ /mL per dose). In high dose situations (more than 100 doses per vial), the same efficacy is yet to be assessed. In view of this, how to develop a replacement of the traditional stabilizer can only maintain the low dose preservation, while using non-toxic, biodegradable and biocompatible stabilizers, the prior art needs to be improved.

In order to overcome the disadvantages of the prior art, the present invention provides a stabilizer composition for a vaccine, in particular a stabilizer composition comprising chitosan and lactose, which enables the present invention to achieve a stable high dose of vaccine titer, Long shelf life, non-toxicity, biodegradability and biocompatibility.

In order to achieve the above object, the technical means employed in the present invention is to provide a stabilizer composition for a vaccine which is based on the total volume (vol%) of the stabilizer composition, and the stabilizer composition comprises 0.2 vol. % to 10 vol% chitosan, 1 vol% to 10 vol% lactose, and 80 vol% to 99.8 vol% water.

Preferably, the chitosan is from 0.25 vol% to 5 vol%.

Preferably, the lactose is 5 vol%.

The present invention further provides a method for preparing a vaccine comprising uniformly mixing the above stabilizer composition with a virus solution at a volume ratio of 10:1 to 1:10 to form a mixed solution; and freezing the mixture at -20oC to -80oC for 12 hours. The vaccine is obtained up to 48 hours; and after the frozen mixture is dried for 12 hours to 48 hours.

Preferably, the stabilizer composition is uniformly mixed with the virus liquid in a volume ratio of 1:1.

Preferably, the virus solution is a swine fever virus solution.

Preferably, the mixture is frozen for 24 hours.

Preferably, the frozen mixture is dried for 24 hours.

The invention further provides a swine fever vaccine comprising an attenuated hog cholera virus and a stabilizer composition as described above.

The advantages of the invention are:

1. When the stabilizer composition for vaccine of the present invention is combined with a specific volume percentage of chitosan and lactose, the vaccine can stably maintain a high dose of vaccine titer, has a long shelf life, and is non-toxic. Biodegradability and biocompatibility are good.

2. A method of preparing a vaccine of the present invention, wherein a vaccine comprising a combination of chitosan and lactose is capable of increasing shelf life via a freeze-drying step.

3. The swine fever vaccine of the present invention achieves the efficacy of maintaining a high dose of vaccine titer by combining with a specific volume percentage of chitosan and lactose.

The technical means adopted by the present invention for achieving the object are further explained below in conjunction with the drawings and the preferred embodiments of the present invention.

Preparation Example 1 Preparation of stabilizers of different dosage forms

Adjust the concentration of the stabilizer according to the ratio of the final total volume of the lyophilized sample. First, add the stabilizer to the 100 ml (mL) serum bottle, and add the appropriate amount of sterilized ultrapure water (Milli-Q ^® ) to dissolve. After stirring for 30 minutes, it was autoclaved, cooled and placed at 4 °C until use. Chitosan is food grade (90% to 95% deacetylated), while solutions containing chitosan components require the addition of acetic acid to dissolve the chitosan and clarify the liquid. Autoclave. Other ingredients such as hydrolyzed milk protein-sucrose (LS), Wembley solution (WBM), and lactalbumin hydrolysate-mannitol (LM) cannot be autoclaved due to protein content. Filter through a 0.2 micron (μm) filter and set at 4oC for use. Table 1, the ingredients of different stabilizers

The composition and ratio of each stabilizer are shown in Table 1. Among them, the stabilizer used in the current commercial swine fever vaccine is lactose with polyvinylpyrrolidone (PVP), which has a good protective effect on the swine fever vaccine, and is called a control group (A dosage form). Stabilizer). The three stable dosage forms of LS (F-type stabilizer), WBM (G-type stabilizer), and LM (H-type stabilizer) are used in the small ruminant vaccine.

Preparation 2 Preparation of Vaccine

The attenuated swine fever virus was purchased from the Animal Health Laboratory of the Agricultural Committee of the Executive Yuan of the Republic of China. The product name is "Dry Rabbit Rabbit Poisonous Toxicosis" Animal Drug Preparation No. 2688, which is easy for those with common knowledge in the field. Acquired. After the obtained virus powder is dissolved, it is cultured in a porcine kidney cell line (PK15) and ready for use. The different formulations of Preparation Example 1 was CSFV stabilizer (Log10 ^6.5 TCID ₅₀ / mL) at a volume ratio of 1: 1 mixed uniformly in a centrifuge tube, and the stabilizer uniformly mixed with the virus solution after aliquoted In the vaccine bottle, the total volume of each bottle is about 2 ml.

Example 1 Evaluation of the effectiveness of different stabilizer formulations

(1) The state after the vaccine freezes and dries

The vaccine prepared by Preparation Example 2 was rapidly frozen at -80 ° C for 24 hours to ensure that the liquid in the vaccine bottle was completely frozen. The fully frozen vaccine bottle was placed in a freeze vacuum dryer for freeze drying, and the freeze drying time was about 24 hours. After the lyophilization time is over, the lyophilized sample is stored at 4oC or room temperature (25oC), and the appearance of the lyophilized sample is observed and photographed. Table 2. Description of the appearance of the swine fever virus mixed with different stabilizers after freeze-drying

It can be seen from Table 2 that the three stabilizers of the F dosage form, the G dosage form, and the H dosage form cannot form a cylindrical shape after freeze-drying of the vaccine product, and use various concentrations of chitosan as a stabilizer component, and the vaccine is generally used. After lyophilization, a cake shape can be formed.

(2) Remelting test of frozen vaccine samples

Injecting a syringe (including a needle) to absorb the live vaccination solution of the swine fever tissue (hog-cholera tissue culture live vaccine, purchased from Taiwan Biopharmaceutical Co., Ltd.) about 2 mL (same freeze-dried The former volume was injected into the lyophilized sample, shaken vigorously for 10 seconds, and then the reconstituted lyophilized sample was allowed to stand on the plane for 5 seconds, 10 seconds, and 15 seconds, and the freeze-dried sample was observed and photographed. After 1 minute, the reconstituted lyophilized sample was aspirated with a syringe to examine whether there was any residue.

Dissolved state of lyophilized sample of A dosage form, B-4 dosage form or C-4 dosage form stabilizer after shaking for 10 seconds: A dosage form stabilizer has the fastest remelting speed and can be completely dissolved within 15 seconds after standing; C-4 dosage form The remelting rate is second only to the A dosage form; while the B-4 dosage form has a slightly slower resolving speed, the concentration of chitosan may be higher, but it can be completely dissolved after being left for 1 minute. Finally, the lyophilized sample is taken back by the injection syringe and left to stand for one minute, and the three dosage forms can be smoothly absorbed, thereby knowing that the lyophilized sample of the A dosage form, the B-4 dosage form or the C-4 dosage form stabilizer is dissolved. The state does not have much effect on the smoothness of the application.

(3) Swine fever virus price test

Porcine kidney cell line (PK-15) was cultured in 96-well plates (2 x 10 ⁴ cells per well) for 24 hours. The dissolved lyophilized samples were serially diluted 10-fold (10 ^-1 to 10 ^-6 ) with minimum essential medium (MEM) containing 5% fetal bovine serum (FBS). One hundred microliters (μL) of serially diluted virus solution was added to a 96-well plate containing cells and cultured in a 37 ° C carbon dioxide incubator. After 72 hours of culture, the waste liquid was removed, and the 96-well plate was washed with a phosphate-buffered saline (PBS), dried, and patted dry with absorbent paper. After adding 100 μL of 10% formalin for 10 minutes at room temperature, 10% of the formalin was removed, and the 96-well plate was washed with 1 time PBS and patted dry with absorbent paper. The pig anti-porcine antibody was entrusted to the Animal Health Laboratory of the Chinese People's Republic of China Executive Yuan, and 50 μL of pig anti-swine antibody (diluted with 1:100 PBS) was applied and incubated in a 37 °C incubator for 50 minutes. The antibody was removed, and the 96-well plate was washed with 1x PBS and patted dry with absorbent paper. Then add 50 μL of goat anti-Swine IgG (purchased from Jackson ImmunoResearch Inc.) (diluted with 1:100 PBS in 1:100) and remove the antibody after 50 minutes in a 37 °C incubator. After washing the 96-well plate with 1x PBS, it was patted dry with absorbent paper. Finally, it was placed under inverted three-eye fluorescence microscope and the 50% tissue culture infective dose (TCID ₅₀ ) method was calculated by Reed-Muench method (Reed, 1938). Fluorescent reaction. Table 3. Preservation efficacy of different dosage forms of tranquilizers in swine fever vaccine "X" is a non-fluorescent reaction; "N" is not good after the first two tests (sample 1 and sample 2), so the third test is not performed.

It can be seen from the above Table 3 that the stability of the three stabilizer viruses using the F dosage form, the G dosage form, and the H dosage form is not good, and the viral power price cannot be detected after freeze-drying, so the stabilizer containing the hydrolyzed milk protein component is not suitable for the piglet. vaccine. The present invention uses different concentrations of chitosan as a stabilizer component, and the virus price analysis results show that when the concentration of chitosan is less than 0.25%, the infectivity of the virus cannot be effectively protected. The concentration of 0.5% and 0.25% of chitosan alone or in combination with PVP can not effectively protect the virus's infectivity. The concentration of 0.5% and 0.25% chitosan is matched with 5% lactose. It has the same viral potency protection utility as the A dosage form of the control group.

Example 2 Humidity Test of Lyophilized Vaccine Samples

The humidity of the freeze-dried vaccine often affects the quality. According to the "Testing Standard for Animal Drugs", it must be tested to have a moisture content of 4% or less. The lyophilized vaccine samples were allowed to stand for 10 hours in a 80 ° C oven for 24 hours before lyophilization, after lyophilization, and after lyophilization, and the test was repeated 10 times. Calculate its moisture content according to the formula below: Table 4, A dosage form and humidity test of B-4 dosage form lyophilized vaccine sample

Please refer to Table 4 to observe the weight change of the lyophilized vaccine sample of the A dosage form and the B-4 dosage form stabilizer before standing in lyophilization, after lyophilization, and after lyophilization in an oven at 80 ° C for 24 hours, such as The results of Table 4) show that the lyophilized vaccine samples obtained using the two stabilizers have a humidity value of <4%, which is in compliance with the humidity acceptable range.

Example 3 Heat-resistance test of lyophilized vaccine samples

The lyophilized samples were placed at 25 ° C for 4 days, 12 days, 16 days, and 20 days, respectively, and placed at 37 ° C for 1 day, 3 days, 5 days, 7 days, and 45 ° C for 6 hours, 12 hours, 18 hours, and 24 hours. The lyophilized samples were taken at different time points and reconstituted with the live vaccination solution of porcine sputum tissue. About 2 mL of each bottle was reconstituted (the volume before lyophilization), and the prion price was measured and the temperature was plotted. Graph.

Referring to Figure 1, the results showed that the lyophilized vaccine sample of the A dosage form stabilizer was replaced at 25oC at 4oC. After 4 days, the viral power price did not decrease, but after 8 days of storage, the viral power price decreased by 1 log value. At this point, the viral price no longer falls to the 20th day of preservation. For the B-4 dosage form stabilizer, the viral power price decreased by about 0.85 log values during the 16th day of storage. However, until the 20th day, the viral price dropped by about 1.25 log values. When the lyophilized vaccine sample was stored at 37oC at 4oC, the lyophilized vaccine of the A dosage form stabilizer retained the original viral power price on the first day, but the viral power price decreased to about 1 log value on the 7th day. The lyophilized vaccine sample of B-4 dosage form stabilizer decreased by 1 log value on the first day of storage at 37oC, but the virus price could be saved to the 7th day. The lyophilized vaccine sample was replaced with a heat resistance test at 45oC at 4oC. The viral power price was maintained for at least 12 hours in the A dosage form and the B-4 dosage form stabilizer, and the viral power price was reduced by 0.5 log value.

The vaccine heat test results show that the B-4 dosage form stabilizer can protect the high dose vaccine to maintain the viral power rate for at least 16 days in the 25oC environment, and the A dosage form stabilizer for at least 4 days. At least 1 day in a 37oC environment and at least 12 hours in a 45oC environment. Generally speaking, the viral power price drops by 0.5 log value is within the normal range. After the A dosage form and the B-4 dosage form stabilizer were stored at 45 ° C for 72 hours, the viral power price was reduced by 2 log values to 2.5 log values (results not shown).

Example 4 Preservation test of lyophilized vaccine samples

The lyophilized vaccine samples were placed at 4oC for long-term preservation testing. After freeze-drying on the first day, 7 days, 15 days, and 1 month, 3 months, 6 months, 10 months, and 12 months, lyophilized samples were taken at different time points and the pig plaque tissue was used for live poisoning. The vaccine solution was reconstituted, and about 2 mL of each bottle was reconstituted (the same volume as before lyophilization), and the price of the swine fever virus was measured. Table 5, A dosage form and B-4 dosage form freeze-dried vaccine sample preservation test

As shown in Table 5, a lyophilized vaccine sample of the A dosage form and the B-4 dosage form stabilizer was placed at 4 ° C for a long-term storage test. Samples were taken for viral power testing on the 1st, 7th, 15th, and 1st, 3rd, 6th, 10th, and 12th months after storage. Based on the viral value of 0.5 log value is within the normal range, B-4 dosage form stabilizer can protect the high-dose vaccine at least 6 months. In the control group, the dosage form stabilizer was maintained at a high dose of the vaccine, and the viral power price was only 1 month. The viral power price had decreased by 2.5 log values until the 12th month. The results showed that the B-4 dosage form stabilizer could be stored for at least 6 months compared with the control group A dosage form stabilizer, and the virus strength was better protected during this period.

Example 5 Animal Experiment - Rabbit Identification Test

White rabbits (about 2 kg each) were used for the rabbit identification test. The rabbits were adapted to the new environment the previous week and the white rabbits were used to measure the rectal temperature at the early (10:00) and late (17:30) mercury thermometers. (anal temperature) After 30 seconds of maintenance and record the current temperature, the rectal temperature should be stable after one week. Then the immunological utility test was started. After the lyophilized vaccine sample was reconstituted using the swine fever tissue vaccine dilution, the dissolved lyophilized sample was subjected to 10-fold serial dilution with MEM medium containing 5% synthetic fetal bovine serum, and placed on ice for use. The blood vessels of the rabbit's ear were injected into the syringe with different concentrations of virus vaccine solution such as Log10 ¹ TCID ₅₀ /mL, Log10 ² TCID ₅₀ /mL, Log10 ³ TCID ₅₀ /mL, Log10 ⁴ TCID ₅₀ /mL, and each concentration. Four white rabbits were injected, and each rabbit was injected with 2 mL of virus vaccine solution, and the rabbits were placed according to the concentration of the injection, and the temperature of the rectum was recorded for the next week. The temperature was 40.5oC, which was the fever temperature. Temperature profile and calculate 50% rabbit infective dose (RID ₅₀ ). Table 6. Rabbit identification test of the lyophilized sample of the application type A Table 7. Rabbit identification test for lyophilized samples of the application of C-4

The results are shown in Table 6 and Table 7. The calcination time of rabbits in each dose was about 2 days after inoculation. The RID ₅₀ of the lyophilized vaccine sample of C-4 dosage form was 10 ^3.5 RID ₅₀ /mL, which was equivalent. The RID ₅₀ of the A-type lyophilized vaccine sample was 10 ^3.55 RID ₅₀ /mL. These results indicate that the C-4 dosage form stabilizer can preserve the efficacy of the vaccine, so the addition of chitosan has potential for the stabilization of the swine fever virus.

Example 6 Sterility test of lyophilized vaccine samples

According to the "Intestinal Standard for Animal Drugs", it may not contain any pathogenic bacteria that may be detected, and it shall be observed and recorded after being cultured in a medium containing nutrients for a certain period of time. Observe that if there is any suspected bacterial development, or if the medium is turbid due to the vaccine itself, it should be re-transplanted into the new medium, cultured at the original temperature, observed and recorded.

(1) Bacterial test of lyophilized vaccine samples

This experiment used LB (Luria-Bertani) medium as a nutrient source for sterility testing. First, LB medium [1% tryptone, 0.5% yeast extract, 0.5% sodium chloride and 1.5% ocean). The vegetable powder) is mixed and dissolved in sterile water and sterilized by autoclaving (121oC) for 20 minutes. The sterilized LB medium is placed in a container. After cooling, LB solid medium is formed and stored in a 4oC refrigerator. Then, in the aseptic processing table, the lyophilized vaccine sample is taken back from the sterilized cotton swab, and the LB solid medium is used in the LB solid medium, which causes the pig to cause liver ulceration and finally causes the muscle layer of the liver surface to be damaged and perforated. Edwardsiella tarda ( E. tarda ), exposed, anal redness, and necrosis of the kidney were in the control group, while sterile water was used as the control group, and was placed at room temperature for 72 hours and observed and photographed.

The lyophilized vaccine sample of the A dosage form and the B-4 dosage form stabilizer is dissolved and applied to the LB solid medium for disc culture, and the bacteria-containing test is performed. The observation showed that no colony was found in the lyophilized vaccine sample and control group of the A dosage form and the B-4 dosage form stabilizer, while the colony grew in the positive control group, which proved that the A dosage form and the B-4 dosage form stabilizer were used. The lyophilized vaccine samples were aseptically contaminated during the procedure.

(2) Test of moldy bacteria in freeze-dried vaccine samples

Since the attenuated vaccine is also often contaminated by mycoplasma, the freeze-dried vaccine samples of the A dosage form and the B-4 dosage form stabilizer are also tested for mycoplasma contamination. Mycoplasma test of lyophilized vaccine samples was performed using a mycoplasma PCR detection kit (purchased from Genlantis). After the lyophilized vaccine sample is uniformly dissolved, take a 100 μL to 1.5 mL centrifuge tube and dry in a dry bath at 95 ° C for 5 minutes. Take 10 μL of the dry bath and freeze the sample to a new tube for PCR amplification. (The primer pairs used in PCR are provided by the previously purchased kit). The amplified PCR product was analyzed by 2% gel electrophoresis, and the mycoplasma DNA size was 500 base pairs (bp).

Mold bacteria test PCR reaction test results shown in Figure 2, A dose and B-4 dosage form stabilizer lyophilized vaccine samples without mold contamination.

Example 7 Vacuum Test of Lyophilized Vaccine Samples

In a dark room, the lyophilized vaccine sample was irradiated with a vaccum tester to lyophilize the sample, and the vacuum of the lyophilized sample was tested by conductive color. A blue-violet light appears in the lyophilized sample vaccine bottle, which is a vacuum.

The results showed that the lyophilized vaccine samples of the A dosage form and the B-4 dosage form stabilizer both stimulated blue-violet light. Therefore, the A dosage form and the B-4 dosage form stabilizer are qualified for the vacuum degree test.

Example 8 Pig Animal Experiment Table 8, Test Group

Eight-week-old second-generation specefic pathogen free (SPF) piglets were divided into three groups (as shown in Table 8), the first group was the control group without vaccine, and the second group was the city of Shidai. The tissue culture vaccines sold (A dosage form) were 1/10 dose and 1/20 dose, Group 3 modified tranquilizer vaccine (C-4 dosage form) 1 dose and 1/100 dose, respectively. All the pigs in the experimental group were immunized in the negative pressure animal room, and on the 10th day after the immunization, the animals were inoculated with 2 ml of 10 ⁴ minimum lethal dose (MLD). The ALD strain was obtained from the Animal Drug Testing Branch of the Animal Health Laboratory of the Agricultural Committee of the Executive Yuan of the Republic of China, and the RP virus of the swine fever vaccine was used in the neck muscles of the pigs in each test group. For 14 days, blood samples were taken before immunization, before challenge, and on the 14th day after challenge. According to the neutralization test method in the national diagnostic criteria for swine fever, the serum of each phase of the test, including all pre-immune, pre- and post-invasion sera, was simultaneously subjected to serum neutralization test. Table 9. Test results and incidence of neutralizing antibody in pigs Note: D stands for death, P stands for tolerance, and NP stands for onset.

As shown in Table 9, the C-4 dosage form stabilizer can only be administered at a dose of 1/100 to achieve the effect of neutralizing antibody titers at a dose of 1/10 or 1/20 of a commercially available vaccine. Therefore, the swine fever vaccine containing the chitosan and the lactose stabilizer of the present invention not only reduces the dosage, but also improves the effect of the neutralizing antibody.

It is apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the invention. Although the present invention has been described in terms of specific preferred specific embodiments, it should be understood that the invention should not be Various modifications that are obvious to those of ordinary skill in the art are also intended to be included within the scope of the appended claims.

Figure 1 is a bar graph showing the change in viral power of lyophilized samples of A dosage form and B-4 dosage form stabilizer after storage at different temperatures and times. The original virus price of the lyophilized vaccine sample was Log10 ^6.5 TCID ₅₀ /mL. 2 is an electrophoresis pattern of the mold test of the lyophilized sample of the A dosage form and the B-4 dosage form of the present invention; M represents a nucleic acid molecular weight standard solution, A represents a dosage form A, B represents a B-4 dosage form, and P represents a positive control group.

Claims (9)

A stabilizer composition for vaccines based on the total volume (vol%) of the stabilizer composition comprising 0.2 vol% to 10 vol% chitosan, 1 vol% to 10 vol % lactose and 80 vol% to 99.8 vol% water. The stabilizer composition of claim 1, wherein the chitosan is from 0.25 vol% to 5 vol%. The stabilizer composition of claim 1 or 2, wherein the lactose is 5 vol%. A method for preparing a vaccine comprising uniformly mixing the stabilizer composition according to any one of claims 1 to 3 with a virus solution at a volume ratio of 10:1 to 1:10 to form a mixed solution; and mixing the mixture at -20 ° C to The vaccine is obtained by freezing at -80oC for 12 hours to 48 hours; and after drying the frozen mixture for 12 hours to 48 hours. The method for producing a vaccine according to claim 4, wherein the stabilizer composition and the virus solution are uniformly mixed at a volume ratio of 1:1. The method for preparing a vaccine according to claim 4, wherein the virus solution is a swine fever virus solution. The method for producing a vaccine according to any one of claims 4 to 6, wherein the mixture is frozen for 24 hours. The method for producing a vaccine according to any one of claims 4 to 6, wherein the frozen mixture is dried for 24 hours. A swine fever vaccine comprising an attenuated swine fever virus and a stabilizer composition according to any one of claims 1 to 3.