KR20050008257A - Preparation and Immunogenicity of the combined vaccine composed of the polysaccharide capsule of Salmonella typhi and Japanese encephalitis virus - Google Patents

Abstract

PURPOSE: Provided are a production method of a conjugate vaccine of polysaccharide capsule of salmonella typhi and japanese encephalitis virus and its immunogenicity. Therefore, tow kinds of antigens are vaccinated at the same time. CONSTITUTION: The conjugate vaccine of polysaccharide capsule of salmonella typhi and japanese encephalitis virus is manufactured by conjugation method of Salmonella typhi Vi and tetanus toxoid. Therefore, it is increases the formation of IgG antibody by about 4-6 times. Also, the addition of aluminium hydroxide adjuvant increases the formation of IgG.

Description

Preparation and Immunogenicity of the combined vaccine composed of the polysaccharide capsule of Salmonella typhi and Japanese encephalitis virus}

Japanese encephalitis is a viral epidemic caused by the Japanese encephalitis virus, which is known to cause more than 35,000 deaths per year in encephalitis mosquito habitats in Southeast Asia, with about 10,000 deaths. In Korea, Japanese encephalitis occurs every year, and is vaccinated for prevention. The encephalitis vaccine is the most widely used vaccine inactivated by culturing viruses in rat brain tissue. The schedule and frequency of Japanese encephalitis vaccines vary from country to country. Korea is vaccinating three times a year and every five years until the age of twelve.

Typhoid fever is a high incidence epidemic in developing countries in Southeast Asia, where water supply management and sewage treatment facilities are inadequate, causing Salmonella typhi to infect the body in a short period of time through the intestinal mucosal tissue, causing systemic outbreaks. More than 12 million cases of illness are reported worldwide each year, of which about 1% are reported to die.

The typhoid vaccine was first used in S. typhi , which was inactivated by heat treatment in England at the end of the 19th century, and is currently using a component vaccine prepared by separating bacterial capsular polysaccharides. These vaccines show 80-90% IgG antibody formation in adults but very low antibody formation in children under 5 years of age. It is known to be very poor.

Recently, one type of antigen that is widely used in the development of component vaccines against bacterial pathogens is a polysaccharide constituting the bacterial capsular membrane. The problem of using bacterial antigenic polysaccharide vaccine is low immunity of polysaccharide antigen. Polysaccharide is a T-independent antigen, which does not stimulate memory B cell, so it does not have immune memory ability. It has a limitation. In particular, these vaccines are known to have little effect under the age of 18 months when the most prophylactic immunity is required.

These problems are common to several polysaccharide vaccines, such as Haemophilus influenza and Shigella dysenteriae, in addition to typhoid fever, and thus the development of a vaccine that combines with a highly immune protein to increase the immunity of the polysaccharide. Proteins can generate both immunity and immune memory by covalently binding proteins to polysaccharides in that antigens are recognized by T-cells and stimulate T-cell dependent immunity. Therefore, in order to increase the immunity of the polysaccharide antigen, development of a mixed vaccine conjugation with a highly immune protein is required. Typhoid fever and Japanese encephalitis are both communicable diseases that are likely to be exposed to children and have similarities in terms of vaccination schedule, three to five years. The present invention is to prepare a mixed vaccine with each vaccine of typhoid fever and Japanese encephalitis as a vaccination vaccine to reduce the number of vaccination of each vaccine, and the possibility of the Japanese encephalitis protein body to improve the restriction of pediatric immunity possessed as typhoid polysaccharide antigen as a mixed vaccine. Therefore, it is an object of the present invention to develop a conjugation of a two vaccines or a method of preparing a mixed type, and to try the possibility of a mixed vaccine.

Materials and methods

In order to achieve the above object, the preparation of Salmonella capsular antigen, activation of Japanese encephalitis antigen protein, activation of Tetanus toxoid, Vi-tetanus conjugation method and the like will be described in detail in the following Examples and Examples.

Reference Example 1 Preparation of Salmonella Capillary Antigens

Salmonella capsular antigen (Vi) was prepared by fermentation of S. typhi Ty2 strain in VaccTech in a semi-synthetic media (Frant'z semisynthetic media) was supplied with a culture medium inactivated cells. The culture solution was centrifuged to recover the cells, homogenized with sterile saline solution, 2.5M calcium solution was added and stirred for 30 minutes to extract the capsular antigen. The extracted antigen was partially purified using ethanol fractionation, recovered by centrifugation, and suspended in sterile saline. The suspension was ultrafiltration with sterile physiological saline, and the precipitated solution was recovered by ethanol addition fractionation to a final alcohol concentration of 80%. The amount of Vi antigen was measured by O-acetyl content test.

Reference Example 2: Activation of Japanese Encephalitis Antigen Protein

Existing Japanese encephalitis vaccine product (J) was centrifuged at 80,000 rpm for 1 hour to precipitate virus particles and suspended in physiological saline. EDAC (1-ethyl-3- (3-dimethylaminopropyl carbodiimide) solution was added to the Japanese encephalitis virus solution, and the mixture was stirred and mixed for 20 minutes at 4 ° C. ADH (adipic acid dihydrazide) solution was adjusted to pH 5.2 and added with EDAC in Japan. The solution was added to the encephalitis solution for 20 minutes while maintaining pH 5.2, after stirring for 3 hours, the pH was adjusted to 7.0 to stop the reaction, and the precipitate formed by adjusting the pH was centrifuged at 40,000 rpm for 30 minutes to remove the precipitate. Were collected, dialyzed on 0.15M NaCl, concentrated dialysate by filtration concentrator, loaded concentrate on Bio-Gel P-10 column equilibrated with sodium phospahte, and collected the collected volumn, ultrafiltration with 0.15M NaCl and concentrated.

Reference Example 3: Activation of Tetanus toxoid

Tetanus toxoid antigen was used by VaccTech. The EDAC solution dissolved in 0.15M NaCl was mixed with tetanus toxoid solution while maintaining pH 6.0. After 20 minutes of mixing, the tetanus toxoid solution was slowly added to ADH solution while maintaining pH 5.2, and after 3 hours of mixing, the pH was adjusted to 6.0. The precipitate was removed by centrifugation. The supernatant was dialyzed in a 10 mM sodium phosphate pH 7.0 solution for 18 hours using a dialysis membrane of fractional molecular weight 5,000, concentrated using a membrane filter system, and the concentrate was desalted by sephadex-G-25 column chromatography (5 X 24 cm). The collected void volume was concentrated with a membrane filter system, and the concentrate was diafiltered with 0.15M NaCl, followed by aseptic filtration with a 0.45㎛ syringe filter.

Reference Example 4: Vi-tetanus conjugation

EDAC solution was added to the purified Vi antigen, followed by mixing. The pH of the reaction solution was maintained at 5.3. Activated tetanus toxoid was added slowly to the Vi antigen solution containing EDAC while maintaining pH 5.3, and then the mixture reaction was further reacted for about 3 hours while maintaining pH 5.3. After 3 hours of reaction, the pH was adjusted to 7.0 and the precipitate formed was removed by centrifugation. The supernatant was dialyzed for 18 hours in a 10 mM sodium-phosphate 0.15 M NaCl pH 7.0 solution using a dialysis membrane of fractional molecular weight 50,000. The dialysate was chromatographed on a sephacryl S-1000 column, the void volume was collected and concentrated, and dialyzed on 0.15 M NaCl solution. Vi content of the conjugate was measured by O-acetyl content measurement and protein content was measured by Lowry method.

Example 1 Antigen Formulation and Antibody Formation Animal Experiment

In animal experiments, 4 week-old female ICR mice were used as experimental animals, and the test sample group was 1 group) sterile physiological saline control group and 2 groups) Vi tablet in which 9 ml of physiological saline was mixed with 1 ml of 50 ㎍ / ml Vi solution. Antigen solution, group 3) 1 ml of 50 µg / ml Vi-tetanus toxoid conjugate antigen solution mixed with 9 ml of saline solution, 4 groups) 25 µg / ml of Japanese encephalitis sample mixed with 9 ml of saline solution , Group 5) Vi-tetanus toxoid conjugate (50 ㎍ Vi / ㎖) and 25 ㎍ / ㎖ JEV antigen 1 ml each mixed with 8 ml of physiological saline. Group 6) 120 μl of aluminum hydroxide gel was added to the prepared sample 5) and adsorbed for 6 hours, and then mixed with 8 ml of physiological saline.

The above antigen formulation samples were intraperitoneally injected into 500 mice at 10 mice per sample group. The inoculations were inoculated twice at 2 week intervals and blood collection was performed 7 days after the final inoculation.

Example 2: Enzyme-linked immunosorbent assay (ELISA) antibody test

To test for antibody formation, 100 μl of Vi purified antigen and encephalitis antigen solution were added to 96-microplate wells and left for 4 to 18 hours for antigen coating. After removing the coating antigen solution and washing with the blocking solution, the blocking solution was added 200 ㎕ per well and allowed to stand at 37 for 1 hour. Remove the blocking solution, dilute the collected serum sample 1,000 times with blocking solution, add to each well, and leave at 37 for 1 hour. After 1 hour, the serum sample solution was removed, washed with a blocking solution, and then 100 μl of conjugated second antibody was added thereto and left for 37 to 30 minutes. After washing with water for 20 minutes by adding a coloring substrate solution, the color intensity is measured with an ELISA reader at 405 nm.

Example 3: plaque reduction test by neutralizing antibody (neutralizing antibody test method)

To test the neutralizing antibody of Japanese encephalitis, Vero cells were used as a virus host cell, which was distributed from ATCC CCL-81 (American Type Culture Collection). Vero cells were cultured in MEM (minimum essential medium) medium containing 5% FBS. The collected mouse serum samples were mixed in each group, sterilized with a 0.22 μm injection filter, and inactivated by heat treatment at 56 for 30 minutes. Each serum sample was diluted 1: 320 and 1: 640 with MEM medium containing 5% FBS, and the same volume of encephalitis virus solution (200PFU / 0.4ml) was added and neutralized for 90 minutes in 37 water bath. Each mixture was inoculated into vero cells cultured in four culture dishes, each 400 μl, and incubated for 37 to 90 minutes. As a control, virus suspension and 5% FBS-containing MEM medium were mixed in the same volume, left for 37 to 90 minutes, then inoculated in 10 culture dishes and incubated for 37 to 90 minutes. After incubation, all culture dishes were layered with 5 ml per culture dish of primary mesial agar medium containing 1: 1 mixture of 10% FBS-containing MEM medium and agarose and incubated for 37 to 72 hours.

4 ml of MEM-agarose mixed secondary media containing 3% neutral red was added and incubated at 37 for 24 hours, and the number of plaques was counted. The reduction rate was calculated by comparing the plaque number of the test group with that of the control group, and the neutralizing antibody titers of the serum samples were calculated.

In the present invention, the first intention was to prepare a conjugate vaccine by conjugating Japanese encephalitis virus and typhoid Vi antigen. In the process of activating the Japanese encephalitis virus antigen solution, when the pH of the reaction solution was adjusted to 5.2, precipitate aggregates were formed. The precipitate aggregates were not dissolved even after the pH was adjusted to 7.0. The precipitates were removed by centrifugation, and the protein content and residual ADH content of the concentrate obtained by dialysis and concentration of the supernatant were measured to be less than 0.0001 mg / ml, and the residual ADH content was also negligible. . These results suggest that denaturation occurs in the Japanese encephalitis virus antigens during the pH adjustment to 5.2 to form aggregate precipitates. Therefore, it was not suitable to use encephalitis virus itself as a carrier protein for conjugation. Therefore, conjugate formation of encephalitis protein and typhoid antigen could not be performed. In contrast, conjugation of Tetanus toxoid protein with Vi antigen was successful. Therefore, mixed vaccine samples were prepared by formulating encephalitis virus and typhoid Vi-TT antigen.

In the case of IgG antibody formation experiment using mice, when Vi-TT antigen and Vi-TT conjugate antigen were compared in the case of typhoid fever, Vi-TT antigen formed about 2 times as many IgG antibodies against Vi as compared to Vi alone administration. The mixed antigen of Vi-TT + JEV produced about four times more IgG antibody than the Vi-TT conjugate antigen, and the sample added with aluminum hydroxide as adjuvant formed about 70% IgG antibody compared to the antigen without adjuvant. Increased. Therefore, when attaching tetanus toxoid as a carrier to Vi, IgG production ability was increased and when mixed with Japanese encephalitis antigen, antibody formation ability was increased.

Table 1. Immunoglobulin G level elicited in mice by Vi antigen.

No. Control Vi Vitt JEV + ViTT AI (JEV + ViTT) One 0.081 0.133 0.140 0.442 0.777 2 0.075 0.126 0.274 0.503 0.862 3 0.081 0.089 0.154 0.896 0.778 4 0.077 0.082 0.191 0.513 1.091 5 0.093 0.150 0.337 0.288 0.768 6 0.092 0.143 0.178 0.183 0.833 7 0.084 0.230 0.581 0.391 0.640 8 0.088 0.160 0.172 0.401 0.459 9 0.097 0.242 0.196 0.463 0.372 10 0.080 0.134 0.259 0.927 0.576 P 0.077 0.118 0.129 0.482 0.735

* P: pooled sample

In the case of Japanese encephalitis, a sample containing a mixture of Japanese encephalitis and Vi-TT conjugate antigen produced IgG antibody values for about 6-fold encephalitis antigen compared to administration of Japanese encephalitis antigen alone. Vi-TT + JEV mixed antigens with aluminum hydroxide as adjuvant formed about 40% more IgG than antigen without adjuvant

Table 2. Immunoglobulin G level elicited in mice by JEV antigen.

No. Control JEV JEV + ViTT AI (JEV + ViTT) One 0.280 0.397 0.877 1.742 2 0.132 0.226 1.689 1.983 3 0.194 0.218 1.155 1.173 4 0.164 0.165 1.321 1.373 5 0.153 0.189 1.594 2.145 6 0.143 0.134 0.824 2.265 7 0.239 0.192 1.479 1.959 8 0.160 0.194 0.729 1.660 9 0.159 0.262 0.470 1.801 10 0.152 0.149 1.788 0.754 P 0.137 0.238 1.414 2.111

* P: pooled sample

In the case of Japanese encephalitis, similar to the ELISA test, JEV and Vi-TT mixed vaccine preparation samples showed higher neutralizing antibody values than in the ELISA test, and samples containing aluminum hydroxide were not added to the mixed samples. It showed a tendency to show higher neutralizing antibody value compared to the sample without.

Table 3.Antibody titer of plaque reduction neutralization test for JEV (titers in log)

Test No. JEV JEV + ViTT AI (JEV + ViTT) One 1.772 2.32 3.168 2 1.668 2.14 2.84 Ave 1.72 2.23 3.004

In the case of IgG antibody formation experiment using mouse, when Vi-TT antigen and Vi-TT conjugate antigen were compared in the case of typhoid fever, the Vi-TT antigen formed about 2 times more IgG antibodies against Vi than that of Vi alone administration. The mixed antigen of TT + JEV produced about four times more IgG antibody than the Vi-TT conjugate antigen, and the sample added with aluminum hydroxide as adjuvant showed about 70% increase in IgG antibody formation compared to the antigen without adjuvant. Indicated. Therefore, when attaching the tetanus toxoid to Vi as a carrier, IgG production ability increased and when mixed with Japanese encephalitis antigen, the antibody formation ability increased. In case of Japanese encephalitis, Japanese encephalitis and Vi-TT conjugate antigen The mixed samples produced IgG antibody values against the encephalitis antigen about 6-fold compared to the administration of the Japanese encephalitis antigen alone. Vi-TT + JEV mixed antigen added with aluminum hydroxide as adjuvant formed about 40% more IgG than antigen without adjuvant, and JEV alone was similar in the case of neutralizing antibody test in Japanese encephalitis. Compared to the samples prepared with JEV and Vi-TT vaccines, the neutralizing antibody values showed higher values, and the samples with aluminum hydroxide added to the mixed samples showed higher values of neutralizing antibodies than the samples without addition.

Claims (2)

Method for preparing a mixed vaccine of typhoid capsular polysaccharide and Japanese encephalitis virus Typhoid Vi and tetanus toxoid conjugation methods