Patent ID: 12252529

MODE FOR THE INVENTION

Hereinafter, the present invention will be described in detail.

However, the following examples are merely for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1. Preparation ofS. pneumoniaeVaccine

S. pneumoniaeculture was performed by methods known to those skilled in the art.S. pneumoniaeof each serotypes (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F) were obtained from depository authorities such as ATCC, JCM, KCCM, schools, and institutions.S. pneumoniaewere identified by capsular and non-motile, gram-positive, lancet-shaped diplococci and hemolysis in the blood agar media. Serotypes were identified using the Quellung test using specific antiserum.

To increaseS. pneumoniaeand remove components of animal origin, seed stocks were cultured in F1, F2, and F3 generations. In addition, the seed stocks were further cultured for two generations. Additional first generations were cultured from F3 vials and subsequent generations were cultured from additional first generation vials. Seed vials were cryopreserved at −70° C. or lower with synthetic glycerol as cryopreservative. For strain bank preparation, all cultures were grown in soy-based medium. Prior to freezing, the strain was concentrated by centrifugation and the medium used was removed, and then the strain pellets were resuspended in fresh medium containing synthetic glycerol.

TheS. pneumoniaeof respective serotypes for preparingS. pneumoniaevaccine were inoculated to the flasks containing a soybean-based medium of pH 7.0±8.2 (pH 8.1±0.1 for serotypes 4, 9N, 9V, 12F, and pH 7.5±0.1 for serotype 14), followed by incubating for 10 to 12 hours in a 36±2° C., 5% CO2incubator without agitation until the absorbance (O.D.600) reaches at least 0.5. After the incubation, the flasks were checked for contamination by a microscope. Thereafter, a seed flask was used to inoculate the culture fermenter containing the soy-based medium. The pH of the culture was maintained using 3N NaOH, and cultured for 4 to 12 hours so that the absorbance (O.D.600) became 0.6 to 1.0 depending on the serotypes ofS. pneumoniae.

37% (v/v) formaldehyde was added so as to have a final concentration of 0.5 to 2% (v/v), homogenized for 30 minutes, and then inactivated at room temperature overnight. The inactivated culture was centrifuged for 30 minutes at 4° C. However, serotype 3 was centrifuged for 40 minutes. Thereafter, the supernatant was discarded and the pellets were collected. Then, Sorensen Buffer (phosphate buffer, formaldehyde) was added and suspended and stored at a temperature of 2 to 8° C.

The suspension of inactivatedS. pneumoniaewas appropriately diluted with 0.9% (w/v) sodium chloride or water for injection to obtain aS. pneumoniaevaccine stock solution of each serotype, and stored at 2 to 8° C. The absorbance (O.D.600) value and dose of theS. pneumoniaevaccine stock solution were recorded, and an inactivation test, gram staining, and swelling test were performed.

S. pneumoniaevaccine for animal inoculation was prepared by diluting theS. pneumoniaevaccine stock solution with Sorensen Buffer such that the absorbance (O.D.600) was 4.0 or less, and stored at 2 to 8° C.

In addition, theS. pneumoniaevaccines for adsorption for each serotype to remove the cross-reactivity of antiserum were prepared by diluting theS. pneumoniaevaccine stock solution with Sorensen Buffer so that the absorbance (O.D.600) was 4.0 or higher and stored at 2 to 8° C.

Example 2. Preparation of Standard Solution for Analyzing Antibody Titer of Antiserum and Cross-Reactivity

In order to confirm the antibody titer and the presence of absence of cross-reactivity of the antiserum, multivalent standard stock solutions and multivalent standard solutions were prepared. The multivalent standard stock solutions were prepared by diluting or mixing monovalent stock solutions having known concentrations, and the multivalent standard solutions were prepared by diluting the multivalent standard stock solutions. The monovalent solutions refer to monovalent bulk produced for each serotype ofS. pneumoniae.

A multivalent (21-valent) standard stock solution was prepared by mixing monovalent stock solutions of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F and 33F, respectively, wherein the multivalent standard stock solution comprises capsular polysaccharide of each serotype at a concentration of 6.6 μg/ml. The amount of monovalent stock solution required for each serotype on the basis of 100 ml was calculated and place it in a plastic container, and then sample dilution buffer (0.85% (v/v) sodium chloride/5 mM succinic acid/0.02% (v/v), polysorbate 80/1 mg/ml aluminum phosphate, pH 5.8) was added so that the total volume became 100 ml. Thereafter, the 21-valent standard solution was stirred and mixed at room temperature for 2 hours or more, and then refrigerated.

In addition, a total of 21 types of monovalent standard stock solutions were prepared by diluting the monovalent stock solutions for each serotype so that the concentration of capsular polysaccharides of each serotype became 6.6 μg/ml. Specifically, after calculating the amount of monovalent stock solution required based on 100 ml and put in a plastic container, the sample dilution buffer was added so that the total volume became 100 ml. At room temperature, the mixture was stirred slowly for 2 hours or more, and then refrigerated.

The monovalent standard solution and the multivalent standard solution were prepared by diluting the monovalent standard stock solution and the multivalent standard stock solution so that the concentrations of the capsular polysaccharides were 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 μg/ml. Each standard solution was prepared by mixing the dilution buffer and standard stock solution, followed by adding sodium hydroxide solution and citric acid.

Example 3: Preparation ofS. pneumoniaeAntiserum

Two or three NewZealand White rabbits (Female, 3-4 kg) were immunized for each serotype. TheS. pneumoniaevaccine for animal inoculation prepared in Example 1 was warmed at 37° C. for 10 minutes before administration to rabbits. The warmedS. pneumoniaevaccine was administered to the ear vein, and 2 ml of blood was collected from the vein before administration of the vaccine. The collected blood was allowed to stand at room temperature for 1 hour and then left at 4° C. overnight. The following day, the collected blood was centrifuged to separate serum. Antibody titer of serum was analyzed by nephelometry after diluting the serum with dilution buffer (Diluent 1, Beckman Coulter) by 1:3, 1:6, 1:10, and 1:30. Six to seven days after the last immunization day, the serum was obtained from the heart when the response value (Rate unit) of 6-fold diluted serum was 20 to 150.

Inoculation schedules and doses are shown in Table 1 below. After 4 weeks, the immune schedule and dose are the same.

TABLE 1Dose (/Head)WeeksMONTUEWEDTHUFRISATSUN10.10.20.2RestRestRestRest20.20.30.3RestRestRestRest30.50.50.7RestRestRestRest4bleeding/1.01.0RestRestRestRest1.0

The final blood collected from each rabbit's heart was allowed to stand at room temperature for 1 hour and then left at 4° C. overnight. The following day, the collected blood was centrifuged to separate serum. The dose of the final blood serum was recorded and heat treated at 56° C. for 30 minutes, then left at room temperature to drop the temperature. A portion of the heat-treated serum was centrifuged to obtain a supernatant. The antibody titer and the cross-reactivity of the final blood serum were analyzed by nephelometry for monovalent standard stock solution (6.6 ug/mL) of each serotype prepared in Example 2.

Specifically, the antibody titer of the separated serum was analyzed by nephelometry using a monovalent standard stock solution of the serotype according to a conventionally known method.

In addition, in order to confirm the cross-reactivity of the preparedS. pneumoniaeantiserum of each serotype, 20 kinds of monovalent standard stock solutions except for the serotype ofS. pneumoniaeadministered to the animals to prepare antiserum were sequentially mixed with each serum. Thereafter, whether antigen-antibody aggregation reaction occurred was analyzed by nephelometry.

If no cross-reactivity was seen, serum of rabbits with an antibody titer of 1 or more was mixed and sodium azide (NaN3) was added at a concentration of 0.0975% (w/v). Thereafter, the mixture was stirred at room temperature for 40 to 80 rpm for 10 to 15 minutes, and then aseptically filtered using a 0.22 μm filter. The filtered antiserum was aliquoted and stored at 2-8° C.

If cross-reactivity occurs, the serum of rabbits having an antibody titer of 1 or more was mixed, and then reacted with theS. pneumoniaevaccine for adsorption of the serotype causing cross-reactivity.S. pneumoniaevaccines for adsorption were prepared at a ratio of 0.5 to 1.0 of the serum dose, and prepared for all serotypes causing cross-reactivity, respectively.S. pneumoniaevaccine for adsorption was centrifugated and the supernatant was removed, and then the antiserum of rabbit was added to the precipitate and suspended uniformly. After stirring for 30 min at 100-150 rpm conditions at room temperature followed by centrifugation, and the supernatant were collected.

The result of cross-reactivity between antiserum of each serotype andS. pneumoniaevaccines of the serotypes except for the serotype above is shown in Table 2.

TABLE 2AB13456 A6 B7 P89 N9 V10 A11 A12 F1415 B18 C19 A19 F22 F23 F33 F12F◯XXX◯◯XXXXXXXXX◯XX◯XA: monovalent stock solution,B: antiserum

If cross-reactivity occurred with two or more serotypes, the same removal process was repeated one by one. The supernatant obtained after the adsorption process was reconfirmed by nephelometry, and the adsorption process was repeated until the cross-reactivity was removed. Finally, the supernatant obtained was analyzed to confirm whether cross-reactivity by nephelometry. The adsorption process was repeated until the response value of nephelometry satisfies (i) less than the average nephelometric result value of blank+(3×standard deviation) or (ii) less than the nephelometric result value of 1.0 ug/ml ofStreptococcus pneumoniaemultivalent standard solution.

When the cross-reactivity fell below the criteria above, sodium azide was added to the antiserum at a concentration of 0.0975% (w/v). Thereafter, the mixture was stirred at room temperature for 40 to 80 rpm for 10 to 15 minutes, and then aseptically filtered using a 0.22 μm filter. The filtered antiserum was aliquoted and stored at 2-8° C.