JPS6231694B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the field of vaccines for immunization against Haemophilus influenzae type B infections, such as meningitis. More specifically, the present invention provides (1) a method for isolating an antigenic polysaccharide, polyribosyl ribitol phosphate (PRP), from a culture solution of Haemophilus influenzae type b;
and (2) a method for producing a conjugate vaccine, ie, a conjugate vaccine of PRP and B pertussis (Bordetella pertussis) antigen. In order to elicit an antibody response in warm-blooded animals, the PRP of the present invention can be used against other non-pathogenic bacterial strains such as E. coli and B. subtilis.
subtilis), S. aureus, B. punilus and L. plantarum.
It also appears to be effective when used with plants such as A. plantarum). Purified polyribosylribitol phosphate obtained from Haemophilus influenzae type b has been investigated as a protective immunogen. However, active antigenic responses in young animals and infants have not yet been achieved. Previous purification techniques used ethanol and cetabron (hexadecyltrimethylammonium bromide). Contaminants, endotoxins and pyrogens were removed using cold phenol or chloroform and tertiary butanol, which would have resulted in loss of antigenicity of the polysaccharide. A new method for the isolation and purification of immunologically active PRP from Haemophilus influenzae type b has been established. The method described here has a distinct advantage over previous methods in that all contaminants (nucleic acids, proteins, and bacterial endotoxins) are removed to the lowest level by treatment with hydroxyapatite. PRP produced by the method described herein is a polysaccharide with a higher molecular weight than previously reported. More importantly, PRP produced using this new procedure is highly immunogenic in warm-blooded animals, contrary to PRP produced using conventional technology procedures. The method for removing impurities (proteins, nucleic acids, and bacterial endotoxins) in polysaccharide PRP is to treat crudely purified polysaccharides with phosphate containing an adsorbent that does not adsorb polysaccharides under specified conditions. The second objective of the present invention is to produce a combined PRP and pertussis vaccine. This vaccine is highly immunogenic in young animals. () Isolation and purification of polyribosyl ribitol phosphate, the capsular polysaccharide of Haemophilus influenzae type B,
Growth Media and Culture Two strains of Haemophilus influenzae type b are used. Rab strains were obtained from Grace Reidy (Infant Hospital, Columbia University, New York City, NY). The CK strain was isolated from a patient at Waterbury Hospital (Waterbury, CT). The fungus passes through the rat as a host several times to ensure its virulence. The fungus was isolated from mouse brain tissue by autopsy and then mixed with 3.7% brain heart infusion (BHI).
(Difco Lab, Detroit, Michigan) Medium or
0.01% nicotinamide adenine dinucleotide (NAD) (PL Biochemicals, Milwaukee, WI) and 1% (v/v)
Supplemented with horse blood from which fibrin was separated (Animal Blood Center, Shiraki Youth, NY)5
%BHI agar medium, and then aliquoted into 1 ml each ampoule, freeze-dried, and stored at -70°C. The basal medium for bacterial growth is 3.7% BHI. Each basal medium was supplemented with 10 mg of NAD and 20 mg of hemin (the hydrochloride salt of heme, a derivative of hemoglobin) (Eastman Kodak, Rochester, NY). 1% horse blood from which fibrin has been separated
(v/v) Add to 50 ml of inoculated culture solution. Replenishment substances are prepared immediately before use and passed through a 0.45μ Naldiene filter unit (Naldiene filter).
Gibron Corporation, Rochester, New York). 14
To grow the bacteria in an incubator, the medium is further supplemented with 0.5% glucose. To prepare a one-flask inoculum, thaw 1 ml of frozen stock culture and add BHI medium enriched with fibrin-separated horse blood and NAD.
Transfer to 50ml. This culture was carried out at 37°C for 8 hours.
This is done in a rotary shaker at 150 revolutions/min. Nutrient-rich BHI gravy 500 containing bacteria in 2 flasks
ml solution at a concentration of 1%, continue to gently shake in a rotary shaker at 37℃, and culture for 8 hours (for the purpose of CRP isolation) or 14 hours (for the purpose of cultivating the inoculum). do. The culture of each lot in 14 incubators is 700% of the inoculum solution.
Begin by aseptically transferring ml into the nutrient-rich BHI broth 7. This nutrient-rich BHI gravy is supplemented with hemin instead of fibrin-separated horse blood. This cultivation is carried out continuously at 37±1°C. The tank was agitated at a rate of 150 times/min, and air was blown into the tank at a rate of 0.25 per minute of thick liquid. During growth, 0.01% silicone antifoam (FD-82, Hodatsuk Chemical Co.) is added as needed. The culture is grown to the terminal logarithmic growth phase (8 to 10 x ¹⁰⁹ viable cells/ml). Growth is usually terminated after about 8 hours by adding 0.4% formaldehyde and leaving at 4°C overnight. Isolation of polyribosyl ribitol phosphate (PRP) The culture solution prepared as above was heated at 27000 x g for 30 minutes.
Centrifuge at 4°C. The cell-free supernatant is collected for further processing. Step 1: Ethanol precipitation Add sodium acetate to the culture supernatant (final concentration 4%)
Add. Adjust the pH of the solution to 6.0 to 6.2, and slowly add 3% ethanol 44 while stirring vigorously at 4°C. The mixture was adjusted to pH 6.8 with glacial acetic acid and left at 3°C for 12 hours. The resulting precipitate is collected by decanting and centrifuged to obtain crude PRP. Step 2: Cetabron (hexadecyltrimethylammonium bromide) treatment The precipitate obtained in Step 1 is dissolved in pyrogen-free distilled water and centrifuged to remove undissolved residue. A clear brown solution was obtained and this was added to Cetavrone.
Gradually add to 100 ml of 10% aqueous solution while stirring (final concentration 0.5%). The mixture is stirred for 1 hour and then centrifuged. The precipitate of nucleic acids and PRP-setabrone complex is mixed with 0.3M saline solution 2. A cloudy solution is obtained, which is centrifuged to remove insoluble matter such as nucleic acid-setabron salts. The supernatant is diluted with an equal volume of water to precipitate the PRP-setabron salt. The resulting mixture is stirred for 1 hour and the precipitate is collected by centrifugation. Then dissolve in 0.3M saline solution 2. Step 3: Ethanol precipitation Setabron and contaminants of nucleic acids and proteins are further removed by ethanol precipitation (at least twice). PRP is precipitated as described in step 1, while cetabron is dissolved in the alcohol solution. The PRP is recovered by centrifugation, dissolved in pyrogen-free distilled water 2, and reprecipitated as described above. Dissolve the final PRP precipitate in 20 mM sodium phosphate solution (PH 6.8). Step 4: Hydroxylapatite treatment Impurities (eg, nucleic acids, proteins, and bacterial endotoxins) in the crudely purified PRP are selectively removed by adsorption to calcium phosphate. Here, calcium phosphate is hydroxyapatite [3・Ca ₃
(PO ₄ ) ₂・Ca(OH) ₂ ]. The present invention provides that the polysaccharide PRP is not adsorbed to a calcium phosphate adsorbent containing a phosphate buffer (20mM) with a pH of about 6.7 to 6.9 or a phosphate buffer (50mM) with a pH of about 5.8, and on the other hand, contaminants (nucleic acid This is based on the discovery that microorganisms such as bacteria, proteins, and toxins inside bacteria are adsorbed. The process of the invention can be carried out in batch or column operations. When carried out in batches, hydroxyapatite is added (at 20mM phosphate, pH 6.9) to the crude PRP product. The mixture is stirred well and centrifuged to remove unwanted solids (adsorbent and impurities adsorbed on it). This treatment is repeated on the supernatant at least twice. The resulting solution was filtered through a Millipore filter,
It is further dialyzed against pyrogen-free distilled water and freeze-dried. For column operation, dissolve crude PRP in 20mM phosphate buffer (PH at least 5.8) and add 20mM
Pour into a column containing the adsorbent hydroxyapatite that has been equilibrated with phosphate buffer (PH 5.8) and then
Elute with sodium phosphate buffer (PH5.8) with a stepwise concentration change from 20mM to 100nM. Each fraction is collected and analyzed for pentose (for polyribosyl ribitol phosphate). Fractions positive for pentoses are dialyzed against pyrogen-free distilled water and lyophilized. () Method for producing a composite vaccine consisting of PRP obtained from Haemophilus influenzae type B and B pertussis antigens To produce a PRP vaccine, freeze-dried PRP (produced as described above) is Dissolve in buffered saline (PBS) to a concentration of 20 μg/ml. This phosphate buffer is an aqueous solution prepared by dissolving 0.113 g of potassium dihydrogen phosphate, 0.83 g of disodium hydrogen phosphate, and 8.5 g of common salt in 1, and has a pH of 7.0 and further contains 0.01% of thimerosal. This vaccine is over-sterilized using a 0.45 μm millipore filter, packaged in glass vials, and stored at 4°C. Concentrated solution of PRP weighs the expected weight of polysaccharide;
1, dissolved 0.113 g of potassium dihydrogen phosphate, 0.83 g of di-sodium hydrogen phosphate, and 8.5 g of common salt (PH7.0), and further dissolved in a phosphate-buffered saline solution containing 0.01% thimerosal. It is made by Next, this PRP concentrated solution is mixed with an appropriate amount of fresh B. pertussis cell solution to prepare a stock solution. The characteristics of B. pertussis strain 138 are described in Bergey's Manual of
Determinative Bacteriology 8th edition Edited by Bukit Yanan Gibbon, Maryland, USA WMS End Wilkins
Published in 1974, page 283. This strain is Amer Type from Maryland, USA.
Available from Culture Coll. Its deposit number is No. 10380. The conjugate vaccine in the stock solution contains 200 μg of PRP per ml and approximately 70 opacity units (OP) of cells per ml. The stock solution is kept at 4°C for 90 days to detoxify the pertussis antigens, after which the final product (vaccine) is manufactured, which contains 10 μg of PRP per 0.5 ml dose and 3.5 OP units of pertussis cells. ). Next, the present invention will be explained with reference to Examples. Example 1 Hydroxyl apatite (e.g. Bio-gel)
2.5 g of HTP, Bio-Rad Laboratories, Inc., Richmond, Calif., and 20 g of PRP crude product.
Add to 250 ml of a solution dissolved in mM sodium phosphate buffer (PH 6.9) and add 1
Mix for an hour. (This crude PRP agent has been treated with cetabron and ethanol, and is approximately PRP.
This mixture (containing 1.0 mg/ml) is centrifuged in a Sorvall RC2-B for 30 minutes at 1600 xg. The supernatant is passed through a 0.65 μm Millipore filter and the above treatment is repeated two more times (each time using 2.5 g of hydroxyapatite). The resulting solution is filtered through 0.65μ and 0.4μ Millipore filters, dialysed against pyrogen-free distilled water, and lyophilized. The lyophilizate shows strong immunological activity (see Combination Vaccines and Animal Studies below) and has a very low content of impurities (such as nucleic acids, proteins and endotoxins) (see Part 1 below). (see table). Obtain approximately 170 mg of lyophilized PRP.
The recovery rate of PRP by this method is about 70% of the starting polysaccharide. This PRP must be stored under suitable conditions such as 4° C. in a desiccator over phosphorous pentoxide and silica gel. Example 2 Crudely purified PRP (300 mg PRP) was dissolved in 100 ml of 20 mM sodium phosphate buffer (PH5.8) (i.e. 3
mgPRP/ml). This solution was heated at room temperature to a column of hydroxyapatite (approximately 250 ml bed volume) (5.0×
45cm). Next, 20m with a stepwise increase in concentration.
Elute with M, 50mM, 100mM sodium phosphate buffer (PH5.8). Among the fractions (200 ml) eluted with 20 mM and 50 mM sodium phosphate buffer (PH5.8), those positive for pentose (pentose was determined by the orcinol method) are collected. It is dialyzed against pyrogen-free distilled water and lyophilized. Approximately 206mg freeze-dried
PRP is obtained. The purity of polysaccharide PRP is analyzed by quantifying the amount of contamination with nucleic acids, proteins, and bacterial endotoxins. Protein concentration is determined by the method of Lowry et al. [J. Biol. Chem. Vol. 193, p. 265 (1951)] using bovine serum albumin as a standard. Nucleic acid is
It is determined by the absorbance of the PRP solution at 260 nm. Light progression: 1cm cell, 1ml water, 50μ nucleic acid
It is calculated assuming that the absorbance when containing g is 1.0.
The molecular size is 1.5 due to Sepharose 4B or 2B.
It is quantified by means of gel filtration on a ×90 cm column (Pharmacia-Fine Chemicals, Piscataway, New Jersey, USA). The partition coefficient Kd value is calculated from the elution pattern developed by the orcinol reaction (Herbert et al., Methods in
Microbiology, Vol. 5B, 285-291).

[Table] Example 1 is Amer Type, Maryland, USA.
The characteristics of the influenza BCK strain available from Culture Coll (accession number 3144) are described. Example 2 is Columbia University, New York, USA.
Influenza B available from Babies Hospital
Describes the characteristics of Rab strains. Example 3 A combined vaccine containing PRP from Haemophilus influenzae type b and B pertussis antigen is produced as follows: 140 mg of PRP (according to the production method of Example 2) is dissolved in sterile phosphate buffered saline. Dissolve in 350 ml of water (PBS) (PBS is 0.113 g of potassium dihydrogen phosphate, disodium hydrogen phosphate
Dissolve 0.83g and 8.5g of sodium chloride (PH
7.0), which is also a solution containing 0.01% thimerosal). Then pass through a 0.45μ Millipore filter. This concentrated PRP solution (400 μg/ml) is used to produce a combined vaccine consisting of PRP and B pertussis antigen. In 150 ml of concentrated PRP solution (400 μg/ml), add a suspension of fresh B. pertussis (strain 138) cells in PBS (PH 7.0, containing 149 opacity units cells/ml) 150
Make the combined vaccine stock solution by adding ml. This stock solution (300 ml) is stored at 4° C. until the level of B. pertussis endotoxin has decreased (at least 90 days). The sterility of this stock solution is tested in thioglycollate medium (32-33°C). The pH of this storage solution is 90
After culturing for one day, the pH is adjusted to 7.0±1. The final product (vaccine) of the stage used in animal experiments is
Made by diluting the combined stock solution with PBS. The solution is PRP 10 μ per 0.5 ml (medicinal dose)
g and 3.5 opacity units of B. pertussis cells. Figures 1 and 2 show the results of antibody production responses to the above-produced conjugate vaccine in young rats.
As shown in the figure. Figure 1 shows the purified product produced in Example 2.
Radioimmunization of anti-PRP antibody production in rats when rats were boosted with PRP alone and when rats were boosted with the (B/pertussis antigen + PRP) combination vaccine produced in Example 3. It is a graph showing the analysis results as weekly geometric mean titer (CPM/100MCL) curves. In the figure, curve A is the case when only the former PRP is boosted, and curve B is the curve when the latter (pertussis antigen + PRP) combination vaccine is boosted. Figure 2 shows the cases in which only the B pertussis antigen was injected into rats (Wistar type), the cases in which the combination vaccine (B pertussis antigen + PRP) was injected once into rats (Wistar type), and the cases in which (B pertussis antigen +
This is a graph showing the results of radioimmunoanalysis of the amount of anti-PRP antibody produced in rats (Wistar series) as a weekly geometric mean titer (cmp/100 mcl) curve when the rats (Wistar type) were boosted with a PRP conjugate vaccine. In the figure, curve C is when only B/pertussis antibody is injected, curve D is when (B/pertussis antigen + PRP) combined vaccine is injected only once, and curve E is when (B/pertussis antigen + PRP) combined vaccine is added. These are the respective curves when immunized. According to these graphs, it can be seen that when the combination vaccine of the present invention is used, the amount of antibody produced is significantly large. The results of radioimmunoassay (RIA) were performed as follows. The outline of the RIA method is as follows:

[Table] Centrifugation
〓 ³ H〓〓PRP bound to antibody
↓
Radioactivity measurements Anti-PRP antibody measurements were performed as follows: a 25-50μ aliquot of each serum sample was placed in a 12mm x 75mm
Contains 2% fetal bovine serum in polyethylene tubes
0.05M phosphate buffered saline (PH7.3) (PBS−
FCS) mixed with 400μ, radioactive [ ^3H ]PRP
(5500cpm) 50μ was added. the contents of the tube
Mixed on a Vortex mixer and then incubated for 1 hour at 37°C. To separate the antibody-[ ³ H]PRP complex from unbound [ ³ H]PRP, 0.5 ml of cold 25% PEG in PBS was added. Vortex
After mixing on the mixer, the tubes were left in the refrigerator (4°C) overnight. The assay mixture was then incubated at 4°C for 20 min.
Centrifugation was performed at 8000×g and the supernatant was discarded by decantation. The pellet was washed once with 1 ml of 12.5% PEG and centrifuged as described above. The tube was cut above the point containing the pellet and the lower portion of the tube (containing the pellet) was placed in a scintillation vial. 0.3 ml of Beckman Histolytic Agent (manufactured by Beckman Instruments, Inc.) was added to each pellet containing the [ ^3H ]PRP-antibody complex. The treated pellets were heated at room temperature for 2 hours.
Incubated for hours. scintillant,
5 ml of Econofluor was then added to each vial and 2 drops of a freshly prepared 15% ascorbic acid solution were added. Radioactivity was measured on a Beckman LS7000 liquid scintillation spectrometer. The percentage of [ ^3H ]PRP bound is calculated using the formula (a-c/b-c)×
Calculate from 100. where a represents the cpm precipitated using the test serum, b represents the total cpm added, and c represents the cpm precipitated non-specifically using PBS-FCS. The amount of anti-PRP antibody produced in the test serum can be read directly from a standard curve obtained from data from a dilution series of a standard serum (S. Klein) containing a known content of anti-PRP immunoglobulin. Example 4 A stock solution of PRP (400 μg/ml) was prepared by dissolving 30 mg of PRP (according to the same method as in Example 1) in 75 ml of sterile phosphate buffered saline (PBS) and passing through a 0.45 μ Millipore filter. Made. Add 25 ml of this PRP concentrated aqueous solution to a PBS suspension of newly prepared B. pertussis (strain 138) cells (PH7.0, 149 opacity unit cells/
A stock solution of the conjugate vaccine is made by adding an equal volume (i.e. 25 ml) of the conjugate vaccine (including ml). This stock solution (50 ml) is kept at 4°C for at least 90 days. The sterility of this stock solution is tested as described above in thioglycollate medium. The pH of the stock solution is 7.0±1. The final product (vaccine) used in experimental animals is prepared by diluting the combined stock solution with PBS. This solution contains 10 μg of PRP and 3.7 opacity units of Bordetella pertussis cells per 0.5 ml (dose). Example 5 PRP stock solution (200μ
g/ml) is prepared in the same manner as in Example 4. pertussis stock solution (containing 75 opacity units/ml) is a new B. pertussis cell suspension (containing 149 opacity units/ml).
ml) by diluting 25 ml of PBS with an equal volume of PBS. Both stock solutions are incubated separately at 4° C. for 90 days or even longer. For the combined vaccine, take 14 ml of PRP stock solution (200 μg/ml) and 14 ml of pertussis antigen (75 opacity units/ml) stock solution (detoxified) and dilute the solution with 112 ml of PBS (final volume 140 ml). made by. The final vaccine used in animal experiments is 0.5ml.
Contains 10 μg of PRP and 3.7 opacity units of pertussis antigen per (dose). The results of antibody production responses to the above-produced conjugate vaccine in young rats are shown in Figures 3 and 2.
Shown in the table. FIG. 3 shows the results when rats were injected with B pertussis antigen alone (curve F) and the case where rats were injected with only B pertussis antigen (curve F) and the case where rats were injected with B pertussis antigen +
FIG. 2 is a graph showing the results of radioimmunoassay of anti-PRP antibody levels when rats were boosted with a PRP conjugate vaccine (curves G and H, respectively) as weekly geometric mean titer (cpm/100 mcl) curves. This graph shows that when the combination vaccine of the present invention is administered, the amount of antibody produced is significantly higher. Note that radioimmunoassay was performed in the same manner as in Example 3.

【table】

【table】 [Brief explanation of the drawing]

FIG. 1, FIG. 2, and FIG. 3 are all graphs showing the results of radioimmunoassay of the amount of anti-PRP antibody produced in rats.

Claims (1)

[Scope of Claims] 1. A young temperature characterized by comprising polyribosylribitol phosphate isolated and purified from capsular polysaccharide of Haemophilus influenzae type B using cetabron and hydroxyapatite treatment, and Bordetella pertussis antigen. A combination vaccine that induces the production of anti-PRP (polyribosyl ribitol phosphate) and anti-pertussis antibodies in blood animals. 2. The combination vaccine according to claim 1, wherein the Bordetella pertussis antigen is obtained from Bordetella pertussis strain 138. 3. Claim 1 in which polyribosyl ribitol phosphate is obtained from Haemophilus influenzae type b Rab strain
Combined vaccine as described in section. 4. The combination vaccine according to claim 1, wherein the Bordetella pertussis antigen is obtained from Bordetella pertussis strain 138 and the polyribosyl ribitol phosphate is obtained from Haemophilus influenzae type b Rab strain. 5. The combination vaccine according to claim 1, wherein the slightly warm-blooded animal is a human child. 6. The combination vaccine according to claim 4, wherein the slightly warm-blooded animal is a human child.