DE2845745C2 - - Google Patents

Description

The invention is in the field of vaccines for immunization against infections caused by Haemophilus influenzae type b, like meningitis. In detail, it relates to a procedure to isolate and clean the antigenic polysac charids polyribosyl ribitol phosphate (PRP) from cultures of Haemophilus influenzae type b and a combination vaccine containing PRP and B. pertussis antigens.

From FR-PS 69 16 279 is a process for the extraction of Glycoproteins by extracting cultures from Haemophilus known influenzae. The glycoproteins have one anti-inflammatory and non-specific antigen effects.

From GB-PS 14 89 896 a vaccine is known which consists of a combination of ribosomal fractions and from the Cell membrane-derived glycoproteins from Haemophilus influenzae exists and acts against bronchial infections.

From GB-PS 11 19 543, US-PS 33 95 219 and US-PS 24 65 078 is the extraction of Bordetella pertussis antigen isolated cell walls of B. pertussis or from suspensions crushed B. pertussis cells known.

The purified polyribitol phosphate (PRP) from Haemophilus influenzae Type b is investigated as a protective immunogen. An effective one However, the antigenic response is still in young animals and children has not been reached. In the known method for cleaning  become ethanol and hexadecyltrimethylammonium bromide (Cetavlon) used. The contaminants - endotoxins and pyrogenic substances - are made by using cold Phenol or chloroform and t-butanol removed, leading to loss the antigenic nature of this polysaccharide.

There has now been a new process for insulation and cleaning of immunologically active PRP from Haemophilus influenzae type b found.

The object of the invention is that specified in claim 1 Method.

This method has compared to the known Working methods have clear advantages because of all impurities (Nucleic acid, proteins and endotoxins) by treatment with hydroxyapatite can be removed. The Process according to the invention for the production of PRP leads to a higher molecular weight polysaccharide than that previously known methods.

The PRP manufactured according to the new process is also more importantly, as opposed to the known one Working methods produced PRP for warm-blooded animals to a high degree immunogenic.

The removal of contaminants (proteins, nucleic acids and endotoxins) of the polysaccharide PRP is treated of the partially purified polyaccharide with a phosphate containing adsorbent achieved under the specified conditions the polysaccharide is not absorbed.

The further object of the invention is to create a combined PRP and pertussis vaccine used in children and has a highly immunogenic effect on young animals.  

The invention furthermore relates to that in claim 3 specified combination vaccine.

(I) isolation and purification of polyribosyl ribitol phosphate, the envelope polysaccharide from Haemophilus influenzae type b Organisms, culture medium and culture

There are two strains of Haemophilus influenzae type b used. The Rab tribe is from Grace Leidy, Babies Hospital, Columbia University, New York City, New York.  

The CK strain is from a patient at Waterbury Hospital, Waterbury, Conn. been isolated. The organisms will subjected to multiple passages through mice to their virulence to guarantee. They are taken from the brain tissue during the autopsy isolated from mice, and for their further breeding One of the following media is used: 3.7 percent brain cardiac infusion (BHI) medium, or 5 percent BHI agar, supplemented with 0.01% nicotinamide adenine Dinucleotide (NAD) and 1% (volume / volume) defibrinated horse blood. For further cultivation, 1 ml portions are distributed to ampoules, lyophilized and stored at -70 ° C.

The basic medium used for the cultivation of the organisms is 3.7 percent BHI. The basic medium is filled with 10 mg NAD and 20 mg hemin supplemented per liter. 50 ml inoculation culture each 1% (volume / volume) is defibrinated Horse blood added. The supplements are fresh prepared and by a 0.45 µ Nalgene filter unit filtered before use. For growing the organism in a 14 l The fermentation vessel also contains 0.5% Glucose supplements.

To make organisms for insemination, 1 ml is frozen Thaw the stock culture and add 50 ml of the enriched BHI Medium supplemented with defibrinated horse blood and NAD is given. The culture is kept at 37 ° C for 8 hours Round shaker bred at 150 revolutions / minute. The organisms are made up as 1 percent inoculum 500 ml each of the enriched BHI medium in 2 l flasks given that then at 37 ° C with moderate shaking from a Circular shaker 8 hours (to isolate PRP) or 14 hours (as seed cultures).  

The fermentation of each portion in a 14 liter fermentation vessel is transferred by aseptic transfer of 700 ml Seed culture to 7 l of the enriched BHI medium, which with Hemin is supplemented instead of defibrinated horse blood, initiated. The culture is kept constantly at 37 ± 1 ° C. With stirring at 150 revolutions / minute there is an air flow maintain 0.25 l air per liter medium and minute. During growth, 0.001% of a silicone defoamer (FD-82, Hodag Chemical Corp.) added as needed. The Cultivation of the cultures continues until only exponing tial growth (8 to 10 × 10⁹ living cells / ml) takes place, usually about 8 hours, and then the breeding is done Add 0.4% formaldehyde and let stand overnight 4 ° C ended.

Isolation of polyribosyl ribitol phosphate (PRP)

The mash obtained as described above is at 4 ° C. 30 minutes at 27,000. g centrifuged. The cell-free Supernatant liquid is separated and as follows treated:

Level 1, ethanol precipitation

The culture supernatant becomes up to one Final concentration of 4% mixed with sodium acetate. To Adjust the pH of the solution to be 6.0 to 6.2 44 l of 3A ethanol are slowly added with vigorous stirring at 4 ° C. The mixture is brought to pH with glacial acetic acid 6.8 and then allowed to stand at 3 ° C for 12 hours. The precipitate formed is decanted and centrifuged fugieren won, whereby raw PRP is obtained.  

Stage 2, treatment with hexadecyltrimethylammonium bromide (Cetavlon)

The precipitate obtained after stage 1 is pyrogen-free distilled water and to remove the residue centrifuged. The clear brown solution then slowly becomes 100 ml of a 10 percent aqueous solution of hexadecyltri methylammonium bromide to a final concentration of 0.5% given with mixing. The mixture is stirred for 1 hour and then centrifuged. Precipitation from nucleic acids and PRP-Hexadecyltrimethylammoniumbromid complex with 2 l 0.3 M sodium chloride mixed. The cloudy solution becomes Removal of insoluble substances such as nuclein-hexadecyltrimethyl centrifuged ammonium bromide salts.

The supernatant liquid is of an equal volume Diluted water, causing the PRP hexadecyltrimethylammonium bromide Salt fails. The mixture is stirred for 1 hour, and the precipitate is obtained by centrifugation and then dissolved in 2 l of 0.3 M sodium chloride.

Level 3, ethanol precipitation

Hexadecyltrimethylammonium bromide and the contaminating Nucleic acids and proteins are replaced by at least two Ethanol precipitation further away. The PRP will, as in stage 1 described, like, while hexadecyltrimethylammonium bromide is dissolved in the alcoholic solution. The PRP will be off-center fugiert, dissolved in 2 l pyrogen-free distilled water and then like again as described above. The finally PRP precipitate obtained is in 20 millimolar sodium phosphate, pH 6.8 dissolved.  

Stage 4, hydroxyapatite treatment

The impurities (for example nucleic acids, proteins and endotoxins) in the partially purified PRP preparations become selective by adsorption on hydroxyapatite [3Ca₃ (PO₄) ₂.Ca (OH) ₂] removed.

The invention is based on the perception that the Polysac charid PRP by the hydroxyapatite, which is a phosphate buffer (20 mM) with a pH of 6.7 to 6.9 or a phosphate buffer Contains (50 mM) with a pH of 5.8, in contrast to contaminants such as nucleic acid, proteins and Endotoxins, is not adsorbed. The invention The process can be carried out batchwise or in column operation be performed. When working in batches the hydroxyapatite to the partially purified PRP preparation (in 20 mM phosphate, pH 6.9). After thorough mixing is used to remove unwanted solids (adsorbent and the impurities adsorbed thereon) centrifuged. The supernatant becomes the above described procedure at least two more times subject. The solution obtained is through Millipor filter filtered, dialyzed against pyrogen-free distilled water and lyophilized.

During column operation, the partially cleaned PRP is in 20 mM Put phosphate buffer (pH at least 5.8) on a column, which contains the adsorbent hydroxylapatite, which with 20mM Phosphate buffer, pH 5.8, and with a gradually changed sodium phosphate buffer (pH 5.8) eluted from 20 mM to 100 mM. The fractions caught are tested for pentose (polyribosyl ribitol phosphate). Those fractions that are positive about pentose are dialyzed against pyrogen-free distilled water and lyophilized.  

(II) Process for the preparation of a combination vaccine PRP from H. influenzae type b and B. pertussis antigens

A PRP vaccine is prepared as described above prepared lyophilized PRP in a concentration of 10 µg / ml in phosphate buffered saline (PBS) (o, 113 g Potassium diphosphate, 0.83 g disodium phosphate and 8.5 g sodium chloride per liter, pH 7.0, containing 0.01% thimerosal) dissolved. The vaccine is separated by 0.45 µ millipore filter units sterile filtered, placed in glass vials and at 4 ° C stored.

A concentrated solution of the PRP is weighed out by dissolving it predetermined amounts of the polysaccharide in phosphate buffered Saline (PBS) (0.113 g potassium diphosphate, 0.83 g disodium phosphate and 8.5 g sodium chloride per liter, pH 7.0, containing 0.01% thimerosal). This concentrated solution from PRP is then mixed with fresh B. pertussis cell suspensions, whereby the stock solution is obtained. The characteristics from B. pertussis strain 138 are in Bergey's Manual of Determinative Bacteriology, VIII, editor Buchanan and Gibbons, WMS and Wilkins Co., Maryland, USA, 1974, on page 283. This strain is in the American Type Culture Collection, Maryland, V. St. A. available under accession number 10380. The combination vaccine in this stock solution contains 200 µg PRP and about 70 opacity units (op) cells / ml. The stock solution is used to detoxify pertussis antigens for 90 days 4 ° C before the finished product is made 10 µg PRP and 3.5 op units of pertussis Contains cells / 0.5 ml dose.

The following examples further illustrate the invention explained.

Example 1

2.5 g of hydroxyapatite are partially purified to 250 ml PRP preparations (after hexadecyltrimethylammonium bromide and ethanol treatments) containing about 1.0 mg PRP / ml placed in 20 mM sodium phosphate buffer, pH 6.9, and 1 hour  mixed in an ice cold water bath (1 to 4 ° C). The Mixing takes 30 minutes at 1600 in the Sorvall RC2-B. g centrifuged. The supernatant liquid is then replaced by a 0.65 µ Millipor filter passed and two more times subject to the operation described above, each Treated with 2.5 g of hydroxyapatite. The received Solution is filtered through 0.65 µ and 0.45 µ Millipor filter, dialyzed against pyrogen-free distilled water and lyophilized. The lyophilized product shows a strong immunogenic Efficacy (see below, combination vaccine and Animal experiment) and contains very little contamination, such as nucleic acids, proteins and endoxins (see Table I). About 170 mg of lyophilized PRP is obtained, which is about 70% of the polysaccharide used corresponds. The PRP must under suitable conditions, for example at 4 ° C, in one Desiccator stored over phosphorus pentoxide and silica gel will.

Example 2

A partially purified PRP with a PRP content of 300 mg is dissolved in 100 ml of 20 mM sodium phosphate buffer, pH 5.8 (i.e. 3 mg PRP / ml) dissolved. This solution is at room temperature on a column (5.0 × 45 cm) of hydroxyapatite (bed volume about 250 ml, with 20 mM sodium phosphate buffer, pH 5.8, equilibrated) and with 20 mM, 50 mM and 100 mM Sodium phosphate buffer, pH 5.8, eluted. The 20 mM and 50 mM Sodium phosphate buffer (pH 5.8) eluted fractions (200 ml), which are pentose positive (pentose determination according to the orcin Method) obtained fractions are against pyrogen-free distilled water dialyzed and lyophilized. It will about 206 mg of lyophilized product, PRP obtained.  

The purity of the polysaccharide PRP is determined by determination the level of contamination with nucleic acid, proteins and endotoxins tested. The protein concentration will according to the Lowry, et al. in J. Biol. Chem. 193: 265 (1951) with bovine serum albumin as the standard. Nucleic acid is determined by the absorption of the PRP solution at 260 nm. The absorption of 50 µg nucleic acid in 1 ml water in a cell with a light path of 1 cm, 1.0 is equated. The molecular weight is determined using Sepharose 4B- or 2B- Gel filtration determined on columns of 1.5 × 90 cm. The values of Partition coefficients (Kd) are obtained from the orcin reaction (Herbert, et al., Methods in Microbiology, Vol. 5B, 285-291) developed elution image.  

Table I

Physico-chemical properties of PRP

Example 3

A PRP combination vaccine from H. influenzae type b and B. contains pertussis antigens is produced as follows: 140 mg of PRP (prepared as described in Example 2) in 350 ml of sterile phosphate buffered saline (PBS) (0.113 g Potassium diphosphate, 0.83 g disodium phosphate and 8.5 g sodium chloride per liter, pH 7.0, containing 0.01% thimerosal) dissolved and filtered through 0.45 µ Millipor filter. This concentrated PRP solution (400 µg / ml) is used to make a combination vaccine used that from PRP and B. pertussis antigens consists.

150 ml of the concentrated PRP solution (400 µg / ml) are added 150 ml of a fresh cell suspension of B. pertussis (strain 138) in PBS, pH 7.0 (containing 149 opacity (op) unit cells / ml) added, which makes the stock solution of the combination vaccine is obtained. This stock solution, 300 ml, is kept at 4 ° C, until the endotoxin level of pertussis is reduced (at least 90 days). The sterility of the stock solution is determined with Thioglycollate media tested at 32 to 33 ° C. The pH of the Stock solution after an incubation of 90 days is checked and set to 7.0 ± 1. The one used for animal experiments Finished product is combined by diluting the Prepared stock solution with PBS and contains 10 µg PRP and 3.5 op units of pertussis cells / 0.5 ml (dose).

The results of the antibody response to this combination vaccine in young rats are shown in FIGS. 1 and 2.

Example 4

The stock solution of PRP (400 µg / ml) is made by dissolving 30 mg of the PRP prepared as described in Example 1 in  75 ml of sterile phosphate buffered saline (PBS) and filter produced by 0.45 µ millipore filter. To 25 ml of this concentrated PRP solution is an equal volume, i.e. H. 25 ml fresh cell suspension of B. pertussis (strain 138) in PBS, pH 7.0 (containing 149 op-units cells / ml), whereby the stock solution of the combination vaccine is obtained. This stock solution (50 ml) is at least 90 days at 4 ° C. ditched. The sterility of the stock solution is as above mentioned, tested with thioglycollate media. The pH of the Stock solution is 7.0 ± 1. The one used for animal experiments The finished product is made by diluting the combination stock solution made with PBS and contains 10 µg PRP and 3.7 op units of pertussis cells / 0.5 ml (dose).

Example 5

The PRP stock solution (200 µg / ml in PBS, pH 7.0) is as in Example 4 prepared. The Pertussis stock solution (containing 75 op units / ml) is made by diluting 25 ml Fresh cell suspension of B. pertussis (149 op units / ml) made with an equal volume of PBS. Both stock solutions are separated from each other, 90 days or a little longer at 4 ° C incubated. The combination vaccine is made using 14 ml PRP stock solution (200µg / ml), 14 ml (detoxified) Stock solution of pertussis antigens (75 op units / ml) and 112 ml PBS prepared (final volume 140 ml). This for animal experiments The vaccine used contains 10 µg PRP and 3.7 op units B. pertussis / 0.5 ml (dose).

The results of the antibody response to this combination vaccine in young rats appear in Fig. 3 and in Table II.

Table II

Immunogenicity of PRP-pertussis complex in young rats)

Claims (3)

1. Process for the isolation and purification of immunologically active polyribosyl ribitol phosphate (PRP), the envelope polysaccharide of Haemophilus influenzae type b, by fermentation of strains of the organism Haemophilus influenzae type b, separation of the PRP by ethanol precipitation and isolation of the PRP as hexadecomidium trimethyl complexium, characterized by that PRP by adding hydroxyapatite [3Ca₃ (PO₄) ₂Ca (OH) ₂] in a 0.02 sodium phosphate buffer at a pH of 6.7 to 6.9, mixing at a temperature of 4 ° C, centrifuging, separating the supernatant liquid and repeating these measures at least twice, filtering the supernatant liquid and then dialyzing against pyrogen-free distilled water and lyophilizing. 2. The method according to claim 1, characterized in that that the PRP by column chromatography in one 0.02 M sodium phosphate buffer at a pH of 5.8 using hydroxyapatite [3Ca₃ (PO₄) ₂Ca (OH) ₂], elution with a graduated 0.02 m up to 0.05 m sodium phosphate buffer at a pH of 5.8, Isolation of fractions by analysis on PRP, dialysis of this Fractions against pyrogen-free distilled water and Lyophilizing is cleaned.   3. Combination substance for the formation of anti-PRP (Polyribosyl ribitol phosphate) - and anti-pertussis anti bodies in young warm-blooded animals, containing as an active ingredient the polyribosyl obtained according to claims 1 and 2 ribitol phosphate obtained from Bordetella pertussis ATCC 10380.