DE2845745A1 - COMBINATION VACCINE - Google Patents

Description

The invention is in the field of vaccines for immunization against Haemophilus influenzae type b infections such as meningitis. Specifically, it relates to (1) a Process for the isolation of the antigenic polysaccharide polyyribosyl-ribitol-phosphate (PRP) from Haemophilus influenzae type b cultures and (2) a process for producing a Combination vaccine containing PRP and B. pertussis antigens. The PRP according to the invention is also in connection with other non-phatogenic strains of bacteria such as E. coli, B. subtilis, S. aureus, B. punilus and L. plantarum are effective for triggering an antibody reaction in warm-blooded animals.

The purified polyribitol phosphate (PRP) from Haemophilus influenzae type b is examined as a protective immunogen. One however, an effective antigen response has not yet been achieved in young animals and children. In the known method ethanol and hexadecyltrimethylammonium bromide are used for cleaning (Cetavlon) is used. The impurities Endotoxins and fumed substances are removed by using cold phenol or chloroform and t-butanol can lead to the loss of the antigenic nature of this polysaccharide.

909819/0640

There has now been a new method for the isolation and purification of immunologically active PRP from Haemophilus influenzae type b found.

The method still to be described has clear advantages over the known working methods, since all impurities (Nucleic acids, proteins and endotoxins) can be removed by treatment with hydroxyapatite. The inventive Process for making PRP results in a polysaccharide having a higher molecular weight than that previously known method.

The PRP produced according to the new process is also, more importantly, in contrast to that according to the known Procedures produced PRP for warm-blooded animals to a high degree immunogenic.

The removal of impurities (proteins, nucleic acids and end toxins) of the polysaccharide PRP is achieved by treatment of the partially purified polysaccharide achieved with a phosphate-containing adsorbent, which under the specified conditions does not adsorb the polysaccharide. '

The further object of the invention is to provide a method for producing a combined PRP and pertussis vaccine, which is highly immunogenic in young animals.

(I) Isolation and purification of polyribosylrxbitolphoshate, the envelope polysaccharide of Haemophilus influenzae type b

Organisms, growth medium and culture

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

909819/0640

The CK strain was isolated from a patient at Waterbury Hospital, Waterbury, Conn. The organisms are subjected to several passages through mice to ensure their virulence. They are isolated from mouse brain tissue at autopsy and one of the following media is used to grow them: 3.7 percent brain heart infusion (BHI) (Difco Lab., Detroit, Mich.) Medium or 5 percent BHI Agar supplemented with 0.01% nicotinamide adenine dinucleotide (NAD) (PL Biochemicals, Milwaukee, Wis.) And 1% (volume / volume) defibrinated horse blood (Animal Blood Center, Syracuse, NY). For further breeding units are distributed by 1 ml into vials, lyophilized and stored at -70 ⁰ C.

The base medium used to grow the organisms is 3.7 percent BHI. The basic medium is 10 mg of NAD and 20 mg Hemin (Eastman Kodak, Rochester, N.Y.) per liter supplemented. 1% (volume / volume) defibrinated per 50 ml inoculum culture Horse blood added. The supplements will be fresh prepared and filtered through a Ο, 45μ Nalgene filter unit (Nalge Sybron Corp., Rochester, N.Y.) prior to use. For growing the organism in a 14 liter fermentation vessel the medium is also supplemented with 0.5% glucose.

To prepare organisms for insemination, 1 ml of frozen stock culture is thawed and added to 50 ml of the enriched BHI medium supplemented with defibrinated horse blood and NAD. The culture is / bred for 8 hours at 37 ⁰ C on a Rundschüttelvorrxchtung at 150 revolutions minute. The organisms are referred to as 1-owned inoculum to portions of 500 ml of BHI-medium placed in 2 1 flasks enriched, then at 37 ⁰ C with moderate shaking on a Rundschüttelvorrxchtung 8 hours (for the isolation of PRP) or 14 hours ( as seed cultures) are incubated.

90981 9 / 06Ä0

The fermentation of each portion in a 14 l fermentation vessel is initiated by aseptic transfer of 700 ml of the seed culture to 7 l of the enriched BHI medium supplemented with hemin instead of defibrinated horse blood. The culture is steadily at 37+; 1 ⁰ C held. An air flow of 0.25 l of air per liter of medium per minute is maintained while stirring at 150 revolutions / minute. During the cultivation, 0.001% of a silicone defoamer (FD-82, Hodag Chemical Corp.) is added as necessary. The cultivation of the cultures is continued until only exponential

9
tial growth (8-10 10 viable cells / ml) occurs, usually about 8 hours, and then cultivation is terminated ^{by adding 0.4% formaldehyde and leaving it to stand at 4 ° C. overnight.}

Isolation of polyribosyl ribitol phosphate (PRP)

The mash obtained as described above becomes ^{27,000 at 4 °} C. for 30 minutes. centrifuged g. The cell-free supernatant fluid is separated and treated as follows:

Stage 1, ethanol precipitation

Sodium acetate is added to the supernatant liquid of the culture up to a final concentration of 4%. After the pH of the solution has been adjusted to 6.0 to 6.2, 44 1 3 A ethanol are slowly added ^{at 4 ° C. with vigorous stirring.} The mixture is adjusted to a pH of 6.8 with glacial acetic acid and then left to stand at 3 ^° C. for 12 hours. The precipitate formed is collected by decantation and centrifugation, whereby crude PRP is obtained.

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Stage 2, treatment with hexadecyltrimethylammonium bromide (Cetavlon)

The precipitate obtained after step 1 is dissolved in pyrogen-free distilled water and used to remove the residue centrifuged. The clear brown solution then slowly becomes 100 ml of a 10 percent aqueous solution of hexadecyltrimethylamreonium bromide added to a final concentration of 0.5% with mixing. The mixture is stirred for 1 hour and then centrifuged. The precipitate of nucleic acids and PRP-hexadecyltrimethylammonium bromide complex is treated with 2 1 0.3m sodium chloride mixed. The cloudy solution is used to remove insolubles such as the nucleic hexadecyltrimethylammonium bromide salts centrifuged.

The supernatant liquid is diluted with an equal volume of water, thereby producing the PRP-hexadecyltrimethylammonium bromide salt fails. The mixture is stirred for 1 hour, and • the precipitate is collected by centrifugation and then dissolved in 2 1 0.3m sodium chloride solution.

Stage 3 , ethanol precipitation

Hexadecyltrimethylamr or-iumbromid and the contaminants Nucleic acids and proteins are further removed by ethanol precipitation at least twice. The PRP is, as in stage 1 described, precipitated while hexadecyltrimethylammonium bromide is dissolved in the alcoholic solution. The PRP is centrifuged off, dissolved in 2 l of pyrogen-free distilled water and then reprecipitated as described above. The one finally PRP precipitate obtained is dissolved in 20 millimolar sodium phosphate, pH 6.8, dissolved.

909819 / 0S40

Stage 4, hydroxyapatite treatment

The impurities (for example, nucleic acids, proteins and endotoxins) in the partially purified PRP preparations are made selective by adsorption on a calcium phosphate-containing adsorbent such as hydroxyapatite / 3Ca-. (PO.) ". Ca (OH) ₂ __ / removed.

The invention is based on the perception that the polysaccharide PRP through the calcium phosphate adsorbent, the phosphate buffer (2O inM) with a pH value of 6.7 to 6.9 or a phosphate buffer (50 mM) with a pH of 5.8, in contrast to the impurities such as nucleic acids, proteins and endotoxins, is not adsorbed under these conditions. The process according to the invention can be operated in batches or in columns be performed. When working in batches, the hydroxyapatite becomes the partially purified PRP preparation (in 20 mM phosphate, pH 6.9). After thorough rental ■ see is centrifuged to remove unwanted solids (adsorbent and the impurities adsorbed on it). The supernatant liquid is subjected to the procedure described above at least two more times subject. The solution obtained is filtered through a Millipore filter and dialyzed against pyrogen-free distilled water and lyophilized.

In column operation, the partially purified PRP in 20 mM phosphate buffer (pH at least 5.8) is applied to a column, which contains the adsorbent hydroxyapatite which has been equilibrated with 20 mM phosphate buffer, pH 5.8, and with a stepwise modified sodium phosphate buffer (pH 5.8) eluted from 20mM to 100mM. The collected fractions are checked for pentose (polyribosylribitol phosphate). Those fractions which are positive for pentose are dialyzed against pyrogen-free distilled water and lyophilized.

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⁴⁹ 28 ^ 5745

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

To produce a PRP vaccine, lyophilized PRP prepared as described above is added at a concentration of 20 μg / Inl 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) dissolved. The vaccine is sterile filtered through 0.45 μ-Millipore filter units, introduced into glass vials and stored ^{at 4 ° C.}

A concentrated solution of the PRP is prepared by dissolving weighed 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) manufactured. This concentrated solution of PRP is then mixed with fresh B. pertussis cell suspensions to give the stock solution. The characteristics of B. pertussis strain 138 are given in Bergey's Manual of Determinative Bacteriology, VIII, Editors Buchanan and Gibbons, WMS and Wilkins Co., Maryland, USA, 1974, at page 283. This strain is with the American Type Culture Collection, Maryland, V-St-A. available under the deposit 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 allowed to stand for detoxification of pertussis antigens 90 days at 4 ⁰ C, before the finished product (Vaccine) is prepared containing 10 ug PRP and 3.5 op units pertussis cells / 0.5 ml dose contains.

The invention is further illustrated by the following examples.

Example 1 1

2.5 g hydroxyapatite (e.g. Bio. Gel HTP, Bio-Rad Laboratories, Richmond, Calif.) Are partially purified to 250 ml

PRP preparations (based on hexadecyltrimethylammonium bromide

and ethanol treatments) containing about 1.0 mg PRP / ml, in 20 mM sodium phosphate buffer, pH 6.9, and 1 hour ^

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mixed in an ice-cold water bath (1 to 4 ⁰ C). The mixture is in the Sorvall RC2-B for 30 minutes at 1600. centrifuged g. The supernatant liquid is then passed through a 0.65 µ Millipore filter and subjected to the procedure described above two more times, each time being treated with 2.5 g of hydroxyapatite. The solution obtained is filtered through 0.65 μ and 0.45 μ Millipore filters, dialyzed against pyrogen-free distilled water and lyophilized. The lyophilized product shows a strong immunogenic activity (see below, combination vaccine and animal experiment) and contains only very few impurities, such as nucleic acids, proteins and endotoxins (see Table I). About 170 mg of lyophilized PRP are obtained, which is about 70%. of the polysaccharide used. The PRP must be stored under suitable conditions, for example at 4 ^° C., in a desiccator over phosphorus pentoxide and silica gel.

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). This solution is' at room temperature on a column (5.0 χ 45 cm) hydroxyapatite (bed volume about 250 ml, with 20 mM sodium phosphate buffer, pH 5.8, equilibrated) abandoned and with 20 mM, 50 mM and 100 mM sodium phosphate buffer, pH 5.8, eluted. The fractions (200 ml) eluted with 20 mM and 50 mM sodium phosphate buffer (pH 5.8), that are pentose-positive (pentose determination according to the orcin method) The fractions obtained are dialyzed against pyrogen-free distilled water and lyophilized. It will about 206 mg of lyophilized product, PRP, was obtained.

909819/0640

The purity of the polysaccharide PRP is determined by determination the level of contamination with nucleic acids, proteins and endotoxins. The protein concentration will according to the method of 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 of nucleic acid in 1 ml of water in a cell with a 1 cm light path, 1.0 is equated. The molecular weight is determined by means of Sepharose 4B or 2B gel filtration on columns of 1.5 × 90 cm (Pharmacia-Fine Chemicals, Piscataway, N.J.) determined. The values of the partition coefficient (Kd) are obtained from that by the orcine reaction (Herbert, et al., Methods in Microbiology, Vol. 5B, 285-291).

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Table I.

Physico-chemical properties of PRP

Endotoxin test

Mol. Size mode of operation (Kd)

Pentose Nucleic Acid Protein Rabbit Pyrogen

test

Limulus Lysate Test ^d

CD O CO CO

example 1

Example 2 0.44

36.0

33.8

0.8

0.3

10.0 10.0

a) using Sepharose 2B and 4B, molecular weight 2 χ

b) Using Sepharose 4B

c) μg PRP / kg body weight (rabbits) that do not cause fever

d) Reciprocal dilution of PRP (100 μg PRP / ml) compared to Bureau of Biologies El (endotoxin standard)

Example 1 relates to the characteristics of Haemophilus influenzae type b CK strain, ATCC 3ISS1

Example 2 relates to the characteristics of Haemophilus influenzae type b Rab strain available from Babies Hosp. Columbia Univ. New York, N.Y., USA

1/400 1/1000

¹ J (OI

Example 3

A combination vaccine containing PRP from H. influenzae type b and B. pertussis antigens is prepared as follows: 140 mg PRP (prepared as described in Example 2) are prepared 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) and filtered through a 0.45 μ millipore filter. This concentrated PRP solution (400 μg / ml ·) is used to make a combination vaccine used, which consists of PRP and B. pertussis antigens.

150 ml of the concentrated PRP solution (400 μg / ml) are mixed with 150 ml of a fresh cell suspension of B. pertussis (strain 138) in PBS, pH 7.0 (containing 149 opacity (op) unit cells / ml), whereby 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 checked ^{with thioglycollate media at 32 to 33 ° C.} The pH of the stock solution after an incubation of 90 days is checked and adjusted to 7.0 + 1. The finished product (vaccine) used for animal experiments is produced by diluting the combined stock solution with PBS and contains 10 μ9 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 as in example. 1 described manufactured PRP in

909819/0640

Prepare 75 ml of sterile phosphate buffered saline (PBS) and filter through 0.45 μ millipore filters. An equal volume, ie 25 ml fresh cell suspension of B. pertussis (strain 138) in PBS, pH 7.0 (containing 149 op-units cells / ml) is added to 25 ml of this concentrated PRP solution, whereby the stock solution of the combination vaccine is obtained will. This stock solution (50 ml) is left to stand at 4 ^° C. for at least 90 days. As mentioned above, the sterility of the stock solution is checked with thioglycollate media. The pH of the stock solution is 7.0 + 1. The finished product (vaccine) used for animal experiments is produced by diluting the combination stock solution with PBS and contains 10 μ9 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 prepared as described in Example 4. The pertussis stock solution (containing 75 op units / ml) is prepared by diluting 25 ml fresh cell suspension of B. pertussis (149 op units / ml) with an equal volume of PBS. Both stock solutions are incubated ^{at 4 °} C. separately for 90 days or a little longer. The combination vaccine is prepared using 14 ml PRP stock solution (200 μg / ml), 14 ml (detoxified) stock solution of pertussis antigens (75 op units / ml) and 112 ml PBS (final volume 140 ml). This vaccine, used for animal experiments, contains 10 μ9 PRP and 3.7 op units of B. pertussis / 0.5 ml (dose).

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

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Table II

Immunogenicity of PRP-pertussis complex in young rats

Vaccine

Single or multiple dose

Anti-PRP antibody activity (cpm H-PRP bound / 50 μΐ serum)

Week after injection 1 2

3rd week after injection (-fold

Increase through pre-immunization

Phosphate Buffered ( _M ) ^ Saline Solution

Pertussis (M)

εο

PRP (K) (example 1)

107 117

158 149

1.6 1.3

PRP (K) (3 mo)

Pertussis (3 mo)
O (example 5)

(M)

(M)

PRP (K) + Perussis (S) (3 mo) (example 4) (M)

148

132

1002

135

751

142

147

1771

140 1410

1.3

1.4

17.5

1.5 11.8

a) 10 μg PRP or 3.5 op units pertissus / dose (0.5 ml)

b) best value 2nd and 3rd week (multiple injections)

c) mean of 10 rats

d) single injection

Claims (12)

Claims 1. Combination vaccine for the formation of anti-PRP (polyribosyl-ribitol-phosphate) - and antipertussis antibodies in young warm-blooded animals, consisting of polyrxbosylribitol phosphate, which is composed of the envelope polysaccharide of Haemophilus infuluenzae type b isolated and. has been purified, and Bordetella pertussis antigens. 2. Combination vaccine according to claim 1, characterized characterized in that the Bordetella pertussis antigens have been obtained from Bordetella pertussis strain 138 are. 3. Combination vaccine according to claim 1, characterized in that the polyribosylribotol phosphate from the Rab strain of Haemophilus influenzae type b. 4. Combination vaccine according to claim 1, characterized characterized in that the polyribosyl ribotol phosphate from the CK strain of Haemophilus influenzae type b. 5. Combination vaccine according to claim 1, characterized in that the Bordetella pertussis antigens from Bordetella pertussis strain 138 and the polyrxbosylribitol phosphate from the Rab strain of Haemophilus influenzae Type b have been obtained. 6. Combination vaccine according to claim 1, characterized in that the Bordetella pertuss'is antigens from Bordetella pertussis strain 138 and the polyrxbosylribitol phosphate from the CK strain of Haemophilus influenzae Type b have been obtained. 909819/0640 ORIGINAL 7. Use of the combination vaccine according to one of the claims 1 to 6 for the active immunization of young warm-blooded creatures against systemic infections by the pathogenic Organism Haemophilus influenzae type b. 8. Use according to claim 7 against meningitis. 9. Use according to claim 7 or 8 in human children. 10. Use according to claim 7 in the case of one caused by the pathogenic organisms Haemophilus influenzae type b and Bordetalla pertussis caused systemic infection. 11. Procedure for the isolation and purification of immunologically active polyribosyl oxbitol phosphate (PRP), the envelope polysaccharide of Haemophilus influenzae type b Fermentation of strains of the organism Haemophilus influenzae type b under known conditions, isolation of the PRP by customary ethanol precipitation and further isolation of the PRP known per se as a hexadecyltrimethylammonium bromide complex, characterized in that PRP by addition of hydroxylapatite / 3Ca_ (PO.) _O Ca (OH) _o / in a 0.02M sodium phosphate buffer at a pH value of 6.7 to 6.9, mixing at a temperature of 4 ⁰ 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. 909819/0640 12. The method according to claim 10, characterized in that the PRP by column chromatography in a 0.02m sodium phosphate buffer at a pH of 5.8 using hydroxyapatite / 3Ca ₃ (PO _{4 I 2} Ca (OH) ₂ _ /, Elution with a graduated 0.02m to 0.05m sodium phosphate buffer at a pH of 5.8, isolation of fractions by analysis for PRP, dialysis of these fractions against pyrogen-free distilled water and lyophilization is purified. 19/0640