NZ188211A

NZ188211A - Isolation of polyribosyl ribitol phosphate(prp)from haemophilus influenzae type b;vaccine comprising prp and bordetellapertussis antigens

Info

Publication number: NZ188211A
Application number: NZ188211A
Authority: NZ
Inventors: J S-C Kuo
Original assignee: American Cyanamid Co
Priority date: 1977-10-28
Filing date: 1978-08-22
Publication date: 1984-09-28
Also published as: NO149611B; NO149611C; GB2007244A; NO783635L; NL7810753A; AU520902B2; FR2407000A1; JPS6231694B2; FI783269A; FR2407000B1; IE47474B1; JPS5480408A; IL55433A; HU178166B; BE871586A; AU4108178A; DE2845745C2; DE2845745A1; DK154327B; SE430753B

Description

<div id="description" class="application article clearfix"> 1 882 1 Priority Datajs): . Complete Specification Filed: C-OTHII ; CiaflM 0*\ Afc.lvV5l\T3S: Class: SEp.igffl PubHcBiJon Data: P.O. jc:<Kc: . . ■ 1 ?^?-v P. & s. x-M NEW ZEALAND PATENTS ACT, 1953 vffi'f C. r* No.: Date: , - COMPLETE SPECIFICATION r&r* fthlS "HAEMOPHILUS INFLUENZAE^VACCINE" XI / We, AMERICAN CYANAMID COMPANY, a corporation organized and existing under the laws o£ the State of Maine, United States of America, and having its executive offices at Wayne, New Jersey, United States of America, hereby declare the invention for whichjfe/ we pray that a patent may be granted to XJg^us, and the method by which it is to be performed, to be particularly described in and by the following statement: - - 1 - (followed by page la) 188211 This invention is in the field of vaccines for immunization against Haemophilus influenzae type b infections, such as meningitis. More specifically, this invention relates to: (1) a method for the isolation of antigenic polysaccharide 5 polyribosyl ribitol phosphate (PRP) from Haemophilus influenzae type b cultures; and, (2) a process for the preparation of a combined vaccine containing PRP and B. pertussis antigens. The PRP of this invention should also be effective when, used in conjunction with other non-pathogenic strains of bacteria, 10 such as E. coli, B. subtilis, S. aureus, B. punilus and L. plantarum, to trigger an antibody response in warm-blooded animals. The purified polyribosyl ribitol phosphate (PRP) from Haemophilus influenzae type b is being investigated as a pro-15 tective immunogen. However, an active antigenic response in young animals and infants has not been achieved. The prior art method for the purification employed ethanol and Cetavlon (hexadecyltrimethyl ammonium bromide). The contaminants, endotoxins, and pyrogenic substances were removed by the use 20 of cold phenol or chloroform and t-butanol which may result in the loss of the antigenic nature of this polysaccharide. A new process for the isolation and purification of immunologically active PRP from Haemophilus influenzae type b has been established. 25 The process to be described has distinct advantages over the prior art procedures in that all contaminants (nucleic acids, proteins and endotoxins) are removed to the minimum level by a treatment with hydroxylapatite. The PRP prepared by the process described here gives a higher molecular 30 weight polysaccharide than those reported previously. la- 188211 10 More importantly, the PRP prepared by this new procedure is highly immunogenic in warm-blooded animals as opposed % to the PRP produced by prior art procedures. A process for removal of contaminants (proteins, nucleic acids and endotoxins) in the polysaccharide PRP is performed by treating the partial purified polysaccharide with a phosphate containing adsorbent which does not absorb the polysaccharide under designated conditions. The second objective of this invention is to provide a process for the preparation of a combined PRP and pertussis vaccine which is highly immunogenic in young animals. DISCLOSURE (I) Isolation and purification of polyribosyl ribitol phosphate, the capsular polysaccharide of Haemophilus -*-5 influenzae type b. Organisms, Growth Medium and Culture Two strains of Haemophilus influenzae type b are used. The Rab strain is obtained from Grace Leidy, Babies Hospital, Columbia University, New York City, New York. The CK strain was isolated from a patient at Waterbury Hospital Waterbury, Conn. The organisms are passed through mice several times to insure their virulence. The organisms are isolated at autopsy from the brain tissue of mice, subcultured on either 3.7% (w/v) Brain Heart Infusion (BHI) (Difco Lab. , Detroit Mich.) medium or on 5% (w/v) BHI agar supplemented with 0.01% (w/v) nicotinamide adenine dinucleotide (NAD) (P-L Biochemicals, Milwaukee, Wis.) and 1% (v/v) defibrinated horse blood (Animal Blood Center, Syracuse, N. Y.) and then distributed ^ in one ml portions in ampules, lyophilized and stored at -70°C. 20 25 - 2 188211 10 Basal medium used for the growth of the organisms is 3.7% (w/v) (BHI). The basalrttedium is supplemented with 10 mg of NAD and 20 mg of hemin (Eastman Kodak, Rochester, N. Y.) per liter. One percent (v/v) defibrinated horse blood is added per 50 ml of seed culture. The supplements are freshly prepared and filtered through a 0.45i/\Nalgene filter unit Nalge Sybron Corp., Rochester, N. Y.) before use. For growth of the organism in a 14 liter fermentor, the medium is further supplemented with 0.5% by weight glucose. ' To prepare a flask of seed organisms, one ml of frozen stock culture is thawed and transferred to 50 ml of the enriched BHI medium supplemented with defibrinated horse blood and NAD. The culture is grown for 8 hours at 37°C on a gyrotory shaker at 150 rpm. The organisms are added as a 1% by -.-.-sight inoculum to 500 ml portions of the enriched BHI broth in 2 liter flasks which are then incubated at 37°C with moderate shaking on a gyrotory shaker for 8 hours (for isolation of PRP) or 14 hours (as seed cultures). The fermentation of each batch in a 14 liter fermentor is initiated by aseptically transferring 700 ml of the seed culture to 7 liters of the enriched BHI broth supplemented with hemin instead of defibrinated horse blood. The culture is continuously maintained at 37°+l°C, The tank is stirred at a rate of 150 rpm and an air flow of 0.25 liter of air per liter of mash per minute is maintained. During the growth 0.001% (w/v) of a silicone antifoam (FD-82, Hodag Chemical Corp.) is added as needed. Cultures are grown to the limit 9 30 logarithmic of growth (8 to 10 x 10 viable cells/ml), usu- 15 20 25 - 3 - 188211 ally about 8 hours and then growth is terminated by adding 0.4% (w/v) formaldehyde and standing overnight at 4 C. Isolation of Polyribosyl Ribitol Phosphate (PRP) Culture broth prepared as described above is centri-fuged at 27,000 x g for 30 minutes at 4°C. The cell free supernatant is collected and treated as follows: Step 1, Ethanol Precipitation To the culture supernatant is added sodium acetate (final concentration 4% (w/v)). The solution is adjusted to pH 6.0-6.2 and 44 liters of formula 3A denatured alcohol are added slowly with vigorous stirring at 4°C. The mixture is adjusted to pH 6.8 with glacial acetic acid and then allowed to stand for 12 hours at 3°C. The resulting precipitate is collected by decantation and then cen-trifugation to afford crude PRP. Step 2, Cetavlon(hexadecyItrimethyl ammonium bromide)Treatment The precipitate from Step 1 is dissolved in pyro-gen-free distilled water and centrifuged to remove the residue. The clear brown solution is then slowly added to 100 ml of a 10% (w/v) aqueous solution of Cetavlon with mixing (final conc. 0.5% (w/v)). The mixture is stirred for one hour and then centrifuged. The precipitate of nucleic acids and PRP-Cetavlon complex is mixed with 2 liters of 0.3M sodium chloride. The cloudy solution is centrifuged to eliminate insoluble materials such as nucleic acid-Cetavlon salt. The supernatant'is diluted with an equal volume of water causing the PRP-Cetavlon salt to precipitate. The mixture is stirred for one hour, the precipitate is collected by centrifugation and is then dissolved in 2 liters of 0.3M sodium chloride. , . I & 188211 Step 3, Ethanol Precipitation Cetavlon and the contaminants of nucleic acids and proteins are further removed by ethanol precipitation (at least 2 times). The PRP is precipitated as described in Step 1 while Cetavlon is dissolved in the alcoholic solution. The PRP is recovered by centrifugation, dissolved in 2 liters of pyrogen-free distilled water and then reprecipitated as described above. The final PRP precipitate is solubilized in 20mM sodium phosphate, pH 6.8. Step 4, Hydroxylapatite Treatment Contaminants (e.g. nucleic acids, proteins and endotoxins) in the partial purified PRP preparations are selectively removed by adsorption on a calcium phosphate containing adsorbent such as hydroxylapatite [3.Ca^(PO^)2.Ca(OH)- This invention is based on the discovery that the polysaccharide PRP is not adsorbed by the calcium phosphate adsorbent containing phosphate buffer (20mM) having a pH of substantially 6.7-6.9; or a phosphate buffer (50 mM) having a pH of substantially 5.8; however, the contaminants (such as nucleic acids, proteins and endotoxins) are adsorbed under these conditions. The process of the present invention may be carried out in batch-wise or column operation. In batch process, the hydroxylapatite is added to the partially purified PRP preparation (in 20mM phosphate; pH 6.9). The mixture is mixed well at a temperature of substantially 1-4°C. and centrifuged to remove non-desired solids (adsorbent and the contaminants adsorbed by the adsorbent). The supernatant fluid is subjected to the foregoing procedure at least 2 more 188211 times. The resulting solution is filtered through millipore filters, dialyzed against pyrogen-free distilled water, and lyophilized. In column operation, the partially purified PRP in 20mM phosphate buffer (pH of at least 5.8) is applied to a column containing the adsorbent hydroxylapatite which had been equilibrated with 20mM phosphate buffer, pH 5.8 and eluted with a stepwise gradient of sodium phosphate buffer (pH 5.3) from 20mM to lOOmM. Fractions are collected and assayed for pentose (for polyribosyl ribitol phosphate). Those fractions which are positive for pentose are dialyzed against pyrogen--free distilled water and lyophilized. (II). A process for preparing a combination vaccine consisting of PRP from H. influenzae type b and B. pertussis antigens. To prepare a PRP vaccine, lyophilized PRP (prepared as aforementioned) is dissolved at a concentration of 20 ng/ml 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% (w/v) thimerosal). The vaccine is sterile filtered through 0. 45y millipore filter units, dispensed into glass vials, and stored at 4°C. A concentrated solution of the PRP is prepared by weighing predetermined amounts of the polysaccharide and dissolving it in phosphate buffered saline (0.113 g potassium diphosphate,, 0.83 g disodium phosphate, and 8.5 g sodium chloride per liter, pH 7.0, containing 0.01% (w/v) thimerosal). This concentrated solution of PRP is then mixed with an appropriate volume of fresh B. pertussis (strain 138) cell suspensions toorepare the , - 1 APR1980 188211 stock solution. The characteristics of B. pertussis (strain 138) are described in Bergey's Manual of Determinative Bacteriology, 8, Buchanan and Gibbons Editors, Wms and Wilkins, Maryland, U.S.A.} 1974,on page 283. This strain is available from the American Type Culture Collection, Maryland, U.S.A., deposit No. 103 80. The combined vaccine in this stock solution contains 200 pg of PRP and approximately 70 opacity units (op) of cells per ml. The stock solution is kept at 4°C for 90 days to allow for detoxification of pertussis antigens before the final product (vaccine) is prepared (which contains 10 yg of PRP and 3.5 op units of pertussis cells/0.5 ml dose). DETAILED DISCLOSURE Example 1 Hydroxylapatite (e.g. Bio. gel HTP, Bio-Rad Laboratories, Richmond, Calif.), 2.5 g is added to 250 ml partially purified PRP preparations (after Cetavlon and ethanol treatments) (containing approximately 1.0 mg PRP/ml) in 20mM sodium phosphate buffer, pH 6.9 and mixed at ice-cold water bath (1-4°C) for one hour. The mixture is centrifuged in the Sorvall RC2-B for 30 minutes at 16,000 x g.. The supernatant fluid is then passed through 0.65 y millipore filter and subjected to the foregoing procedure (each time treated with 2.5 g hydroxylapatite) 2 more times. The resulting solution is filtered through 0.65 y and 0.45 y millipore filters, dialyzed against pyrogen-free distilled water and lyophilized. The lyophilized product exhibits strong immunogenic activity (see below combined vaccine and animal experiment) and contains very low contaminants (such as nucleic acids, proteins and endotoxins) (see Table I below). Approximately 170 mg of the lyophilized PRP is obtained. The recovery of PRP by this process is approximately 70% of the starting polysaccharide. The PRP should be stored under suitable conditions, such as at 4°C in a desiccator over phosphorus pentoxide and silica gel. - 7 - 1 882 1 1 Example 2 A partially purified PRP (300 mg PRP) is dissolved in 100 ml of 20mM sodium phosphate buffer, pH 5.8 (i.e. 3 mg PRP/ml). This solution is applied to a column at ambient temperature (5.0 x 45 cm) of hydroxylapatite (approximately 250 ml bed volume which has been equilibrated with 20mM sodium phosphate buffer, pH 5.8 and eluted with a stepwise gradient of 20mM, 50mM, lOOmM sodium phosphate buffer, pH 5.8. The individual fractions (200 ml) eluted with 20mM and 50mM sodium phosphate buffer (pH 5.8), positive for pentose (pejitose determination by the orcinal method) are collected, dialyzed against pyrogen-free distilled water and lyophilized. Approximately 206 mg of the lyophilized product, PRP, is obtained. The purity of the polysaccharide, PRP is assayed by estimating to what extent they are contaiminated with nucleic acids, proteins and endotoxins. Protein concentration is determined by the method of Lowry, et al^. in J. Biol. Chem. 193:265 (1951) with boving serum albumin as standard. Nucleic acid is measured gy the absorption of the PRP solution at 260nm. The absorbance of 50ug of nucleic acid in one ml of water in a cell of 1-cm light path is assumed to be equal to 1.0. Molecular size is estimated by means of Sepharose 4B or 2B gel filtration on columns 1.5 x 90 cm (Pharmacia-Fine Chemicals, Piscataway, N. J.). Partition coefficient (Kd) values are calculated from the elution pattern developed by the orcinal reaction (Herbert, et al., Methods in Microbiology, Vol. 5B, 285-291. Table I Physical-Chemical Characteristics of PRP Prefered Assay Endotoxin Procedure Mol. Size (Kd) Pentose (%) Nucleic Acid (%) Protein (%) Rabbit Pyrogen Testc Limulus Lysate Test^ Example 1 oa 36.0 0.8 0.7 10.0 1/400 Example 2 0.44k 33.8 0.3 0.7 10.0 1/1000 a) Using both Sepharose 2B and 4B, molecular weight> 2 x 10 b) Using Sepharose 4B c) vg PRP/kg rabbit body weight not yielding fever d) Reciprocal dilution of PRP (100 Ug PRP/ml) when compared to Bureau of Biologies El (endotoxin standard) Example 1 utilises PRP derived from Haemophilus influenzae type b CK strain, available from the American Type Culture Collection, Maryland, U.S.A., deposit No. 31441. Example 2 utilises PRP derived from Haemophilus influenzae type b Rab strain available from Babies Hospital, Columbia University, New York, U.S.A. 1 882 1 1 Example 3 A combination vaccine containing PRP from H. influenzae type b and B. pertussis antigens is prepared as follows: 140 mg PRP (as prepared in Example 2) is dissolved in 350 ml 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 filter through 0.45H millipore filter. This concentrated PRP solution (400-VJg/ml) is used to prepare a combination vaccine which consists of PRP and B. pertussis antigens. To 150 ml of the concentrated PRP solution (400 £g/-ml) add 150 ml of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (containing 149 opacity; op, unit cells/ml) to prepare the stock solution of the combined vaccine. This stock solution (300 ml) is kept at 4°C until the endotoxin level of the pertussis is decreased (at least 90 days). The sterility of the stock solution is tested with thioglycollate media (at 32-33°C). The pH of the stock solution after 90 days incubation + is checked and adjusted to pH 7.0 -1. The final product (vaccine) used for animal experiments is prepared by diluting the stock combined solution with PBS and it contains 10 ^g of PRP and 3.5 op units of pertussis cells/0.5 ml (dose). The results of the antibody response to this combined vaccine in young rats appears in Figures 1 and 2 as shown below. - 10 - 1 882 Example 4 The stock solution of PRP (400 jjg/ml) is prepared by dissolving 30 mg PRP (as prepared in Example 1) in 75 ml sterile phosphate buffered saline (PBS) and filtered through 0.45 amillipore filter. To 25 ml of this concentrated PRP is added an equal volume (i.e. 25 ml) of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (containing 149 op units cells/ml) to prepare the stock solution of the combined vaccine. This stock solution (50 ml) is kept at 4°C for (at least) 90 days. The sterility of the stock solution is tested as aforementioned with thioglycollate media. The pH of the stock solution is pH 7.0+1. The final product (vaccine) used for animal experiments is prepared by diluting the stock combined solution with PBS, and it contains 10 Jig of PRP and 3.7 op units of pertussis cells/0.5 ml (dose). Example 5 The PRP stock solution (200 ug/ml in PBS (pH 7.0) is prepared as in Example 4. The pertussis stock solution (containing 75 op units/ml) is prepared by diluting 25 ml of fresh B. pertussis cell suspension (149 op units/ml) with an equal volume of PBS. Both stock solutions are incubated separately at 4°C for 90 days or slightly longer. The combined vaccine is made by taking 14 ml of PRP stock solution (200 Ug/ml), plus 14 ml (detoxified) stock solution of pertussis antigens (75 op units/ml) and diluting it with 112 nil PBS (final val. 140 ml). The final vaccine used for animal experiments contains 10 vg PRP and 3.7 op units pertussis/-0.5 ml (dose). The results of the antibody response to this combined vaccine in young rats appears in Figure 3 and in Table II. - 11 - TABLE II Immunogenicity of PRP-Pertussis Complex in Young Ratsa Anti -PRP Antibody Activity (cpm 3H-PRP Bound/50tJl Serum) Dose (single or Week Post- -Injection 3rd Week Post-injection (fold increase from Vaccine multiple) 0 1 2 3 preimmunization) Phosphate Buffered Saline (M)b 99° 116 107 158 1.6 Pertussis (M) 113 108 117 149 1.3 PRP (K) (Example 1) (M) 110 136 148 142 1.3 PRP (K) (3 mo) + Pertussis (3 no) (Example 5) PRP (K)+Pertussis (3 mo) (Example 4) (S)d 108 136 132 147 1.4 (M) (S) (M) 101 93 119 135 146 154 1002 135 751 1771 140 1410 17.5 1.5 11.8 a) 10 ug PRP or 3.5 op units pertussis/dose (0.5 ml). b) Booster - 2nd and 3rd week respectively (multiple injection) c) Average 10 rats. d) Single injection. I382U wherein said polyribosyl ribitol phosphate is obtained from Haemophilus influenzae type b Rab strain. wherein said Bordetella pertussis antigens are obtained from Bordetella pertussis strain 13 8 and said polyribosyl ribitol phosphate is obtained from Haemophilus influenzae type b Rab strain. 15. A combined vaccine according to Claim 11> wherein said young warm-blooded animal is a human child. 16. A combined vaccine according to Claim 14^ wherein said young warm-blooded animal is a human child. 14. A combined vaccine according to Claim 11 , AMERICAN CYANAMID COMPANY By t their authorised Agents., Per - 16 - (8821J 10. In the method of isolating and purifying immunologically active polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, by fermenting a strain of the organism Haemophilus influenzae type b under art recognized conditions, isolating the PRP by conventional ethanol precipitation, and further isolating the PRP as a hexadecyltrimethyl ammonium bromide complex by conventional procedures, the improvement comprising: 20 mM sodium phosphate buffer at a pH of substantially 5.8 through hydroxylapatite /3Ca.j (PO^) ^ •Ca (OH) 2J' eluting with substantially 5.8, isolating fractions by analysis for PRP, dialyzing the PRP - containing fractions against pyrogen-free distilled water and then lyophilizing the product. levels of anti-PRP (polyribosyl ribitol phosphate) and anti-pertussis antibody formation in young warm-blooded animals which consists of polyribosyl ribitol phosphate, isolated from the capsular polysaccharide of Haemophilus influenzae type b and purified by adding hydroxylapatite in a substantially 20 mM phosphate buffer at a pH from substantially 6.7 to substantially 6.9, mixing at a temperature of substantially 1 to 4°C., centrifuging, and removing the supernatant and repeating the foregoing procedure at least 2 more times, filtering the supernatant through suitable filters, dialyzing against pyrogen-free distilled water, and then lyophilizing the product; and Bordetella pertussis antigens. wherein said Bordetella pertussis antigens are obtained from Bordetella pertussis strain 138. purifying the PRP by column chromatography in a 20 mM and 50 mM sodium phosphate buffer at a pH of 11. A combined vaccine that elicits effective 12. A combined vaccine according to Claim 11 > 13. A combined vaccine according to Claim 11 15 I 88*21I 9. In the method of isolating and purifying immunologically active polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, by fermenting a strain of the organism Haemophilus influenzae type b under art recognized conditions, isolating the PRP by conventional ethanol precipitation, and further isolating the PRP as a hexadecyltrimethyl ammonium bromide complex by conventional procedures, the improvement comprising: purifying the PRP by adding hydroxylapatite /3~ .Ca^ (P04) 2Ca- (OH) 2_7 in a 20 mM sodium phosphate buffer at a pH from substantially 6.7 to substantially 6.9, mixing at a temperature of substantially 1-4°C, centrifuging, removing the supernatant and repeating the foregoing procedure at least two more times, filtering the supernatant through suitable filters, dialyzing against pyrogen-free distilled water, and then lyophilizing the product. </div>

Claims

1882t 1 WHAT WE CLAIM IS:

1. A combined vaccine that elicits anti-PRP (polyribosyl ribitol phosphate) and antipertussis antibody formation in young warm-blooded animals which consists of polyribosyl ribitol phosphate, isolated and purified from the capsular polysaccharide of Haemophilus influenzae type b, and Bordetella pertussis antigens.

2. A combined vaccine according to Claim 1, wherein said Bordetella pertussis antigens are obtained from Bordetella pertussis strain 138.

3. A combined vaccine according to Claim 1, wherein said polyribosyl ribitol phosphate is obtained from Haemophilus influenzae type b Rab strain.

4. A combined vaccine according to Claim 1, wherein said polyribosyl ribitol phosphate is obtained from Haemophilus influenzae type b CK strain.

5. A combined vaccine according to Claim 1, wherein said Bordetella pertussis antigens are obtained from Bordetella pertussis strain 13 8 and said polyribosyl ribitol phosphate is obtained from Haemophilus influenzae type b Rab strain.

6. A combined vaccine according to Claim 1, wherein said Bordetella pertussis antigens are obtained from Bordetella pertussis strain 138 and said polyribosyl ribitol phosphate is obtained from Haemophilus influenzae type b CK strain.

7. A combined vaccine according to any one of Claims 1 to 4, wherein said young warm-blooded animal is a human child.

8. A combined vaccine according to Claim 5 or Claim 6, wherein said young warm-blooded animal is a human child.