CA1117866A

CA1117866A - Haemophilus influenzae vaccine

Info

Publication number: CA1117866A
Application number: CA000310218A
Authority: CA
Inventors: Joseph S.-C. Kuo
Original assignee: American Cyanamid Co
Current assignee: Wyeth Holdings LLC
Priority date: 1977-10-28
Filing date: 1978-08-29
Publication date: 1982-02-09
Also published as: FR2407000B1; PH15882A; IE782146L; AU520902B2; FI63674B; PL210545A1; FR2407000A1; FI783269A; BE871586A; DK154327B; NL7810753A; AU4108178A; IL55433A; NZ188211A; IT7851621A0; AR218932A1; DE2845745A1; JPS6231694B2; NO149611B; IT1107570B

Abstract

ABSTRACT
A process for the isolation and purification of immunologically active polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b.
The PRP has been purified with ethanol, Cetavlon (hexadecyl-trimethyl ammonium bromide) and a phosphate containing adsor-bent, hydroxylapatite. The contaminants (nucleic acid, pro-teins and endotoxins) are removed to the minimum level by a treatment with hydroxylapatite. Also described is a process for the preparation of a combined vaccine containing the PRP (prepared as forementioned) and B. pertussis antigens.
The vaccine elicits anti-PRP antibody and anatipertussis anti-body (as measured by microagglutination) formations in young animals. This sera with anti-PRP antibody exhibits a strong bactericidal activity.

Description

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This invention is in the field o~ vaccines for immunization against Haemophilus influenzae type b infections, . _ such as meningitis. More specifically, this invention relates to: (1) a metnod for the isolation of antigenic polysaccharid 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 wlth other non-pathogenic strains of bacteria, such as E. coli, B. subtilis, S. aureus, B. punilus and L.
plantarum, to trigger an antibody response in warm-blooded animals.
The purified polyribitol phosphate (PRP) from Haemophilus influenzae type b is being investigated as a pro-tective immunogen. However, an active antigenic response in young animals and in~ants has not been achieved. The prior art method ~or the purification employed ethanol and Cetavlon (hexadecyltrimethyl ammonium bromide). The contaminants~
endotoxins, and pyrogenic substances were removed by the use 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 puriication of ; immunologically active PRP from Haemophilus influenzae type b has been established.
The process to be described has distinct advantages over the prior art procedures in that all contaminants ~nu-cleic acids, proteins and endotoxins) are removed to the mini-- mum level by a treatment with hydroxylapatite. The PRP pre-pared by the process described here gives a higher molecular weight polysaccharide than those reported previously.

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More importantly, the PRP prepared by this new pro-cedure is highly immunogenic in warm-blooded animals as opposed to the PRP produced by prior art procedures.
A process for removal of contaminants (proteins, nu-cleis acids and endotoxins) in the polysaccharide PRP is per-formed by treating the partial purified polysaccharide with a phosphate containing adsorbent which does not absorb the poly-saccharide under desiganted 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 phos-phate, the capsular polysaccharide of Haemophilus lnfluenzae type b.
Organisms, Growth Medium and Culture . _ . . . .. . .
Two strains of _emophilus influenzae type b are used. The Rab strain is obtained from Graae Leidy, Babies Hospital~ Columbia University, New York City, l~ew ~or~. The CK strain was isolated from a patient at Waterbury Hospital Waterbury, Conn. The organisms are passed through mice sev-eral times to insure their virulence. The organisms are iso-lated at autopsy from the brain tissue of mice, subcultured on either 3.7~ Brain Heart Infusion (BHI) ~Difco Lab., Detroit Mich.) medium or on 5% BHI agar supplemented with 0.01% nico-tinamide adenine dinucleotide (NAD) (P-L Biochemicals, Milwaukee, Wis.) and 1~ (v/v) defibrinated horse blood (Animal Blood Center, Syracuse, N. Y.) and then distributed 30) in one ml portions in ampulesj lyophilized and stored at -70C.

1~178G~i Basal medium used for the growth of the orga~isms is 3.7% (BHI). The basal medium 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 0.45~ 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% glucose.
- To prepare a flask of seed organisms, on~ 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 37C on a gyrotory shaker at 150 rpm. The organisms are added as a 1~ inoculum to 500 ml portions of the enriched BHI broth in

2- liter flasks which are then incubated at 37C 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 fer- 0 mentor is initiated by aseptically transferring 700 ml of the seed culture to 7 liters of the enriched BHI broth supple-mented with hemin instead of defibrinated horse blood. The culture is continuously maintained at 37~1C. 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% of a silicone antiform (FD-82, Hodag Chemical Corp.) is added as needed. Cultures are grown to the late logarithmic of growht (8 to 10 x 109 viable cells/ml), usu-

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ally about 8 hours and then growth is terminated by adding 0.4% formaldehyde and standing overnight at 4C.
Isolation of Polyribosyl Ribitol Phosphate ~PRP) Culture broth prepared as described above is centri-fuged at 27,000 x g for 30 minutes at 4C. The cell free supernatant is collected and treated as follows:
~Step 1, Ethanol Precipitation To the culture supernatant i8 added sodium acetate (final concentration 4%). The solution is adjusted to pH 6.0-fi~.2 and 44 liters of 3A ethanol are added slowly with vigorous stirrin~ at ~C. The ~i~tur~ is ~dju~ted to ~H 6.8 T~ith ~lacial acetic acid and then allot~ed to stand ~or 12 hour3 at 3C. The re ultin~ precipitate is collected b~ decantation and then cen-t~i~ugation to afford crude PRP.

St~p 2, Cetavlon(hexadecyltrimethyl ammonium bromide)Treatment The pr~cipitate 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~ aqueous solution of Cetavlon with mixing ~final conc. 0.5~). The mixture si stirred for one hour and then centrifuged. The precipitate of nucleic acids and PRP-Cetavlon complex is mixed with 2 liters of ~.3M sodium chloride. The cloudy solution is centrifuged to eliminate insoluble materials such as nucleic-Cetavlon salt.
The supernatant is diluted with an equal volume of water causing the PRP-Cetavlon salt to precipitate. The mix-ture is stirred for one hour, the precipitate is collected by centrifugation and is then dissolved in 2 liters of 0.3M
sodium chloride.

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Step 3, Ethanol Precipltatlon 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 de-scribed above. The final PRP precipitate is solubilized in 20mM sodium phosphate, pH 6.8.
Step 4, Hydroxyla~atite ~eat~
.
Contaminants (e.g. nucleic acids, proteins and endo-toxins) in the partial purified PRP preparations are selec-tively removed by adsorption on a calcium phosphate containing adqorbent such as hydroxylapatite [3.Ca3(PO4)2.Ca~OH)2].
This inventlon i5 based on the discovery that the polysaccharide PRP iq not adsorbed by the calcium phosphate - adsorbent containing phosphate buffer (20mM) having a pH of about 6.7-6.9; or a phosphate buffer (50 mM) having a pH of about 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 hydrox-ylapatite is added to the partially purified PRP preparation (in 20mM phosphate; pH 6.9). The mixture is mixed well 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 L7~66 times. The resultlng 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.~) -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 ug/ml in phosphate buffered saline (PBS) (0.113 g potasslum diphos-phate, 0.83 g disodium phosphate, and 8.5 g sodium chloride per liter, pH 7. 0, containing 0.01~ thimerosal). The vaccine is sterile filkered through 0.45~ millipore filter units, - dispensed into glas~ vials, and stored at 4C.

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 chlor-ide per liter, pH 7.0, containing 0.01% thimerosal). This concentrated solution of PRP is then mixed with an appropriate volume of fresh B. pertussis cell suspensions to prepare the 86~

stock solution. The characteristics of B. pertussis strain 138 are desc~ibed in Bergey's Manual of Determinative Bacteriology, 8, Buchanan and Gibbons, editors, Willams and ~ilkins Co., Maryland U.S.A. 1974, on page 283. This strain is available from the American Type Culture Collection, hlaryland, U.S.A. under deposit No. 10380. The combined vaccine in this stock solution contains 200 ~ g of PRP and approximately 70 opacity units (op) of cells per ml. The stock solution is kept at 4C for 90 days to allow for detoxi-fication of pertussis antigens before the final product (vaccine) is prepared (which contains lO~g of PRP and 3.5 op units of pertussis cells/0.5 ml dose).
DETAILED DISCLOSURE
. _ .
Example 1 Hydroxylapatite (e.g. Bio. gel HTP7 Bio-Rad Laboratories, Richmond, Calif.), 2.5 g is added to 250 ml partially purified PRP prepara-tions ~after Cetavlon and ethanol treatments) (containing approximately 1.0 mg PRP/ml) in ?OmM sodium phosphate buffer, pH 6.9 and mixed at ice-cold water bath ~1-4DC) 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 ~ 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~ and 0.45~ millipore fil-ters~ dialyzed agannst 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, pro-teins and endotoxins) (see Table I below). Approximately 170 mg of the lyo-philized PRP is obtained. The recovery of PRP by this process is approximate-ly 70% of the starting polysaccharide. The PRP should be stored under suit-able conditions, such as at 4C in a desiccator over phosphorus pentoxide and silica gel.

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Example 2 A partially purified PRP (300 mg PRP) is dissolved in lO0 ml of 29mM sodium phosphate buffer, pH 5.8 (i.e. 3 mg PRP/ml). This solution is applied to a column at ambient S 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 (pe~tose determination by the orcinal method) are collected, dialyzed against pyrogen-free distilled water and lyophilized.
Approximately 206 mg of the lyophilized product, PRP, is ob-tained.
The purlty 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 mathod 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 50 ~ g of nucleic acid in one ml of water in a cell of l-cm light path is assumad to be equal to 1 0. Molecular size is estimated by means of Sepharose 4B or 2~ gel filtration on columns 1.5 x 90 cm (Pharmacia-Fine Chemicals, ~iscataway, N. J.~. Partition coefficient ~Kd) values are calculated from the elution pattern daveloped by the orcinal reaction (Herbert, et al., Methods in Microbio-logy, Vol. 5B, 285-291.

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Exa~ple 3 A combination vaccine containing PRP ~rom H.
influenzae type b and B. pertus~is anti~ens i5 prepared as follows: 140 mg PRP (as prepared in Example 2) i5 dissolved in 350 ml sterile phosphate buffered saline (PBS) (0.113 g potassium diphosphate, 0.83 ~ disodium phosphate, and 8.5 g sodium chloride per liter, pH 7.0, containing 0.01% thimero-sal), and filter through 0.45~ millipore filter. This concen-trated PRP solution (400 ~ g/ml) is used to prepare a combination vaccine which consists of PRP and ~. pertuss s antigens.
To 150 ml of the concentrated PRP solution (400 ~g/-ml) add 150 ml of fresh B~ pertussis (strain 138) cell suspen-sion in PBS, pH 7.0 (containing 149 opacity; op, unit cell-s/ml3 to prepare the stock solution of the combined vaccine. This stock solution (300 ml3 is kept at 4C until the endotoxin level of the pertu~sis is decreased (at least 90 days). The sterility of the stock solution is tested with thioglycollate media (at 32 33C). The pH of the stock solution after 90 days incubation is checked and adju~ted to pH 7.0 ~1. The final product (vac- -cine3 used or animal experiments is prepared by diluting the stock combined solution with P~S and it contains 10 yg of PRP
and 3.5 op units of pertussis cells/0.5 ml (dose).
The result~ of the antibody response to this combined vaccine in young rats appears in Figures 1 and 2 as shown below.

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~,xample 4 The stock solution of PRP (400 vg/ml) is prepared by dissolving 30 mg PRP (as prepared in Example 1) in 75 ml sterile phosphate buffered saline (PBS) and filtered through 0-45D millipore 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, p~l 7.0 (containing 149 op units cells/ml) to prepare the stock solution of the com-bined vaccine. ThiY stock solution (50 ml) is kept at 4C
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 p~ 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 3.7 op units of pertussis cells/0.5 ml (dose).
Example 5 The PRP stock solution (200 vg/ml in PBS (pH 7.0) i~ prepared as in Example 4. The pertussis stock solution (containing 75 op uni~s/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 4C for 90 days or slightly longer. The com-bined vaccine is made by taking 14 ml of PRP stock solution (~00 ~g/ml), plus 14 ml (detoxifiedl stock solution of per-tussis antigens (75 op units/ml) and diluting it with 112 ml PBS (final val. 140 ml). The final vaccine used for animal experiments contains 10 ~g PRP and 3.7 op units pertussis/-0.5 ml (dose).
The results of the antibody response to this com-bined vaccine in young rats appears in Figure 3 and in Table II.

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Claims

26,910 THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OF PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

WE CLAIM:

1. A combined vaccine that elicits anti-PRP (poly-ribosyl ribitol phosphate) and antipertussis antibody for-mations in young warm-blooded animals which consists of poly-ribosyl ribitol phosphate, isolated and purified from the capsular polysaccharide of Haemophilus influenzae type b.
and Bordetella pertussis antigens obtained from Bordetella pertussis strain 138.

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 Bordetelle pertussis antigens are obtained from Borde-tella pertussis strain 138 and said polyribosyl ribitol phos-phate is obtained from Haemophilus influenzae type b Rab strain.

5. A combined vaccine according to Claim 1, wherein said young warm-blooded animal is a human child.

6. A combined vaccine according to Claim 4, wherein said warm-blooded animal is a human child.

7. A method for the preparation of a combined vaccine consisting of anti-PRP (polyribosyl ribitol phosphate) and antipertussis antibody which comprises the steps:
(a) purifying the PRP by adding hydroxylapatite [3-Ca3(PO4)2Ca(OH)2] in a 0.02M sodium phosphate buffer at a pH from about 6.7 to about 6.9, mixing at a temperature of about 4°C., cen-trifuging, removing the supernatant and re-peating the foregoing procedure at least two more times, filtering the supernatant through suitable filters, dialyzing against pyrogen-free distilled water, and then lyophilizing;
(b) dissolving the purified PRP in sterile phosphate buffered saline solution PBS pH 7 and adding a fresh B.pertussis cell suspension in PBS at pH 7Ø

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

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

10. A combined vaccine that elicits effective levels of anti-PRP (polyribosyl ribitol phosphate) and anti-pertussis antibody formations in young warm-blooded animals which consists of polyribosyl ribitol phosphate isolated and purified from the capsular polysaccharide of Haemophilus influenzae type b by adding hydroxylapatite in about 2 to about 20 millimolar phosphate buffer at pH
from about 6.5 to about 7.0, mixing at a temperature of about 2 to 10°C., centrifuging, and removing the super-natant and repeating the foregoing procedure at least 2 more times, filtering the supernatant, dializing against pyrogen-free distilled water, and then lyophilizing; and Bordetella pertussis antigens.

11 A combined vaccine according to Claim 10, wherein said Bordetella pertussis antigens are obtained from Bordetella pertussis strain 138.

12. A combined vaccine according to Claim 10, wherein said polyribosyl ribitol phosphate is obtained from Haemophilus influenzae type b Rab strain.

13. A combined vaccine according to Claim 10, wherein said Bordetella pertussis antigens are obtained from Bordetella pertussis strain 138 and said polyribosyl ribitol phosphate is obtained from Haemophilus influenzae type b Rab strain.

14. A combined vaccine according to Claim 10, wherein said young warm-blooded animal is a human child.

15. A combined vaccine according to Claim 13, wherein said young warm-blooded animal is a human child.