CA1339187C - Bacterial antigens, antibodies, vaccines, and methods of manufacture - Google Patents

Abstract

Antigens and vaccines containing purified oligomers (1-50 units) of the repeating pentasaccharide unit of type III Group B Stretococcus (III GBS) polysaccharide capsule. Methods of making the antigen by recovering polysaccharide from cultured III GBS or medium and digesting the polysaccharide with a specific endo-B-galactosidase. Enzymatic cleavage of bacterial polysaccharide to make purified oligomers. A purified trypsin-resistant C surface protein, m.w. about 14,000 and vaccine. Passive immunization using the above vaccines. Immunoassays for GBS immunodeterminants or anti-GBS antibodies.

Description

1 33?1 ~7 BACTERIAL ANTIGENS, ANTIBODIES, VACCINES, AND METHODS OF MANUFACTURE
Background of the Invention This invention relates to antigens having immunogenic determinants of Group B Streptococcus bacteria, to vaccines protecting against those bacteria, to methods of making such vaccines, and to methods of 5 preparing oligosaccharides from bacterial polysaccharides. As used in this application, the term Group B Streptococcus (or GBS) bacteria is used as understood by those in the field, particularly with reference to Lancefield, J. Exp. Med. 108:329-341 (1938) 10 and subsequent work further characterizing Group B
serotypes, e.g. Russell-Jones, J. Exper. Med. 160:1476 (1984). The term specifically includes bacteria taxonomically designated Streptococcus agalactiae.
GBS are a recognized etiological agent for 15 bacteremia and/or meningitis in infants, and for infections in adults. Baker, "Group B Streptococcal Infections" in Advances in Internal Medicine, 25:475-500 (1980). Accordingly, it is important to develop rapid and definitive assays for diagnosis of GBS infection, 20 and methods of generating protection against GBS, particularly in infants and compromised individuals.
The GBS capsular polysaccharides are known to be important to GBS virulence and immunity. Baker, cited above. Moreover, the recognized GBS types and 25 subtypes have chemically related but antigenically distinct capsular polysaccharides having a repeating structure composed of galactose, glucose, N-acetyl glucosamine, and N-acetyl-neuraminic (sialic) acid.
Baker, cited above. Type III GBS capsular 30 polysaccharide is composed of a backbone made up of ~L

133~l87 repeating branched pentasaccharide units.
One study of type III GBS polysaccharides suggests that the natural immunodeterminant site is located at the side chain-backbone junction. Jennings et al., Biochemistry 20:4511-4518 (1981). The presence of the side chain terminal N-acetyl-neur-aminic acid residues reportedly was critical for immunodeterminant expression.
Studies of GBS protein immunogenicity are also reported.
Lancefield et al., J. Exp. Med. 142:165-179 (1975) report that the so-called C proteins of GBS are capable of inducing protective antibodies when present as an immunogen on a whole organism. The nomenclature "C proteins" is used as defined by Henrichsen et al., Int. J. of Systematic Bacteriol. 34:500 (1984), and the term includes proteins formerly designated Ibc proteins.
Molecular studies of C proteins have shown that these substances constitute a complex group of proteins of variable molecular weight. Wilkinson et al., Infect. Immunol. 4:596-604 (1971); Bevanger et al., Acta Path. Microbiol. Scand. Sect. B, 87:51-54 (1979); Russell-Jones et al., J. Exp. Med. 160:1476-1484 (1984). Bevanger et al., Acta Path. Microbiol. Scand. Sect. B, 89:205-209 (1981).
Wilkinson et al. disclose that hot acid extracts of whole type Ic GBS yields 13 proteins of varying molecular size (determined by polyacrylamide gel electrophoresis) having sero-logical reactivity in agar gel against type Ic antisera. Bevanger et al. (1979) disclose GBS acid extract containing nine trypsin sensitive proteins and five trypsin resistant but pepsin 133~187 sensitlve proteins. Bevanger et al. (1981) disclose immunogenicity of partially purifled acld extracted proteins.
Russell-Jones et al. disclose extracts containing up to 30 proteins varying in molecular weight from 20,000 to 130,000, the 130 kd protein being predominant. Russell-Jones et al. also report that multiple proteins reacted by im~uno (Western) blot to a single monoclonal antibody, suggesting that some proteins were breakdown products of others. Using another monoclonal antibody, the 130,000 MW proteins and 12 other proteins spaced between 10,000 and 120,000 MW appeared to share a common epitope.
Insel and Andersen, J. EXP. Med. 163:262 (1986) disclose conjugatinq Haemoplllus influenzae capsular polysaccharide to a protein carrier in an effort to convert the capsule to a more thymus-dependent immunogen.
Summary of the Invention We have discovered that oligomers of the repeating pentassaccharide unit of the type III Group B Streptococcus (III-GBS) polysaccharide capsule ~and even the single unit alone) are antigenic. Antigens containing the unit, particularly antigens containing repeated units, can be used as a component of a III-GBS vaccine. Accordingly, one aspect of the invention features an immunogenic substance, raising an antibody selectively immunoreactive with type III Group B Streptoccocus (III-GBS) bacteria, said immunogenic substance comprising a purified oligosaccharide hapten, and a carrier associated with said oligosaccharide hapten, said oligosaccharlde hapten having 133~87 the formula:
~4)~-D-Glcp(1~6)~-D-GlcpNAcp(1~3)~-D-Galp(l~

~-D-Galp ~-D-NeuNAc n in which n=1-50. GlcNAcp represents _-acetyl glucosamine (in the pyranose form), Galp represents galactose (in the pyranose form), Glcp represents glucose (in the pyranose form), and ~-D-NeuNAc represents N-acetyl neuraminic (sialic) acid.
"Purified" means substantially separated from the various protein, lipid, and carbohydrate components that naturally occur with the oligosaccharide. In particular, purified oligosaccharide is substantially free from intact III-GBS polysaccharide capsule, or fragments of it having a molecular weight above 100,000. Whatever traces of foreign components are in the purified oligosaccharide do not interfere with the use of the purified material in a vaccine or as an antigen. The term "purified" is not intended to exclude synthetic oligosaccharide preparations retaining artifacts of their synthesis; nor is the term meant to exclude preparations that include some impurities, so long as the preparation exhibits reproducible oligosaccharide characterization data, for example molecular weight, sugar residue content, sugar linkages, 133918l chromatographic response, and lmmunogenic behavior.
In a second aspect, the invention features a vaccine capable of eliciting protection against III-GBS, comprising a pharmaceutically acceptable vehicle and the above described antigen, optlonally conjugated to a carrier.
A third aspect of the invention features a method of making the immunogenic substance of claim 1 comprising:
a) culturing a type III group B Streptococcus bacterium;
b) recovering polysaccharide capsule in said medium or bacterial cells, said polysaccharide capsule having a backbone and side chains;
c) digesting said polysaccharide with an endo-~-galactosidase specific for cleaving one linkage on the backbone of said III-GBS polysaccharide without cleaving side chain linkages of said III-GBS polysaccharide and without cleaving other backbone linkages of said III--GBS polysaccharide; and d) recovering sald oligosaccharide hapten.
The invention further features a method of making the above-described antigen by: 1) culturing III GBS bacteria in a suitable medium; 2) recovering polysaccharide in the medium or from the bacteria cells; 3) digesting the polysaccharide with an endo-~-glactosidase specific for cleaving the linkage Sl(1~3)-~-gal(1~4)S2, where S1 and S2 are independently selected from glucose, glucosamine, or N-acetyl glucosamine; and 5) recovering the oligosaccharide antigen from the medium or from the bacteria.

5a 60412-1633 Optionally, the oligosaccharide is conjuqated to a carrier as described below.
In preferred embodiments of any of the first three aspects, the oligosaccharide antigen is produced by enzymatic hydrolysis of III-GBS capsular polysaccharide having a backbone and side chains using an endo-~-galactosidase specific for cleaving one linkage on the backbone of said III-GBS poly-saccharide without cleaving side chain linkages of sald III-GBS
polysaccharide and without cleaving other backbone linkages of said III-GBS polysaccharide. One such endo-~-galactosidase is found in Flavobacterium keratolyticus. Also preferably, the oligosaccharide is covalently bound (e.g., via a secondary amine link) to a carrier such as a protein, particularly a bacterial toxoid or a bacterial surface protein, comprlsing a bacterial immunodeterminant site.
In a fourth aspect, the invention more generally features enzymatic cleavage of a bacterial polysaccharide having the above-described linkage, in a method of making a purified oligosaccharide; specifically a bacteria having a surface poly-saccharide (e.g. a polysaccharide capsule or membrane) having atleast one linkage gal D-Galp(l~ S where S is selected from the group consisting of glucose, glucosamine, and N acetyl glucos-amine, is cultured, and the polysaccharide is extracted, after which it is digested with the above-described enzymes and the oligosaccharide is recovered. Preferably the bacteria is gram positive, e.g., type III-GBS; alternatively the bacteria can be a 1 33q 1 87 5b 63884-33D
specles having a surface capsule polysaccharide, such as type 14 Streptococcus pneumoniae, e.g., ATCC No. 6314.
In another aspect there is provided a commercial package comprising a vaccine as defined above together with instructions for the use thereof in the treatment or prevention of a group B
Streptococcus infection.
In a fuLther aspect, the invention features a su:~stantially purified trypsin-resistant C surface protein of type Ia~c Group B Streptococcus, the protein , .,:

~339~p~7 having a molecular weight about 14,000, and being non-cross-immunoreactive with Group B Streptococcus bacterial polysaccharides, yet cross-immunogenic with type Ia/c GBS. In other aspects, the above described protein is used in a vaccine that elicits protection against type Ia/c (and type Ib/c) GBS, which vaccine comprises the protein (optionally conjugated to an oligosaccharide such as the III GBS oligosaccharide described above) and a pharmaceutically acceptable carrier. The resulting conjugate provides broad protection against GBS, in that it protects against III
GBS and against GBS having C protein.
In yet another aspect, the invention features a gamma globulin fraction capable of passive protection against GBS, the fraction being produced by immunizing a mammal with one of the above-described vaccines. The fraction is then adminstered to an individual to provide protection against GBS infection or to treat on-going infection.
Finally, the invention features a method of assaying a sample for anti GBS antibody by adding to the sample the oligosaccharide or protein antigen described above, and then detecting the formation of an immunocomplex; alternatively a sample is assayed for the presence of a GBS immunodeterminant by raising an antibody to the oligosaccharide or protein antigen, adding the antibody to the sample, and detecting the formation of an immunocomplex.
The enzymatic selective hydrolysis of the backbone glycosidic bonds is superior to simple acid hydrolysis, because the latter technique results in the loss of the side chain sialic acid residues, and cleaves bonds non-selectively, yielding a heterogeneous mixture 133ql~7 of reduced immunogenicity. In contrast, enzymatic digestion preserves the sialic acid residues and produces backbone cleavage only at the gal-B 1-4-glc bonds, which occur once per backbone repeating unit.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims.
Description of the Preferred Embodiments We now describe preferred embodiments of the invention, first briefly describing the drawing.
I. Drawing.
Fig. 1 is a diagram of a repeating pentasaccharide of the III GBS capsular polysaccharide.
II. Type III GBS Antigen And Vaccine A. Oligosaccharide The specific pentasaccharide described above is obtained by culturing type III GBS and extracting the polysaccharide capsule from the supernatant broth or from the bacterial cells by the general method of Jennings et al., Canadian J. Biochem. 58:112-120 (1980). The polysaccharide extract is digested by an endo-n-galactosidase that specifically cleaves the structure Sl(1~3)Bgal(1~4)BS2, where Sl and S2 are independently selected from glucose, glucosamine, or N-acetyl glucosamine. The above structure occurs once per repeating unit of the III GBS
polysaccharide capsule, and the resulting cleavage products are oligosaccharides having one or more of the above-described pentasaccharide repeating units.
The enzyme is obtained from bacteria known to produce an enzyme catalyzing the requisite specific cleavage. One such preferred bacteria is Flavobacterium keratolyticus. Another bacterium thought to produce an enzyme with the requisite specific activity is Bacteroides fragilis. Kitamikado et al., cited below, report that the requisite endo-~-galactosidase activity is also present in Escherichia freundii, Pseudomonas sp., and Cocobàcillus sp.
when induced on keratan sulfate. See Nakagawa et al. J. Biol.
Chem. 250:912-917; and Hirano et al. Connect. Tissue Res.
2:1-10. A suitable F. keratolyticus can be obtained from the Institute of Fermentation (IFO) Osaka, Japan, under the designation IFO #14087. A suitable general procedure for preparing endo-~-galactosidase is given by Kitamikado et al., J. Biol. Chem. 2:3906-3909 (1981).
The extracted III GBS polysaccharide capsule prepared as described above is added to a buffered enzyme preparation, and incubated (e. g., 37~C, 48 hrs.). After digestion, the oligosaccharides are separated by molecular weight by gel filtration chromatography, e. g., Sephadex G75 (Pharmacia), and purified by anion exchange chromatography, e. g., anion exchange high performance liquid chromatography (HPLC), Mono Q
(Pharmacia), or QAE Sephadex (Pharmacia).
A purified oligosaccharide comprising between about 1 and 50 (and most preferably between about 2 and 30 penta-saccharide units) is selected from chromatographic group and then conjugated to a protein for use in a vaccine. The protein may be an inactive carrier, or it can be selected to provide some protection in its own right, particularly protection that complements the protection induced by the oligosaccharide, for example, by inducing protection against other types of GBS or Trade-mark - 8a - 1 339 1 87 other bacteria. The non-toxic diphtheria toxin analog, CP~
197, may be used as described by Insel et al. (1986) J. Exp.
Med. 163:262 1 ~39~ 87 g and Andersen et al. J. Clin. Invest. 76:52 (1985).
Other toxoids that can be used include tetanus or standard diphtheria toxoids available from the Massachusetts State Laboratory, South Street, Jamaica Plain, MA.
Protein-oligosaccharide conjugation is achieved by a coupling reaction, such as the general method of Swartz and Gray, Arch. Bioch. Biophys. (1977) 181:542, which couples a reducing sugar directly to a protein.
Oligosaccharide fragments produced by deamination can be directly linked via cyanoborhydride to the carrier protein. In the case of III GBS, a secondary amine is formed by bonding between a nitrogen on the protein and an aldehyde group on 2,5-anhydromannose formed by deamination of the oligosaccharide. This aldehyde is available for direct coupling using cyanoborhydride.
The resulting oligosaccharide-protein conjugate is suspended in pyrogen-free saline or any other physiologically acceptable non-toxic vehicle or medium, which may contain any conventional stabilizers or other additives as desired. The concentration of antigen is not critical and may be varied over a wide range, but for many purposes a range of 10-1000 ~g/ml is convenient and suitable. In this form it is stable during prolonged storage at 4~C. and is sterile, non-toxic, and non-pyrogenic when subjected to animal tests as prescribed by Food and Drug Administration Regulations (Title 21, Sec. 610.11-610.13). Suitable volumes (e.g. about 0.5 ml) of the above-described saline suspension are administered (e.g. by subcutaneous injection) to induce III GBS protection.
The invention also features a method of passive immunization, used particularly for infants or compromised adults, by injecting the oligosaccharide-protein conjugate vaccine into a human to raise antibodies thereto in high titer, separating the antiserum from the blood of the human, and fractionating the antiserum to produce a gamma globulin fraction containing the antibodies, which can be used for passive immunization. This method of establishing donors for passive immunization is useful because, although occasional non-immunized human adults have very high levels of type-specific GBS antibody in their sera, it would be necessary to screen very large populations to select those individuals whose plasma could be pooled to make sufficiently high titered globulin fractions to be useful for passive immunizations. Several lots of pooled human gamma globulin from non-immunized adults have been assayed for anti-Types Ia and Ic, anti-Type II, and anti-Type III polysaccharide antibody and have been found to contain only 5-20 ~giml of serum.
Immunizations by intravenous injection of such low titer globulins would require prohibitively large doses with attendant risk of adverse reactions.
For passive immunization, pools of human sera from selected individuals vaccinated with the above-described conjugated polysaccharide vaccine can be concentrated and fractionated by conventional procedures to provide a globulin fraction containing most of the type-specific antibody and of sufficiently high activity so that the hyperimmune globulin is effective in small doses of 0.3-1 ml, preferably about 0.5 ml, when administered in a suitable physiologically acceptable càrrier such as normal saline either intravenously or intramuscularly. The concentration of the globulin in the carrier may be from 5 to 20% by weight. The hyperimmune globulin can be administered either to pregnant women prior to delivery, to neonates, or to immunologically compromised individuals, to provide passive immunization or therapy.
III. Oligosaccharidè Antibodies Oligosaccharides generally can be recovered from endo-~-galactosidase digestion of bacterial polysaccharides.
The above description concerns III GBS oligosaccharides and their use in vaccines, however, the oligosaccharides also can be used to elicit antibody in experimental mammals, e. g., by challenging the mammal, e. g. by injecting the oligosaccharide protein conjugates, and recovering the resulting antibody.
Also, monoclonal antibodies can be generated by standard techniques. The resulting antibodies are useful, e. g. in immunoassays for GBS, or III GBS in particular. For example, the antibody to the oligosaccharide can be used for immuno-assays such as competitive or sandwich immunoassays known in the field.
The following examples illustrate the oligosaccharide antigen, the method of isolating the oligosaccharide, the vaccine, and the method of passive immunization.
Example 1: Preparation of endo-~-galactosidase F. keratolyticus is cultured overnight and used to inoculate a modified trypticase peptone broth (3~ trypticase peptone from BBL Microbiology Systems, Cockeysville, MD); 0.1%
yeast extract (Difco Laboratories, Detroit, MI); 0.2~ NaCl, pH7Ø The broth is incubated for 18 hours (Biolafitte fermentor, Lafitte, France), while controlling aeration (15 *

Trade-mark ,X 63884-33D

13391~7 l/min), stirring (150 rpm), temperature (25~C), and pH (7.0).
Bacteria are removed by centrifugation and the supernatant *

concentrated (Pellicon Cassette System, Millipore Corp., Bedford, MA). Solid (NH4)2S04 is added to 75% saturation and the solution is allowed to stand overnight at 4~C.
Precipitate is removed by centrifugation and dissolved in lOmm sodium acetate, 0.2M NaCl, pH6Ø The solution is loaded onto a 5 x 90 cm Sephadex G-100 column (Pharmacia Fine Chemicals, Piscataway, NJ) and eluted with the same buffer at 40ml/hr, 4~C. Active fractions are identified by assaying for endo-~-galactosidase activity and then pooled.
Specifically, the fractions are incubated with bovine cornea keratan sulfate (Sigma Chemical Co., St. Louis, MO) overnight at 37~C. Production of reducing sugar is measured by the general method of Park and Johnson, J. Biol. Chem. 181:149-151 (1949).
Pooled active fractions are dialyzed, lyophilized, and dissolved in buffer (50mM sodium acetate, 2mM CaCl2, pH6.0).
The buffered enzyme preparation is loaded onto a 2.5 x 14 cm column containing DEAE Sephacel (Pharmacia) in the top half and BioRex 70 (BioRad Laboratories, Rockville Center, NY) iIl the bottom half. The enzyme elutes with 250ml of the above buffer.
Residual bound protein is eluted with l.OM MaCl, and active pooled fractions are dialyzed against lOmM sodium acetate, 2mM

CaCl2, pH7.0, and lyophilized.
Example 2: Polysaccharide Digestion GBS type III polysaccharide is extracted by the general technique of Jennings (1980) cited above. The lyo-Trade-mark 1 33~ 87 philized endo-~-galactosidase preparation from one DEAE
*

Sephacel /BioRex 70 column run is dissolved in 10ml 10mM sodium acetate, 2mM calcium chloride, pH7.0 and dialyzed against 2 liters of the same buffer overnight at 4~C with one bath exchange. Twenty mg of purified type III G~S capsular poly-saccharide is added to the enzyme preparation. The mixture is filter sterilized, and incubated at 37~C for 48 h, with stirring.
Example 3: Oliqosaccharide Purification and Characteriza~ion After digestion, oligosaccharides are separated into large versus small molecular weight pools by dialysis against water through a 12,000-14,000 dalton pore size membrane (Spectrum Medical Industries, Los Angeles, CA). Oligo-saccharides corresponding to one (pentasaccharide) and two (decasaccharide) repeating units are purified by anion exchange chromatography. Small amounts of oligosaccharides (1 mg or less) can be purified by aniGn exchange high perfGrmance liquid chromatography (HPLC). Five hundred ~g of diqestion mixture (small molecular weight pool) are loaded on a 5 x 50 mm Mono Q column (Pharmacia) and one and two repeating unit oliqo-saccharides are eluted isocratically with 20mM Tris HCl buffer, pH7.2. Larger oligosaccharides can be eluted ~-ith higher salt concentrations. For purification of large amounts of oligo-saccharides, a 1.2 x 10 cm column filled with QAE Sephadex A50 (Pharmacia) is used. Samples of 2-8 mg are loaded OntG this column in a starting buffer of 20mM N-methyl diethanolamine acetate, pH9.6. The column is washed with 30 ml startinq Trade-mark 6388~-33D

buffer at 1 ml/min, then the single repeating unit oligo-saccharide is eluted with 50 ml of starting buffer containing 15mM Na acetate. TWG ml fractions are collected, neutralized with lM acetic acid, and analyzed by thin layer chromatography (TLC). Fractions containing the single repeating unit oligosaccharide are pooled, lyophilized, dissolved in 2 ml water, and desalted on a 1.6 x 25 cm column CGntaining Sephadex G15 (Pharmacia).
The major digestion product corresponds to a band migrating between tetrasaccharide and decasaccharide standards, and has a molecular size compatible with that predicted for the pentasaccharide unit of Fig. 1. Diphenylamine and resorcinGl staining of this band is consistent with a sialylated sugar.
Methylation analysis confirms that structure is a complete pentasaccharide repeating unit.
One and two repeating-unit oligosaccharides are purified by anion exchange chromatography. Anion exchange HPLC
is a quick and convenient method for purifying small amounts of oligosaccharides (1-2 mg or less). The single repeating unit oligosaccharide elutes in the void volume, while the two repeating unit oligosaccharide elutes later in the isocratic phase. Larger oligosaccharides or native polysaccharide, are retained in the isocratic phase, but are readily eluted from this column with 0.5 M sodium chloride. This method can be used to purify tritiated one and two repeating unit oliqo-saccharides for use in radioantibody binding assay (RABA) studies. See Schifferle (1985) J. Immunology 135:4164. For purification of larger amounts of single repeating unit X

13391~7 oligosaccharide, an open anion exchange column can be used to provide a larger binding capacity. Using this ~AE Sephadex A50 column, the single repeating unit, as well as larger oligo-saccharides are retained in the starting buffer. The single repeating unit is eluted, free of larger oligosaccharides, with the starting buffer containing 15mM sodium acetate. Larger oligosaccharides remain bound, and can be eluted with increased salt concentration. From digestion of 20 mg of type III native polysaccharide, 6 mg of purified single repeating unit oligo-saccharide are obtained. The oligosaccharide corresponding toa minor band migrating just distal to the single repeating unit cn TLC is also retained somewhat more avidly by the anion exchange column. Although complete separation is not achieved with preparative runs, the single repeating unit oligosaccharide purified in this manner is estimated to be 95% pure by TLC and methylation analysis. Larger oligosaccharides are also fractionated by chromatography, e. g., a Sephadex G75 column.
Example 4: Binding Assay for Oligosaccharide The chromatogram binding assay is used to examine directly the ability of the oligosaccharide bands visualized by TLC to bind antibody directed aaainst the native poly-saccharide. Antibody binding to the single repeating unit band is clearly evident. Although the assay is not quantitative, it is clear from the relative intensities of the bands on the autoradiograph that the native polysaccharide binds antibody more efficiently. This assay is highly specific for native type III polysaccharide -- cross reactions are not seen with the desialylated core polysaccharide, with the group B poly-saccharide, nor with capsular polysaccharides from other GBS

Xl ~ 3 39 I R7 serotypes.
The oligosaccharide antigen described above is then conjugated to a protein as described above, and suspended in pyrogen-free saline solution, as described above, to form a vaccine.
The single repeating pentasaccharide (Fig. 1) is weakly antigenic using radioantibcdy binding assay inhibition TLC binding assay, and direct radioantibody binding assay.
Increasing oligcsaccharide size to two repeating units results in an eight-fold increase in antigen binding. Most Preferably, the immunogen includes 2-50 units.
IV. GBS Protein Antigen An antigenic GBS C protein is isolated from type Ic GBS culture supernatants as illustrated by the following example. The protein is immunologically reactive with antisera (e. g., mouse antisera) to a P10 GBS protein fracticn, i. e., a fraction including proteins of MW between 10,000 and 30,000.
The purified protein elicits rabbit antisera that protect mice against challenge with type Ib GBS. This protective ability is not affected by incubations with GBS polysaccharides.
Example 5: Production and Purification of Protein Antigens Type Ia/c GBS (e. g. Channing Laboratory Strain A909 Rockefeller University, N. Y., N. Y., ATCC 27591) are cultured into late log phase in a dialysate of Todd Hewett Broth (THB, Difco Laboratories, Detroit, MI), and then resuspended in 0.3 formalin in 0.15M sodium phosphate buffer (pH7.4), by the general method of Levy et al. J. Infect Dis. 149:851-868 (1984).

X, A one liter log phase culture Gf type Ic is added to 15 liters of the dialysate of THB (dialyzed on an exclusion membrane of 10,000 MW), supplemented with 10% qlucose, and grown into late log phase maintaining neutral pH by titration in a fermentor (Biolafitte Model BL 20.2, LSL Biolafitte Inc., Princeton, NJ). After removal of the bacteria by pelleting at 10,000 g, the supernatant is concentrated to 100 ml on a Pellicon apparatus (Millipore Corp., Bedford, MA) containing a 10,000 MW exclusion membrane, dialyzed against H2O extensively and frozen at -20~C.
The culture supernatant is separated by approximate molecular size using ultrafiltration. TWG fractions are obtained, one retained by a PM30 membrane (P30; greater than 30,000 MW), and another retained by a PM10 membrane but not retained by a PM30 (P10; greater than 10,000 ~W and less than 30,000 MW). The P10 is dialyzed against 0.05 M sodium phosphate buffer, pH 7.4 with 0.15 M NaCl and fractionated on a Sephadex G-75 column (1.6 x 82 cm) (Pharmacia). The protein content of fractions is monitored by absorpticn at 750 nm in the Lowry assay, J. Biol. Chem., 193:265-275 (1951).
Example 6: SDS-PAGE and Western Blot Samples are analyzed by SDS-PAGE following the methcd of Laemmli, Nature 227:680-684 (1970). Gradient gels either 5-15% or 10-20% are employed depending on the molecular weiqht of the proteins. Samples for electrophoresis are diluted in a buffer cGntaining 0.1 M Tris, pH8, 1% SDS, 4 M urea and 60 mM
iodoacetamide as final concentration. When reduction of the samples is required, 50 mM dithiothreiGtol is included in the buffer. One hundred ~1 samples containing 100 ~g protein are 1 3391 ~7 placed into each well. After electrophoresis the proteins are visualized by Coomassie brilliant blue stain.
To identify immunologically reactive antigens, Western blot is used. Proteins are first subjected to electro-phoresis and the SDS-PAGE gel washed in Buffer A composed of 0.02 M Tris buffer, pH 8.5 with 0.15 M glycine and 20%
methanol. The proteins are transferred to nitrocellulose sheets (Schleicher and Schuell, Keene, NH) by electrophoresis (150 mA, 20 h) in Buffer A and washed in 0.01 M Tris buffer, pH 7.4 with 0.5 M NaCl and 0.1% Brig 58 (Buffer B). The sheet is then incubated with antiserum for 2 h at 22~C and washed in Buffer B. After incubation with I-protein A for 30 min at 22~C, the sheet is washed in Buffer B, blotted dry and exposed to Kodak XAR 5 film.
Example 7: Mouse Protection Studies Male outbred CD-l mice weighing 22-24 gr are obtained from Charles River Breeding Laboratories (Wilmington, ~).
Sera obtained from the mice contained no detectable antibody to type Ia/c GBS polysaccharide antigen as tested by RABA.
Each experimental group contained between 5 and 10 animals.
For mouse protection studies, mice are injected IP with 1 ml mouse or rabbit antiserum and 24 h later challenged IP with 1 x 10 CFU of type Ib/c GBS. Preimmune rabbit or mouse serum is used as the control. Mice are examined every 24 h and the number of surviving mice is calculated 48 h after bacterial challenge.
For absorption of protective antibodies from antisera, GBS polysaccharides or protein fractions in concentrations of 1 mg per ml are used. One ml of antiserum and one ml of poly-saccharide or protein is incubated for 2 h at 37~C and then overnight at 4~C. The mixture is then pelleted at 13,000 g for 3 min and the supernatant was diluted in 0.15 M NaCl for protection studies.
After partial purification by column chromatography, the 14,000 MW protein was reisolated from preparative SDS-PAGE
gel and used to elicit antiserum in a rabbit. In mouse protection studies this rabbit antiserum protected mice (89~) against Ia/c GBS challenge.
To investigate whether the P10 fraction could elicit mouse protective antibodies against a heteroloqous GBS strain, mice were immunized with the type Ia/c P10 fraction. The post immunization antisera were then pooled and examined for its capacity to protect mice against challenge with type Ib GBS.
Mice were injected IP with various dilutions of pooled mouse antisera to the P10 fraction followed 24 h later by challenge with the type Ib/c strain. The protective capacity of the sera begins to dilute out at a 1:80 dilution of the sera. Therefore the sera were used at an optimal dilution of 1:40 for these experiments. In mouse protection studies the immune sera protected mice against challenge with the type Ib strain (P < 0.001) compared with the normal mouse sera at the same dilution. This pooled sera contained no detectable antibody to the type Ia polysaccharide as measured in the RABA.
Furthermore absorption of the pooled antisera with the type Ia or Ib polysaccharide did not significantly alter the protective level. The protective efficacy of mouse antisera to P10 fraction from type Ic contained non-capsular antigens which ~.

elicited antibody in mice which passively protected mice against challenge with the type Ib GBS.
Specifically, rabbit antiserum to the SDS-PAGE
eluted 14,000 MW protein from type Ic GBS was next used in mouse protection studies. Mice were injected with antiserum IP and challenged 24 hours later with type Ib GBS. The anti-serum elicited to the SDS-PAGE eluted 14,000 MW protein was-protective in mice at dilutions of 1:5 to 1:10. The differ-ence between the normal rabbit serum and the antiserum elicited to the SDS-PAGE eluted 14,000 MW protein was significant.

V 63884-33D~ I

Claims (8)

1. A substantially purified trypsin-resistant C surface protein of type Ic group B Streptococcus bacteria, said protein eliciting protection against type Ib/c group B and type Ia/c group B Streptococcus bacteria, said protein having a molecular weight about 14,000, said protein being non-crossimmunoreactive with group B Streptococcus bacteria polysaccharides, said protein being crossimmunoreactive with type Ib/c group B Streptococcus bacteria.

2. A vaccine that elicits protection against type Ia/c group B Streptococcus bacteria comprising a pharmaceutically acceptable vehicle and an immunogen comprising the protein of claim 1 or a fragment comprising an immunodeterminant thereof, said protein or fragment being optionally conjugated to a carrier.

3. A use of a gamma globulin fraction of serum from an animal immunized with a vaccine according to claim 2 to prepare a therapeutic suitable for administration to an individual to provide passive immunization or therapy against type Ib/c group B Streptococcus bacteria.

4. A use of a vaccine according to claim 2 in the treatment or prevention of a group B Streptococcus infection.

5. A gamma globulin fraction capable of providing an individual passive protection against type Ib/c group B and type Ia/c group B Streptococcus, said gamma globulin fraction being produced by immunizing a mammal with the vaccine of claim 2, and recovering gamma globulin from said mammal.

6. A method of assaying a sample of anti group B
Streptococcus antibody comprising adding to said sample the protein of claim 1 and detecting formation of an immunocomplex.

7. A method of assaying a sample for the presence of a group B Streptococcus immunodeterminant comprising raising an antibody to the protein of claim 1, adding said antibody to said sample, and detecting the formation of an immunocomplex.

8. A commercial package comprising a vaccine according to claim 2 together with instructions for the use thereof in the treatment or prevention of a group B Streptococcus infection.