CLONING OF NON-IgA Fc BINDING FORMS OF THE GROUP B STREPTOCOCCAL BETA ANTIGENS

Background of the Invention Group B streptococci (GBS) are important human pathogens. These bacteria are increasingly being recognized as disease causing agents in adults, particularly in immunocompromised individuals; however, it is as the infectious agent of over 40% of all cases of neonatal sepsis in the U.S. which caused GBS to be recognized by the

National Academy of Sciences in 1985 as the fourth most important cause of preventable infectious morbidity in this country. There are over 12,000 cases of GBS sepsis in the U.S. annually, resulting in over 2,500 infant deaths and 1,350 cases of permanent neurologic damage. In addition, pregnancy-related morbidity occurs in nearly 50,000 women each year. One recent review article estimated the direct cost per year of GBS disease in this country at over $726 million. No GBS vaccine is currently available, yet it has been estimated that over 94% of the cost due to group B streptococcal disease could potentially be avoided by the development of an effective maternal vaccine. In addition to the group B specific carbohydrate antigen which delineates GBS from other streptococcal species, these bacteria are serotyped based on the presence of one of seven known type-specific carbohydrate antigens expressed on their surfaces. These are called la, lb, II, HI, IV, V, and VI. In addition, a number of protein antigens known collectively as C proteins have been identified. These are designated as alpha, beta, gamma, and delta. The genes encoding the alpha and beta antigens have been cloned (Cleat and Timmis, 1987; Michel et al, 1991) and sequenced (Jerlstrom et al, 1991; Heden et al, 1991; Michel et al, 1992), and the beta antigen has been shown by a number of researchers to interact specifically, but in a non- immune manner, with the Fc region of human IgA (Russell-Jones and Gotschlich, 1984; Russell- Jones et al, 1984; Brady et al, 1989; Anthony et al, 1990; Lindahl et al, 1990; Kvam et al, 1992). The distribution of specific C protein antigens among

strains of particular carbohydrate serotypes has been partially described in the literature and is complex.

A number of research groups have reported that greater than half of all cases of neonatal sepsis are caused by type IJJ organisms, whereas type m organisms account for less than 25% of the organisms isolated from healthy colonized infants and pregnant women. There is a greater interest in protection against serotype m GBS, although none of the serotypes are considered to be benign. The only C protein antigen commonly associated with type IE GBS is the delta antigen (Brady et al., 1989; Chun et al., 1991). Low levels of maternal IgG antibodies to GBS serotype-specific carbohydrate antigens have been shown to be correlated with disease susceptibility in neonates (Baker et al, 1978; Fisher et al, 1983). Unfortunately, many carbohydrate antigens are poorly immunogenic in humans. This is known to be true of GBS type specific carbohydrates with the possible exception of the type II polysaccharide. Development of a vaccine that is effective against multiple serotypes of GBS is considered to be of paramount importance in disease prevention. The full-length GBS beta antigen is a polypeptide of approximately 130,000 daltons. It has been reported to be immunogenic and to elicit the formation of protective antibodies in animal models (Michel et al, 1991; Madoff et al., 1992). However, the potential for the use of the β antigen as a vaccine is substantially compromised because of its undesirable property of interacting with high affinity and in a non-immune manner with the Fc region of human IgA. Truncated forms of the beta antigen are secreted by certain strains of GBS in the absence of cell surface expression of the antigen, and both IgA Fc binding and non-binding forms are observed (Brady et al., 1989). There is evidence that high levels of maternal antibodies against GBS can be passed to and protect the newborn via the placenta. Therefore, there is a great deal of interest in developing a maternal GBS vaccine. Although the beta antigen is known to be immunogenic (i.e., it induces the formation of protective antibodies) in rabbits and mice, it would be dangerous to include in a human vaccine component which can bind to a human protein.

Therefore, an object of the subject invention is to provide a non-IgA Fc binding form of the beta antigen of GBS.

Brief Summary of the Invention

The subject invention pertains to the genetic manipulation of the gene encoding a GBS surface protein called the beta antigen so that it is no longer able to bind to human IgA. Specifically, a portion of the beta antigen gene essential for IgA binding by the encoded protein has been identified and deleted. The novel protein encoded by the altered beta antigen gene does not bind to IgA but does immunoreact with monospecific anti-beta antigen antisera raised against the natural beta antigen protein.

This will allow the genetically engineered beta antigen of the subject invention to be used as a component in a human vaccine to protect against the serious health threat of GBS infections.

Brief Description of the Drawings Figure 1. The DNA sequence of the beta antigen gene is shown. The positions of forward (a and c) and reverse (b and d) oligonucleotide primers used for the polymerase chain reaction are indicated. The location of restriction endonuclease sequences engineered into the oligonucleotide primers are also indicated (BamΗI and Sail). The region of DNA between reverse primer b and forward primer c was deleted by the cloning strategy described in the text and in Figure 2.

Figure 2. The construction of the truncated beta antigen gene is shown. PCR generated DNAs (952 base pairs for pJB2a and pAHIO and 2,385 base pairs for pAH5 and pJB48) were ligated into the TA cloning site of the 3,932 base pair pCR™π vector. The positions of the BamΗI (B) and Sail (S) restriction endonuclease sites engineered onto the ends of the GBS sequences and the orientation of the cloned insert DNAs are indicated. GBS-derived DNA is indicated by a bold line. Figure 3. A BstXL restriction digest of plasmid DNA from clones JB2a (lane

1), AH10 (lane 2), AH5 (lane 3), JB48 (lane 4), and 806-2 (lane 5) is shown. The approximate size of the DNA standards indicated are 20, 5.0, 3.5, 2.0, 1.9, 1.6, and 1.3 kilobases.

Figures 4A-4E. The sequence of the GBS strain HG806 derived insert DNA from plasmid pJB2a is shown aligned with the corresponding regions of the published beta antigen gene sequences (Jerlstron et al, 1991; Heden et al, 1991).

Figure 5. Western immunoblot analysis of concentrated LB broth culture supernatants (Panels A and B) or cell extracts (Panels C and D) of E. coli probed with anti-beta antiserum (Panels A and C) or biotin-labelled myeloma IgA kappa (Panels B and D) are shown. Lanes 1 through 7 correspond to E. coli INVαF' harboring plasmids pJB2a, AH5, 806-2, pAHIO, pJB48, p618-12, or pCR™π, respectively.

Clones JB2a, 806-2, AH10, 618-12, and 618-18 all contain the GBS promoter DNA for the beta antigen gene and detectable levels of beta antigen expression are consistently observed for these clones.

Detailed Disclosure of the Invention

The subject invention concerns the identification and deletion of the IgA binding portion of the group B streptococcal (GBS) beta antigen. The IgA Fc binding domain of the GBS beta antigen was located by comparison of the activities of two truncated beta antigen polypeptides. The «55,000 dalton polypeptide secreted by GBS strain 2AR binds to the Fc region of human IgA while the «38,000 dalton polypeptide secreted by strain HG806 does not. Both polypeptides are reactive with rabbit anti- beta antiserum and were demonstrated to share the same amino-terminus as the mature full-length wildtype beta antigen protein. It was deduced, therefore, that either the IgA Fc binding activity of the beta antigen resides directly within the carboxy-terminal 17,000 daltons of the polypeptide expressed by strain 2AR or this region is necessary to confer IgA Fc binding activity in conjunction with the amino-terminal portion of the molecule.

A specific aspect of the subject invention concerns the construction of a novel recombinant beta antigen gene lacking that portion of DNA which encodes the IgA binding activity of the wild-type beta antigen protein. A cloning strategy was developed to construct a gene which lacked that segment of DNA believed to encode the portion of the beta antigen polypeptide necessary for non-immune binding of human IgA. Oligonucleotide primers were designed to amplify two specific segments of beta antigen DNA using the polymerase chain reaction (PCR). 0.95 kilobases (kb) of beta antigen DNA upstream as well as 2.4 kilobases of DNA downstream of the putative IgA Fc binding domain were amplified and cloned (see Figure 1). Chromosomal DNAs from two GBS strains were used as templates for the PCR.

Strain HG806 expresses the truncated «38,000 dalton non-IgA Fc binding molecule, while strain ss618C expresses full-length («130,000 dalton) IgA Fc binding beta antigen. A Sail restriction endonuclease site was engineered into the reverse and forward primers used to generate the «0.95 kb and «2.4 kb DNA segments, respectively, so that once cloned, the two segments could be ligated in frame to result in a final polypeptide product lacking approximately 150 amino acid residues in close proximity to the IgA Fc binding domain of the beta antigen. The PCR amplified gene segments for each strain were cloned into the commercially available vector pCR™π. This vector is specifically designed to accept PCR-generated DNA. Lastly, pCR™π plasmids harboring the 0.95 kb and 2.4 kb gene segments for each strain were double digested with Sail and Tt ΩIi restriction endonucleases. The appropriate size fragments were recovered and ligated to fuse the beta antigen gene segments in frame, as well as to reconstitute a single copy of the pCR™II vector (see Figure 2). BamΗI restriction endonuclease sequences were engineered into the forward and reverse primers used to generate the «0.95 and «2.4 kb gene segments, respectively.

Therefore, the beta antigen gene constructs lacking DNA necessary to encode a functional IgA Fc binding domain can be excised from the vector by digestion with BamΗI. This enables transfer of these gene constructs to any vector of choice with a BamΗI sequence in its multiple cloning site. The «0.95 and «2.4 kb gene segments can be excised from their respective plasmids either by double digestion with Sail or

BamΗ or by digestion with BsiXl, which cleaves on either side of the insert in the vector DNA.

The subject invention further concerns the expression of a novel non-IgA binding polypeptide using the recombinant beta antigen gene constructs containing the region deleted by the cloning strategy. Successful PCR amplification of both the 0.95 kb and 2.4 kb beta antigen gene fragments from strain HG806 indicates that despite the expression of a markedly truncated polypeptide by this strain, no major deletions exist in the gene to account for the observed phenotype. A likely explanation for the expression of a truncated product is the existence of a nonsense mutation in this particular strain's beta antigen gene resulting in a premature stop codon. As expected, there were no premature stop codons found during sequencing of HG806-derived DNA located upstream of the putative IgA binding domain. The genetic lesion present in

HG806 is most likely present in that portion of its beta antigen gene eliminated by the cloning strategy described above. Such a deletion in HG806 would therefore allow for reexpression of carboxy-terminal beta antigen. This indeed seems to be the case as the polypeptide product of the «3.3 kb fused gene construct is reactive with anti- beta antibodies and is substantially larger than the product of the «0.95 kb gene segment.

Elimination of the DNA encoding the IgA Fc binding domain results in an obliteration of IgA Fc binding activity by the gene construct derived from strain ss618C. Appropriate size gene constructs (3.3 kb) have been derived from both strains HG806 and ss618C. The polypeptides expressed by the «3.3 kb fused gene constructs derived from both strains HG806 and ss618C can be detected by Western immunoblotting using polyclonal rabbit antiserum recognizing the GBS beta antigen, yet no interaction of these polypeptides with biotin-labelled human myeloma IgA kappa protein has been demonstrated. These results indicate that the segment of DNA necessary for IgA Fc binding has been sufficiently disrupted to eliminate this property of the beta antigen, while the antigenic nature of the polypeptide has not been sufficiently disturbed to preclude its interaction with specific anti-beta antibodies. Since it is unacceptable to use, as a component of a vaccine, any molecule which can specifically bind with high affinity to a host protein, e.g., an immunoglobulin molecule, the construction of specifically engineered GBS beta antigen genes which eliminate this undesirable property will allow its use as both a carrier and immunogen in a GBS vaccine preparation. Therefore, the subject invention further relates to the use of the non-IgA binding beta antigen disclosed herein as an immunogenic composition to raise an immune response. Because of the redundancy of the genetic code, a variety of different polynucleotide sequences can encode the polypeptides disclosed herein. It is well within the skill of a person trained in the art to create alternative polynucleotide sequences encoding the same, or essentially the same, polypeptides of the subject invention. These variant or alternative polynucleotide sequences are within the scope of the subject invention. As used herein, references to "essentially the same" sequence refers to sequences which encode amino acid substitutions, deletions, additions, or insertions which do not materially alter the immunological reactivity of the encoded

polypeptide with anti-beta antigen antisera. In addition, the scope of the subject invention encompasses all or part of the nucleotide sequences disclosed herein, provided that the polypeptide encoded by the polynucleotide sequence does not bind to IgA but does immunoreact with the anti-beta antigen antisera. Fragments and variants of the claimed polypeptides which do not bind to IgA but retain immunological reactivity with anti-beta antigen antisera are within the scope of the subject invention. As a person skilled in the art would appreciate, certain amino acid substitutions within the amino acid sequence of the polypeptide can be made without altering the immunological reactivity of the polypeptide. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions, whereby an amino acid of one class is replaced with another amino acid of the same class, fall within the scope of the subject invention so long as the substitution does not materially alter the immunological reactivity of the polypeptide. Non-conservative substitutions are also contemplated as long as the substitution does not significantly alter the immunological reactivity of the non-IgA binding polypeptide.

The polypeptide specifically exemplified herein encompasses amino acids 1- 209 and 353-1127 of the full-length wild-type GBS beta antigen as a single fusion product. As the skilled artisan will readily appreciate, the deleted region of the beta antigen could be somewhat smaller or larger than that which is exemplified herein.

Variant polypeptides would be within the scope of the subject invention as long as the polypeptide did not bind to IgA but did immunoreact with anti-beta antigen antisera. For example, using the teachings provided herein, a person skilled in the art could readily prepare a polypeptide that varied from 1 to about 60 amino acids, added to or removed from, either end of the deleted region exemplified herein. For example, in a preferred embodiment, amino acids starting at about 250 can be deleted up to about amino acid 350. Preferably, any added amino acids would be the same as the corresponding amino acids of the wild-type beta antigen polypeptide. Also within the scope of the subject invention are polypeptides having amino acids added to or deleted from either the amino-terminus or carboxy-terminus of the polypeptide specifically exemplified herein. Such additions or deletions would be readily apparent to a person of ordinary skill in the art.

The polynucleotides of the subject invention can be used to express the recombinant beta antigen. They can also be used as a probe to assay for GBS infection. The polynucleotides can also be used as DNA sizing standards.

The polypeptides of the subject invention can be used to raise an immunogenic response to GBS. They can also be used as a molecular weight standards, or as an inert protein in an assay. The polypeptides can also be used to detect the presence of antibodies immunoreactive with GBS.

The polynucleotide sequences of the subject invention may be composed of either RNA or DNA. More preferably, the polynucleotide sequences are composed of DNA.

Materials and Methods Bacterial strains and plasmids. Isolates of group B streptococci (GBS) from the clinical laboratories of Shands Hospital, J. Hillis Miller Health Science Center, University of Florida, Gainesville, Florida were used in this study. Strain ss618 was obtained from the Centers for Disease Control (Atlanta, GA). Strain ss618C was selected for high levels of expression of the GBS beta antigen and IgA Fc binding activity as previously described (Brady et al, 1989). For use in serological tests, GBS were grown to a stationary phase in Todd-Hewitt broth (BBL Microbiology Systems, Cockeysville, MD) for 18-24 hours at 37°C. Stock cultures were stored in glycerol at — 70°C. The plasmid vector pCR™II (InVitrogen Corp., San Diego, CA) was used for cloning fragments of the GBS beta antigen gene generated using the polymerase chain reaction. Ligated pCR™π and PCR-generated beta antigen DNAs were used to transform E. coli INVαF' ("ONESHOT", InVitrogen Corp.) in accordance with the manufacturer's instructions. E. coli were grown in Luria-Bertani (LB) broth supplemented with 50 μg/ml ampicillin or kanamycin at 37°C with aeration.

Antibodies. Rabbit antibody to type la, lb, II, and DI carbohydrate antigens, as well as rabbit antibody to the c-protein marker, were provided by Dr. R. Facklam (Centers for Disease Control, Atlanta, GA). Monospecific antiserum recognizing the GBS beta antigen was prepared by selective adsorption of the anti-c protein serum with appropriate strains expressing the alpha, gamma, and delta antigens as previously

described (Brady et al, 1989). Peroxidase conjugated goat anti-rabbit IgG (whole molecule) was purchased from Cappel (Organon Teknika Corp., Westchester, PA).

Restriction endonucleases. Restriction endonucleases A vNl, BspHH, BstXl, Dram, Hindm, Kpril, Sail (New England BioLabs, Beverly, MA), Bam HI, Bgtt., BglE, EcoRV (Promega, Madison, WI), Clal (Bethesda Research Laboratories, Gaithersburg,

MD), /hπii (International Biotechnologies, Inc., New Haven, CT), and Xmril (Stratagene, La Jolla, CA) were used according to the manufacturer's instructions.

Biotinylation of human IgA. Chromatographically purified human myeloma IgA kappa was purchased from Cappel (Westchester, PA). Protein was resuspended to a concentration of 5 mg/ml in 0.01 M sodium phosphate buffer, pH 7.3. One milligram of IgA was reacted with 250 μg (10 mg/ml in dimethyl sulfoxide, Fisher Scientific, Fair Lawn, NJ) of biotin-N-hydroxysuccinimide ester (Sigma Chemical Co., St. Louis MO). The reaction was performed in 0.1 M sodium borate buffer, pH 8.8, in a 1.0 ml reaction volume. The mixture was rotated end over end for four hours at 4°C. The reaction was stopped by the addition of 20 μl of 1.0 M NH ₄ C1 and incubation at ambient temperature for 10 minutes. The uncoupled NHS-biotin was separated from the conjugated protein by passage over a column of "SΕPHADΕX" G- 25 M (PD-10, Pharmacia, Piscataway, NJ). The IgA-biotin conjugate was buffer exchanged into PBS and stored in aliquots at — 20°C. Peroxidase-avidin was purchased from Sigma Chemical Co.

Dot blot assay for detection of group B streptococcal surface and secreted antigens. All isolates used for this study were confirmed as GBS by screening with the "PHADΕBACT" streptococcus test (Pharmacia Diagnostics, Piscataway, NJ). Bacteria were typed using a modification of a previously described method (Brady et al, 1988. Briefly, the bacteria were grown to stationary phase at 37°C («18 hours) in 10 ml Todd-Hewitt broth, harvested by centrifugation (8 minutes at 100 X g), washed once with 5 ml of 0.15 M phosphate buffered saline (PBS), pH 7.4, and resuspended in 2 ml of PBS. This bacteria suspension was subjected to an additional 1:40 dilution in PBS. Culture supernatants were filtered using 0.2 micron disposable filters ("ACRODISC," Gelman Sciences, Ann Arbor, MI) and concentrated approximately 20-fold using "MINICON" Macrosolute Concentrators (Amicon, Beverly, MA). Fifty microliter samples of each GBS cell suspension and 100 μl of

each corresponding culture supernatant were dotted in duplicate onto a nitrocellulose membrane ("TRANSBLOT" transfer medium Bio-Rad Laboratories, Hercules, CA) using a "MINIFOLD I" microsample filtration manifold (Schleicher & Schuell, Keene, NH). Wells were washed twice with 200 μl of PBS and the filter removed from the apparatus. Nitrocellulose filters were blocked by washing four times (15 minutes per wash, approximately 2 ml per cm ² filter area) with PBS containing 0.25% gelatin and 0.25% "TWEEN-20" (PBS-Gel-Tw) at room temperature. Filters were then reacted for 1-3 hours with type-specific antibody (0.1 ml per cm ² ) diluted 1:500 in PBS-Gel- Tw and washed another four times with PBS-Gel-Tw as described above. Filters were probed overnight with peroxidase conjugated goat anti-rabbit IgG (0.1 ml per cm ² ) diluted 1:1000 in PBS-Gel-Tw. Filters were washed twice (15 minutes each) with PBS-Gel-Tw and twice with PBS prior to development. Reactivity was visualized by development at ambient temperature for 30 minutes in 0.1 ml per cm ² of 4-chloro-l- naphthol solution (7 ml of PBS, 1 ml of 4-chloro-l-naphthol [Sigma Chemical Co.; 3 mg/ml in ice cold methanol], and 8 microliters of 30% hydrogen peroxide [Fisher

Scientific]). Bacterial suspensions and culture supernatants were tested for reactivity with each GBS type-specific antiserum and monospecific anti-beta antiserum. All strains which demonstrated reactivity with anti-lb carbohydrate typing antiserum and/or with anti-beta antiserum were subsequently tested for IgA Fc binding activity. Dot blot assay for detection of human IgA Fc binding activity. GBS were screened for IgA Fc binding activity using the same dot blot procedure described above except that biotin-labelled human myeloma IgA kappa (1:500 dilution) was substituted for the primary antibody in the first stage of the assay and peroxidase- avidin (1:1000) was substituted for the peroxidase-conjugated secondary antibody prior to development.

Amino-terminal sequencing of truncated beta antigen polypeptides. Concentrated Todd-Hewitt broth culture supernatants containing truncated beta antigen polypeptides from GBS strains 2AR and HG806 were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described below using 0.2 M Tris, pH 8.9 as the anode buffer and 0.1 M Tris, 0.1 M Tricine, 0.1%

SDS as the cathode buffer. Proteins were transferred to a PVDF membrane ("IMMOBILON-P," Millipore Corp., Bedford, MA) by electroblotting using 20%

methanol, 10 mM MES buffer, pH 6.0 (2-[N-morpholino]ethanesulfonic acid, Sigma Chemical Co.). The membrane was stained with Coomassie Brilliant Blue and the blotted beta antigen band was excised and sequenced using an Applied Biosystems 470A Protein Sequencer (Foster City, CA). Preparation of chromosomal DNA. GBS were grown overnight at 37°C in 5 ml of Todd-Hewitt broth containing 20 mM DL-threonine. The next morning, 10 ml of fresh broth was added and the culture was grown for an additional 45 minutes. Then, 0.75 g of glycine was added and the culture grown for another 60 minutes. Cells were harvested by centrifugation at 7,500 X g for 10 minutes and resuspended in 1 ml of sterile distilled water. The cell suspension was transferred to an Eppendorf tube and the cells pelleted by centrifugation in an Eppendorf centrifuge on high speed for 3 minutes. The cells were resuspended in 0.5 ml 5 mM EDTA, 10 mM Tris, pH 8.5, containing 25% sucrose. Six microliters of RNAse (10 mg/ml) and 70 μl of lysozyme (15 mg/ml) were added and the cells were incubated at 37°C for 1 hour. Cells were lysed by the addition of 40 μl of 10% SDS and incubation for 20 minutes at room temperature. The mixture was vortexed briefly, followed by three extractions with 0.6 ml phenol/chloroform/isoamyl alcohol (25:24:1). The phases were separated by 5 minutes of low speed spinning in an Eppendorf centrifuge. Three addition extractions were performed with 0.5 ml chloroform/isoamyl alcohol (24:1) to remove residual phenol. The DNA containing aqueous phase was dialyzed overnight against

10 mM Tris, 2 mM EDTA, pH 8.0 at 4°C. DNA was precipitated by the addition of 1/10 volume 3 M sodium acetate and 2 volumes of 95% ethanol. The pellet was washed with 70% ethanol and the DNA was resuspended in sterile distilled water to a concentration of 1 mg/ml. Polvmerase chain reaction. Oligonucleotide primers employed for the PCR corresponded to base positions 121-139 (forward primer a) and 1491-1509 (forward primer c) and complementary nucleotides corresponding to base positions 1039-1057 (reverse primer b) and 3841-3859 (reverse primer d) of the previously published sequence of the gene encoding the GBS beta antigen (Jerlstrom et al., 1991). Added to the 5' ends of forward primer a and reverse primer d were restriction sequences for

BamΗI, while Sail restriction sequences were added to the 5' ends of reverse primer b and forward primer c. The positions of these oligonucleotide primers are shown

schematically in Figure 1. The PCR primer sequences with restriction sequences underlined and the beta antigen DNA shown in boldface are as follows:

Forward primer a: 5 '-GCGGATT£GCTTATGTGACAT CATC-3 '

Reverse primer b: 5 '-GCGTC JA£AACCTTΓA<7ITCGGCATC-3 '

Forward primer c: 5 '-GCGICGACCTAGAAGAGGAAGCTCAT-3 '

Reverse primer d: 5 '-GCGGATCCATCAAATGCTAGATATCG-3 '

PCR was carried out using approximately 50 to 100 ng of template DNA, 1 μm of each primer, and reagents included in the "TA CLONING KIT" (InVitrogen Corp.) according to the manufacturer's instructions. The reaction was carried out for 33 cycles using a Coy "TEMPCYCLER" (Coy, Ann Arbor, MI) with GBS strains HG806 and ss618C chromosomal DNA as templates and with the following parameters: (i) denaturation, 96°C, 30 seconds; (ii) primer annealing, 56°C, 1 minute; (iii) primer extension, 72°C, 2 minutes. An additional 5 minute primer extension step was performed after the final cycle. DNA fragments of 952 base pairs and 2,385 base pairs including the new BamΗI and Sail restriction sites were predicted to be produced from this process. Products of the PCR were analyzed by electrophoresis through 0.7% agarose to confirm their size prior to cloning directly into the pCR™II vector as described below.

Cloning of PCR-generated DNA fragments. The 952 and 2,385 base pair beta antigen gene fragments produced by PCR using HG806 and ss618C chromosomal

DNA as templates were ligated into the pCR™π vector. This vector is supplied in linear form with overlapping thymidine residues that are ligated to the overhanging adenosine residues on the DNA fragments that result from the PCR process. The ligated DNAs were used to transform E. coli INVαF' ("ONESHOT," InVitrogen Corp.) according to the manufacturer's instructions. Clones were screened by blue- white selection on LB agar supplemented with 50 μg ampicillin or kanamycin and 0.75 μg of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-GAL) per ml. White

colonies were picked, and plasmid DNA was prepared by the Mini-Prep procedure described below. Clones were screened for incorporation of appropriate sized inserts by digesting Mini-Prep DNA with BstXl. There are two -BstXl restriction endonuclease sites approximately 20 bases upstream and downstream of the TA cloning site in the pCR™II vector. There were no BstXl sites found within either of the sequenced beta antigen genes (Jerlstrom et al, 1991; Heden et al., 1991). The orientation of insert DNA with respect to the vector DNA was determined by restriction endonuclease analysis. Bglll and EcoRV were used for those clones having 952 bp inserts, while .Drαiπ, _9.spHI, and Hindm were used for those clones with 2,385 bp inserts. The following clones were chosen for use in construction of a gene encoding a non-IgA Fc binding form of the beta antigen, JB2a, AH5, AH10, and JB48. pJB2a and pAH5 represent plasmids containing 952 bp and 2,385 bp inserts derived from strain HG806, respectively; while pAHIO and pJB48 represent plasmids containing 952 bp and 2,385 bp inserts derived from ss618C, respectively. The 952 bp inserts derived from each streptococcal strain would be predicted to contain the putative promoter DNA for the beta antigen gene. The insert DNA for each of the four plasmids chosen for further work was in the opposite orientation (3' to 5') with respect to the pCR™II vector DNA (5' to 3') (see Figure 2).

Large scale preparations of each of the four plasmids were made as described below, and each plasmid was digested with Sail and TthUli restriction endonucleases.

Sail sites were engineered into one end each of the 952 and 2,385 bp inserts, while there is a single TtMHi site within the pCR™π vector at nucleotide position 1567. This is illustrated schematically in Figure 2. Digestion of pJB2a and pAHIO would be predicted to result in fragment sizes of approximately 2.2 and 2.7 kb and digestion of pAH5, and pJB48 would be predicted to result in fragment sizes of 1.2 and 5.1 kb.

Ligation of the 2.2 and 5.1 kb fragments results in an in-frame fusion of the beta antigen gene segments, as well as the reconstitution of a single copy of the pCR™II vector. Approximately 20 μg of each plasmid DNA was first digested with Sail, followed by digestion with 77AIIIi according to the manufacturer's instructions. The double digestions were run out on 0.7% agarose prep gels, the appropriate sized bands were excised, and the DNA was recovered by placing each gel slice in a "MICROFILTΕRFUGΕ" tube (Rainin, Woburn, MA), freezing at -70°C for 30

minutes, spinning on high speed in an Eppendorf centrifuge at 4°C for 30 minutes, and precipitating the DNA at — 70°C with 1/10 volume 3 M sodium acetate and 2 volumes of 95% ethanol. The 2.2 kb fragment from pJB2a was ligated with the 5.1 kb fragment from pJB48. The ligated DNAs were used to transform E. coli INVotF' according to the manufacturer's instructions. Clones were screened as described above. Mini-Prep DNAs were digested with BstXl to check for the presence of a 3,337 bp insert. This represents the fusion of the 952 bp and 2,385 bp fragments by means of the Sail site engineered into each. The following clones contained appropriate-sized DNA inserts and were selected for further analysis. p806-2 represents the fusion product of pJB2a and pAH5 plasmid DNAs, while p618-12 and p618-18 represent the fusion product of pAHIO and pJB48 plasmid DNAs. Large scale plasmid preparations of p806-2, p618-12, and p618-18 were prepared as described below and each plasmid was checked against the predicted beta antigen gene sequence by restriction enzyme analysis as described below. In addition, the insert DNA from pJB2a was subjected to DNA sequence analysis at the University of

Florida ICBR DNA Sequencing Core Facility using Ml 3 forward and reverse sequencing primers.

Preparation of plasmid DNA. Large quantities of plasmid DNA were prepared using an alkaline lysis, PEG precipitation procedure. Briefly, E. coli harboring recombinant plasmids were grown overnight at 37°C with aeration in 30 ml of Terrific

Broth (12 g/1 bacto-tryptone, 24 g/1 bacto-yeast extract, 4 ml/1 glycerol, 17 mM KH ₂ PO ₄ , 72 mM K ₂ HPO ₄ ) containing 50 μg/ml ampicillin or kanamycin. Bacterial cells were harvested by centrifugation and resuspended in 4 ml of GTE buffer (50 mM glucose, 25 mM Tris, pH 8.0, 10 mM EDTA, pH 8.0). Cells were lysed by addition of 6 ml of freshly prepared 0.2 N NaOH containing 1% SDS and incubation on ice for 5 minutes. The solution was neutralized by the addition of 6 ml of 3.0 M potassium acetate, pH 4.8, and incubation on ice for 5 minutes. Cellular debris was removed by centrifugation at 12,000 X g for 10 minutes at room temperature, and the supernatant was transferred to a clean tube. RNAse was added to a final concentration of 20 μg/ml and incubated at 37°C for 20 minutes. The supernatant was then extracted twice with 1/2 volume chloroform/isoamyl alcohol (24:1) and the aqueous phase transferred to a clean tube. Total DNA was precipitated by adding an equal

volume of 100% isopropanol and centrifugation at 12,000 X g for 10 minutes at room temperature. The DNA pellet was washed with 70% ethanol, dried under vacuum, and resuspended in 600 μl of sterile distilled water. Plasmid DNA was precipitated by first adding 160 μl of 4 M NaCl and then adding 800 μl of 13% PEG ₈₀₀₀ . After thorough mixing, the sample was incubated on ice for 20 minutes and the plasmid

DNA was pelleted by centrifugation at 12,000 X g for 15 minutes at 4°C. The pellet was washed with 70% ethanol, dried under vacuum, and resuspended to a concentration of 1 mg/ml in sterile distilled water.

Mini-Prep DNA was prepared to screen plasmid DNA isolated from white colonies by inoculating each colony into 2 ml of Terrific Broth containing 25 μg/ml of kanamycin and incubating overnight at 37°C with aeration. One and a half milliliters of each overnight culture were transferred to an Eppendorf tube and the bacteria harvested by centrifugation in an Eppendorf centrifuge for 1 minute on high speed at room temperature. The supernatant was discarded and the bacteria resuspended in 100 μl lysis buffer (8% sucrose, 10 mM Tris, pH 8.0, 50 mM EDTA, pH 8.0, and 0.5% Triton X-100). Ten microliters of fresh lysozyme (10 mg/ml) and 2 μl of RNAse (10 mg/ml) were added to the cell suspension and mixed. The cells were boiled for 30 seconds and the bacterial debris pelleted by centrifugation for 5 minutes on high speed at room temperature. The pellet was removed with a sterile toothpick and the DNA precipitated by the addition of 100 μl of isopropanol at room temperature. DNA was pelleted by centrifugation for 15 minutes on high speed at room temperature. The supernatant was decanted and the pellet dried under vacuum.

The DNA pellet was resuspended in 10 μl TE (10 mM Tris, 2 mM EDTA, pH 8.0).

Restriction endonuclease digestion of clones. Plasmids were purified from seven clones of interest: JB2a, AH5, AH10, JB48, 806-2, 618-12, and 618-18. Each plasmid was subjected to restriction endonuclease analysis with the enzymes listed below to confirm the digestion pattern predicted based on the published sequences of the beta antigen gene (Jerlstrom et al, 1991; Heden et al, 1991) and the pCR™π vector. pJB2a and pAHIO were digested with Kpril, BgR, A lwNl, Xmril, and ClallBssEl; pAH5 and pJB48 were digested with Kpril, Dram, A IwNl, and HwdUI; and p806-2, p618-12, and p618-18 were digested with Kpril, Bg , BgHI, A lwNl, and ClallBssEl.

Sodium dodecyl sulfate-poivacrylamide gel electrophoresis and Western immunoblotring. Protein samples were denatured by boiling for 5 minutes in 2% (wt/vol) sodium dodecyl sulfate, 10% glycerol (wt/vol), 0.5 M Tris-HCl, pH 6.8, 0.01% bromphenol blue. Denatured proteins were electrophoresed on 7.5% or 10% polyacrylamide gel slabs at 25 mA per slab for 1 hour by the method of Laemmli

(1970). Prestained molecular weight markers (Sigma Chemical Co.) were run in one lane of each gel for determination of estimated molecular weights. The proteins on the gels were transferred electrophoretically in nitrocellulose ("TRANSBLOT" transfer medium, Bio-Rad) by the method of Towbin et al. (1979). The gels and nitrocellulose filters were presoaked in 25 mM Tris, 192 mM glycine, 20% methanol (pH 8.3), assembled into a "TRANSBLOT SD" Semi-Dry Transfer Cell (Bio-Rad) and electrophoresed for 30 minutes at 15 V. Blots were blocked and probed with rabbit anti-beta antiserum and peroxidase goat anti-rabbit IgG or biotin-labelled human myeloma IgA kappa and peroxidase-avidin as described above for the dot blot assays. Protein samples were prepared as follows: Supernatants from GBS strains were prepared as described above for the dot blot assay. Fifteen microliters of each concentrated GBS culture supernatant was loaded per lane. Cell extracts of E. coli were prepared by growing 10 ml of bacteria overnight at 37°C with aeration in LB broth containing 50 μg/ml ampicillin or kanamycin. The bacteria were harvested by centrifugation at 2,000 X g for 10 minutes at room temperature. The cells were washed once with 5 ml of PBS and once with 1 ml of PBS. The cells were resuspended in 200 μl of SDS-sample buffer and boiled for 5 minutes. Cellular debris was removed by centrifugation at high speed in an Eppendorf centrifuge for 10 minutes. Fifty microliters of each cell extract were loaded per lane. The residual LB broth culture supernatant was concentrated approximately 40-fold as described above for GBS Todd-Hewitt broth culture supernatant and 50 μl of each concentrated E. coli culture supernatant were loaded per lane.

Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 — Identification of Beta Antigen Expression/IgA Fc Binding Activity by GBS

Fifty-three strains of group B streptococci were identified which either expressed the beta antigen or the type lb carbohydrate on their surfaces when tested by dot blot assay. All of these strains were tested for secretion of the beta antigen into culture supernatants and were screened for surface and/or secreted IgA Fc binding activity. None of these strains were shown to express the beta antigen in the absence of IgA Fc binding activity. Therefore, the previously identified GBS strains 2AR and HG806, which secrete truncated M_«55,000 IgA Fc binding and M.«38,000 non-IgA Fc binding forms of the beta antigen, respectively, in the absence of surface expression, were chosen for further characterization. The GBS strain ss618C, which expresses high levels of full-length IgA Fc binding beta antigen was also chosen as a candidate strain for cloning experiments.

Example 2 — Amino-Terminal Sequencing of Truncated Forms of the Beta Antigen

In order to determine the approximate location of IgA Fc binding activity within the GBS beta antigen protein, amino-terminal sequencing was performed on the two truncated beta antigen polypeptides secreted by GBS strains 2AR. and HG806.

The ten amino-terminal residues of the M^ 5,000 IgA Fc binding polypeptide expressed by strain 2AR. corresponds to the amino-terminal sequence predicted for the mature full-length beta antigen protein following cleavage of a thirty-seven amino acid residue signal sequence (Jerlstrom et al., 1991; Heden et al., 1991). The amino- terminal residues of the M _j «38,000 non-IgA Fc binding polypeptide expressed by strain HG806 were also the same. It is therefore reasonable to conclude that the IgA Fc binding domain of the GBS beta antigen lies within the carboxy-terminal 17,000 daltons of the polypeptide expressed by strain 2AR. Alternatively, the IgA Fc binding domain may lie at least in part within the 38,000 dalton polypeptide expressed by HG806, but the additional 17,000 daltons expressed by 2AR may be necessary to achieve the proper conformation to confer IgA Fc binding activity.

Example 3 — Construction of a Gene Encoding a Non-IgA Fc Binding Form of the GBS Beta Antigen

Oligonucleotide primers were designed so that DNA upstream and downstream of the putative IgA Fc binding domain would be amplified by the polymerase chain reaction (PCR). The location of these primers is shown in Figure 1. Also illustrated is the DNA segment which was eliminated from the beta antigen gene by this cloning strategy. Added to the 5' ends of forward primer a and reverse primer d were restriction sequences for BamEI, while Sail restriction sequences were added to the 5 ' ends of reverse primer b and forward primer c. The positions of these engineered restriction sites are also indicated in Figure 1.

Chromosomal DNA from GBS strains HG806 and ss618C were used as templates for the PCR reactions. A DNA fragment of 952 base pairs including the

BamEI and Sail restriction sites was predicted to result from the use of forward primer a and reverse primer b, while a fragment containing 2,385 base pairs was predicted to result from the use of forward primer c and reverse primer d. These two fragments were successfully generated by PCR using chromosomal DNA from both GBS strains as templates. The products of the PCR were analyzed by electrophoresis through

0.7% agarose gel to confirm their sizes prior to cloning directly into the pCR™π vector.

The 952 bp and 2,385 bp PCR-generated DNAs were ligated to the linear pCR™U vector and used to transform E. coli INVocF'. Clones were screened by blue- white selection. White colonies were picked and screened by Mini-Prep for the presence of insert DNA. Those clones containing inserts were subjected to restriction analysis with BstXl, which cuts on either side of insert DNA in the pCR™II vector. Those clones with appropriate-sized inserts, approximately 0.95 kb or 2.4 kb, were subjected to further restriction endonuclease analysis. Clones with »0.95 kb inserts were mapped with BgH and EcoRV restriction endonucleases, while clones with «2.4 kb inserts were mapped with HinUlI, Dram, and BspEI restriction endonucleases. This enabled the determination of the orientation of the insert DNA with respect to the vector DNA in each clone. Four clones were selected for further genetic manipulation. JB2 and AH10 represented the «0.95 kb clones derived from GBS strains HG806 and ss618C, respectively. AH5 and JB48 represented the «2.4 kb clones derived from GBS strains HG806 and ss618C, respectively. The insert DNA

in all four of these clones was found to be in the opposite orientation (3' to 5') as the vector DNA (5' to 3').

The strategy for ligation of the «0.95 kb and «2.4 kb DNA fragments and reconstruction of a single copy of the pCR™II vector is shown in Figure 2. Plasmid DNAs from each of the four clones were digested with both Sail and rtΛJJIi restriction endonucleases. Appropriate digestion fragments from plasmids derived from GBS strains HG806 and ss618C were purified and the «0.95 kb and «2.4 kb gene segments were ligated in frame via the Sail site engineered into one end of each. Ligation via the rtAH-i site at the other end of the restriction fragment resulted in reconstitution of an intact pCR™II vector. Restriction fragments from pJB2a («0.95 kb, HG806) and pAHIO («0.95 kb, ss618C) were ligated to pAH5 (*2.4 kb, HG806) and pJB48 («2.4 kb, ss618C), respectively. The ligated DNAs were again used to transform E. coli INVαF', and white colonies were screened by Mini-Prep and BstXl digestion for the presence of appropriate-sized inserts (3,337 base pairs). Three clones which contained «3.3 kb inserts were chosen for further study. These included 806-2, constructed by the fusion of pJB2a and pAHIO, and 618-12 and 618-18, constructed by the fusion of ρAH5 and pJB48.

Example 4 — Restriction Endonuclease Analysis of Plasmid DNA Plasmids were purified from the seven selected clones using an alkaline lysis/PEG precipitation procedure. Figure 3 shows each plasmid (except p618-12 and p618-18) digested with BstXl to separate the insert DNA from the vector DNA. The pCR™π vector contains 3,932 base pairs and has a BstXl site approximate 20 base pairs upstream and downstream of the TA cloning site. There are no BstXl sites within the beta antigen gene based on the two published sequences (Jerlstrom et al.,

1991; Heden et al, 1991). All plasmids show an «3.9 kb fragment which represents the linear vector, and either an «0.95 kb, «2.4 kb, or «3.3 kb fragment which represents cloned GBS DNA.

This figure shows that identical and appropriate sized fragments were generated by PCR using oligonucleotide primers a and b (lanes 1 and 2) or c and d (lanes 3 and

4) for both strains HG806 (lanes 1 and 3) and ss618C (lanes 2 and 4) and were successfully cloned in the pCR™II vector. The successful ligation of the «0.95 kb

and «2.4 kb PCR generated DNAs to create an «3.3 kb beta antigen gene insert is shown for the strain HG806 derived clone, 806-2, in lane 5. Identical results were observed when p618-12 and p618-18 were digested with BstXl.

For additional confirmation that the GBS DNA contained within the clones was representative of the published beta antigen gene sequences, each plasmid was analyzed using a panel of restriction endonucleases. The predicted approximate fragments sizes, based on the published sequences of the pCR™π vector and the beta antigen gene, are listed in parentheses after each enzyme name. pJB2a and pAHIO were digested with Kpril («4.9 kb), BglR («1.7 and »3.2 kb), /VNI (»2.3 and «2.5 kb), Xmril (*2.4, «0.5, and *2.0 kb), and OdUBssBB. (»1.7 and *3.2 kb). pAH5 and pJB48 were digested with Kpril («6.3 kb), Dram (»1.9, «0.07, and *4.3 kb), _4/wNT (*2.3, *2.5, and »1.5 kb), and Hinάm (*0.02, *0.02, «0.08, and «5.5 kb). p806-12, p618-12, and p618-18 were digested with Kpril («7.3 kb), BgH («2.3 and *5.0 kb), BglΩ. (*1.7 and *5.6 kb), ΛvNI (*2.3, «1.5, and *3.4 kb), and CldUBssΕΩ. (*1.7 and «5.5 kb). The predicted digestion pattern was demonstrated in each case.

Example 5 — Sequencing of GBS Insert DNA from Plasmid JB2a

In addition to restriction endonuclease analysis, one of the clones (JB2a), harboring DNA derived from GBS strain HG806, was subjected to DNA sequence analysis. Forward and reverse Ml 3 sequencing primers were employed as these sequences are engineered into the pCR™II cloning vector. The DNA sequence of the

JB2a insert DNA is shown in Figures 4A-4E. This sequence is shown aligned to the corresponding regions of the two previously-published beta antigen gene sequences

(Jerlstrom et al., 1991; Heden et al, 1991).

Example 6 — Analysis of Clones for Beta Antigen Expression and IgA Fc Binding

Activity

Cell extracts (boiling preps) and concentrated culture supernatants from each of the seven clones were tested for reactivity with rabbit anti-beta antiserum and biotin-labelled human myeloma IgA kappa by Western immunoblot analysis. Samples prepared using E. coli harboring only pCR™π vector DNA were included in these experiments as negative controls. The results (excluding p618-18) are shown in Figure

5. Molecules reactive with anti-beta antibodies were seen in the culture supernatants (Panel A) of clones JB2 (lane 1) and 806-2 (lane 3), and in the cell extracts (Panel C) of clones JB2a (lane 1), AH10 (lane 4), and 618-12 (lane 6). There was no IgA Fc binding activity observed for any of the polypeptides that reacted specifically with the anti-beta antibodies (Panels B and D). Although some non-specific IgA binding activity was observed in E. coli culture supernatants and cell extracts, the pattern of reactivity was the same in the test samples as the pCR™π negative control (lane 7) and hence cannot be attributed to the beta antigen. The pattern of reactivity observed for clone 618-18 is similar to that demonstrated for 618-12.

Example 7 — Vaccines

The novel beta antigen polypeptide described herein can be used advantageously in an immunogenic composition such as a vaccine. Such a composition, when administered to a person or animal, raises antibodies or other immune responses which reduce the susceptibility of that human or animal to GBS infection.

Vaccines comprising the beta antigen polypeptide disclosed herein, and variants thereof having antigenic or immunogenic properties, can be prepared by procedures well known in the art. For example, such vaccines can be prepared as injectables, e.g., liquid solutions or suspensions. Solid forms for solution in, or suspension in, a liquid prior to injection also can be prepared. Optionally, the preparation also can be emulsified. The active antigenic ingredient or ingredients can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Examples of suitable excipients are water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants such as aluminum hydroxide or muramyl dipeptide or variations thereof. Also, cholera toxin subunit B or other agents which stimulate antibody production at mucosal sites can be used. In the case of peptides, coupling to larger molecules such as KLH or tetanus toxoid sometimes enhances immunogenicity. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional

formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers include, for example, polyalkalene glycols or triglycerides. Suppositories can be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%, preferably about 1 to about 2%. Oral formulations can include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain from about 10% to about 95% of active ingredient, preferably from about 25% to about 70%.

The compounds can be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered can depend on the subject to be treated and the degree of protection desired. Advantageously, methods known to promote mucosal immunity can be combined with systemic immunity promoters to maximize protection against GBS. Also, the beta antigen polypeptide of the subject invention may be combined with carbohydrate antigenic components to enhance the immunogenic response and provide a broader range of protection. The combination may be, for example, through chemical coupling. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and can be peculiar to each individual. However, suitable dosage ranges are of the order of about several hundred micrograms active ingredient per individual. Suitable regimes for initial administration and booster

shots are also variable, but are typified by an initial administration followed in one or two week intervals by a subsequent injection or other administration.

Advantageously, the vaccines of the subject invention can be formulated and administered in a manner designed specifically to induce mucosal immunity.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

References

Anthony, B.F., N.F. Concepcion, S.M. Puentes, N.R. Payne (1990) "Nonimmune binding of human immunoglobulin A to type II group B streptococcus," Infect. Immun. 58:1789- 1795.

Baker, C.J., (1990) "Immunization to prevent group B streptococcal disease: Victories and vexations," J. Infect. Dis. 161:917-921.

Baker, C.J., M.S. Edwards, D.L. Kaspar (1978) "Immunogenicity of polysaccharides from type II, group B streptococci," J. Clin. Invest. 61:1107-1110.

Brady, L.J., U.D. Daphtary, E. Ayoub, M.D.P. Boyle (1988) "Two novel antigens associated with group B streptococci identified by a rapid two-stage radioimmunoassay," J. Infect. Dis. 158:965-9772.

Brady, L.J., M.D.P. Boyle (1989) "Identification of non-immunoglobulin A Fc binding forms and low molecular weight secreted forms of the group B streptococcal beta antigen," Infect. Immun. 57:1573-1581.

Chun, C.S.Y., L.J. Brady, M.D.P. Boyle, H.C. Dillon, E.M. Ayoub (1991) "Group B streptococcal C protein-associated antigens: association with neonatal sepsis," J. Infect. Dis. 163:786-791.

Cleat, P.H., K.N. Timmis (1987) "Cloning an expression in Escherichia coli of the Ibc protein genes of group B streptococci: Binding of human immunoglobulin A to the beta antigen," Infect. Immun. 55:1151-1155.

Coleman, R.T., D.N. Sherer, W.M. Maniscalco (1992) "Prevention of neonatal group B streptococcal infections: Advances in maternal vaccine development," Obstetrics and Gynecology 80:301-309.

Committee of Issues and Priorities for New Vaccine Development, Institute of Medicine (1985) "Comparisons of disease burdens and costs, and prospects for immunizing against streptococcal group B," In New Vaccine Development: Establishing Priorities. Vol. 1. Diseases of importance in the United States. Washington, D.C.: National Academy press, pp. 39-58 and 424-439.

Dillon, H.C, S. Khare, B.M. Gray (1987) "Group B streptococcal carriage and disease: a six- year prospective study," J. Pediatr. 110:31-36.

Fisher, G., R.E. Horton, R. Edelman (1983) "From the National Institute of Allergy and Infectious Diseases: Summary of the National Institutes of Health workshop on group B streptococcal infection," J. Infect. Dis. 148:163-166.

Heden, L.-O., E. Frithz, G. Lindahl (1991) "Molecular characterization of an IgA receptor from group B streptococci: sequence of the gene, identification of a proline-rich

region with unique structure and isolation of N-terminal fragments with IgA-binding capacity," Eur. J. Immunol. 21:1481-1490.

Jerlstron, P.G., G.S. Chatwall, K.N. Timmis (1991) "The IgA binding antigen of the C protein complex of group B streptococci: sequence determination of its gene and detection of two binding regions," Mol Microbiol. 5:843-849.

Lindahl, G., B. Akerstrom, J.-P. Vaerman, L. Stenber (1990) "Characterization of an IgA receptor from group B streptococci: specificity for serum IgA," Eur. J. Immunol.

20:2241-2247.

Kvam, A.I., O.-J. Iverson, L. Bevenger (1992) "Binding of human IgA to HCl-extracted C protein from group B streptococcus (GBS)," APMIS 100:1129-1132.

Madoff, L.C., J.L. Michel, E.W. Gong, A.K. Rodewald, D.L. Kaspar (1992) "Protection of neonatal mice from group B streptococcal infection by maternal immunization with beta C protein," Infect. Immun. 60:4989-4994.

Michel, J.L., L.C. Madoff, D.E. Kling, D.L. Kaspar, F.M. Ausubel (1991) "Cloned alpha and beta C protein antigens of group B streptococci elicit protective immunity," Infect. Immun. 59:2023-2028.

Michel, J.L., L.C. Madoff, K. Olson, D.E. Kling, D.L. Kaspar, F.M. Ausubel (1992) "Large, identical, tandem-repeating units in the C protein alpha antigen gene, bca, of group B streptococci," Proc. Natl. Acad. Sci. USA 89:10060-10064.

Paoletti, L.C, M.R. Wessels, F. Michon, J. DiFabio, H.J. Jennings, D.L. Kaspar (1992) "Group B streptococcus type II polysaccharide-tetanus toxoid conjugate vaccine," Infect. Immun. 60:4009-4014.

Rainard, P. (1992) "Isotype antibody response in cows to Streptococcus agalactiae group B polysaccharide-ovaϊbumin conjugate," J Clin. Microbiol. 30:1856-1862.

Russell-Jones, G.J., E.C Gotschlich (1984) "Identification of protein antigens of group B streptococci with special reference to the Ibc antigens," J. Exp. Med. 160:1476-1484.

Russell-Jones, G.J., E.C. Gotschlich, M.S. Blake (1984) "A surface receptor specific for human IgA on group B streptococci possessing the Ibc protein antigen," J. Exp. Med. 160:1467-1475.

Wessels, M.R., L.C. Paoletti, D.L. Kaspar, J.L. DiFabio, F. Michon, K. Holme, H.J. Jennings (1990) "Immunogenicity in animals of a polysaccharide-protein conjugate vaccine against type HI group B streptococcus," J. Clin. Invest. 86:1429-1433.

Wessels, M.R., L.C Paoletti, A.K. Rodewald, F. Michon, J. DiFabio, HJ. Jennings, D.L. Kaspar (1993) "Stimulation of protective antibodies against la and lb group B streptococci by a type la polysaccharide-tetanus toxoid conjugate vaccine," Infect. Immun. 61:4760-4766.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT INFORMATION:

Applicant Name(S): UNIVERSITY OF FLORIDA

Street address: 186 Grinter Hall

City: Gainesville state/Province: Florida country: us

Postal code/Zip: 32611

Phone number: 904-392-8929 Fax number: 904-392-6600 Telex number:

(ii) TITLE OF INVENTION: Cloning of Non-IgA Fc Binding Forms of the Group B Streptococcal Beta Antigens

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Saliwanchik & Saliwanchik

(B) STREET: 2421 N.W. 41st Street, Suite A-l

(C) CITY: Gainesville

(D) STATE: FL

(E) COUNTRY: USA

(F) ZIP: 32606

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patent n Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/242,932

(B) FILING DATE: 16-MAY-1994

(C) CLASSIFICATION:

( iii) ATTORNE /AGENT INFORMATION:

(A) NAME: Saliwanchik, David R.

(B) REGISTRATION NUMBER: 31,794

(C) REFERENCE/DOCKET NUMBER: UF142

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 904-375-8100

(B) TELEFAX: 904-372-5800

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4200 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: AAGCTTATGC TTGTCAATAA TCACAAATTT GTAGATCACT TCCTTTTTAG GACTGTAAAG 60 CATCCTAATT ACTTTTTAAA TATATTACCA GAACTAGTTG GTTTGGCCCT GGTGAGTCAT 120

RECTIFIED SHEET (RULE 91) ISA/EP

GCTTATGTGA CATTCATCTT TATTTTTCCT GTCTATGCGG TTATTCTTTA TCAAAGAATA 180

GCAGAGGAAG AAAAATTATT GCAGGAAGTT ATTATTCCGA ATGGAAGAAT GAAAGGTTAA 240

AAATAATATA CCCAATTTAA TATGCAGTTC ATATTGGAAG GGTATACTGT AGATAAATAA 300

AATATTGGAG GATATCGATA TGTTTAAATC TAATTATGAA AGAAAAATGC GTTATTCCAT 360

TCGTAAATTT AGTGTAGGAG TAGCTAGTGT AGCGGTAGCT AGTTTGTTCA TGGGAAGCGT 420

TGCTCATGCA AGTGAGCTTG TAAAGGACGA TAGTGTGAAG ACTACCGAGG TTGCAGCTAA 480

GCCCTATCCA AGTATGGCTC AAACAGATCA AGGAAATAAT TCATCATCCT CGGAACTTGA 540

GACAACAAAG ATGGAAATTC CTACAACAGA CATAAAAAAA GCTGTTGAAC CGGTCGAGAA 600

AACAGCTGGG GAAACATCTG CCACTGATAC TGGAAAACGA GAGAAACAAT TACAACAATG 660

GAAAAATAAT CTAAAAAATG ATGTGGATAA CACAATTCTA TCTCATGAAC AGAAAAATGA 720

GTTTAAAACA AAAATTGATG AAACAAATGA TTCTGATGCA TTATTAGAAT TAGAAAATCA 780

ATTTAACGAA ACTAATAGAC TGTTACACAT CAAACAACAT GAAGAAGTTG AGAAAGATAA 840

GAAAGCTAAG CAACAGAAAA CTCTGAAACA GTCAGATACG AAAGTAGATC TAAGCAATAT 900

TGACAAAGAG CTTAATCATC AAAAAAGTCA AGTTGAAAAA ATGGCAGAGC AAAAGGGAAT 960

CACAAATGAA GATAAAGATT CTATGCTGAA AAAAATCGAA GATATTCGTA AACAAGCTCA 1020

ACAAGCAGAT AAAAAAGAAG ATGCCGAAGT AAAGGTTCGT GAAGAACTAG GTAAACTCTT 1080

TAGTTCAACT AAAGCTGGTC TGGATCAAGA AATTCAAGAG CATGTGAAGA AAGAAACGAG 1140

TAGTGAGGAA AATACTCAGA AAGTTGATGA ACACTATGCT AATAGCCTTC AGAACCTTGC 1200

TCAAAAATCT CTTGAAGAAC TAGATAAGGC AACTACCAAT GAACAAGCTA CACAAGTTAA 1260

AAATCAATTC TTAGAAAACG CTCAAAAGCT CAAAGAAATA CAACCTCTTA TCAAAGAAAC 1320

GAATGTGAAA TTGTATAAGG CTATGAGTGA GAGCTTGGAG CAGGTTGAGA AGGAATTAAA 1380

ACATAATTCG GAAGCTAATT TAGAAGATTT GGTTGCGAAA TCTAAAGAAA TCGTAAGAGA 1440

ATACGAAGGA AAACTTAATC AATCTAAAAA TCTTCCAGAA TTAAAGCAAC TAGAAGAGGA 1500

AGCTCATTCG AAGTTGAAAC AAGTTGTGGA GGATTTTAGA AAAAAATTTA AAACGTCAGA 1560

GCAAGTGACA CCAAAAAAAC GTGTCAAACG AGATTTAGCT GCTAATGAAA ATAATCAACA 1620

AAAGATTGAG TTAACAGTTT CACCAGAGAA TATCACTGTA TATGAAGGTG AAGACGTGAA 1680

ATTTACAGTC ACAGCTAAAA GTGATTCGAA GACGACGTTG GACTTCAGTG ATCTTTTAAC 1740

AAAATATAAT CCGTCTGTAT CAGATAGAAT TAGTACAAAT TATAAGACTA ACACGGATAA 1800

TCATAAGATT GCCGAAATCA CTATCAAGAA TTTGAAGCTA AATGAAAGTC AAACAGTGAC 1860

TCTAAAAGCT AAAGATGATT CTGGCAATGT AGTTGAAAAA ACATTCACTA TTACAGTGCA 1920

AAAGAAAGAG GAGAAACAAG TTCCTAAAAC ACCAGAGCAG AAAGATTCTA AAACGGAAGA 1980

AAAGGTTCCT CAAGAACCAA AATCAAATGA CAAGAATCAA TTACAAGAGT TGATTAAATC 2040

AGCTCAACAA GAACTGGAAA AGTTAGAAAA AGCAATAAAA GAATTAATGG AGCAACCAGA 2100

GATTCCATCC AATCCAGAGT ATGGTATTCA AAAATCTATT TGGGAGTCAC AAAAAGAGCC 2160

TATCCAGGAA GCCATAACAA GTTTTAAGAA GATTATTGGT GATTCATCTT CAAAATACTA 2220

CACAGAGCAC TATTTTAACA AATATAAATC TGATTTTATG AATTATCAAC TTCATGCACA 2280

AATGGAGATG CTGACTAGAA AAGTGGTTCA GTATATGAAC AAATATCCTG ATAATGCAGA 2340

AATTAAAAAG ATATTTGAGT CAGATATGAA GAGAACGAAA GAAGATAATT ACGGAAGTTT 2400

AGAAAATGAT GCTTTGAAAG GCTATTTTGA GAAATATTTC CTTACACCAT TTAATAAAAT 2460

TAAGCAGATT GTAGATGATT TGGATAAAAA AGTAGAACAA GATCAGCCAG CACCAATTCC 2520

GGAAAATTCA GAAATGGATC AGGCTAAGGA AAAGGCTAAG ATTGCTGTAT CGAAGTATAT 2580

GAGTAAGGTT TTAGATGGAG TTCATCAACA TCTGCAGAAG AAAAATAACA GTAAAATTGT 2640

TGATCTTTTT AAGGAACTTG AAGCGATTAA ACAACAAACT ATTTTTGATA TTGACAATGC 2700

AAAGACTGAA GTAGAGATTG ATAACTTAGT ACACGATGCA TTCTCAAAAA TGAATGCTAC 2760

TGTTGCTAAA TTTCAAAAAG GTCTAGAGAC AAATACGCCA GAAACTCCAG ATACACCGAA 2820

GATTCCAGAG CTACCTCAAG CCCCAGATAC ACCGCAGGCT CCAGACACAC CGCATGTTCC 2880

GGAATCACCA AAGGCCCCAG AAGCACCGCG TGTTCCGGAA TCACCAAAGA CTCCAGAAGC 2940

ACCGCATGTT CCGGAATCAC CAAAGGCCCC AGAAGCACCG CGTGTTCCGG AATCACCAAA 3000

GACTCCAGAA GCACCGCATG TTCCGGAATC ACCAAAGACT CCAGAAGCAC CAAAGATTCC 3060

GGAACCCCCT AAGACTCCAG ACGTCCCTAA GCTTCCAGAC GTCCCTAAGC TTCCAGACGT 3120

CCCTAAGCTT CCAGATGCAC CGAAGTTACC AGATGGGTTA AATAAAGTTG GACAAGCAGT 3180

ATTTACATCA ACTGATGGAA ATACTAAGGT TACGGTTGTA TTTGATAAAC CTACAGATGC 3240

TGATAAGTTA CATCTCAAGG AAGTAACGAC GAAAGAGTTG GCTGATAAAA TTGCTCATAA 3300

AACAGGAGGA GGAACAGTTC GTGTGTTTGA CTTATCTCTT TCTAAAGGAG GCAAGGAAAC 3360

ACATGTCAAT GGAGAACGAA CTGTTCGGCT CGCGCTTGGG CAGACTGGCT CAGATGTTCA 3420

CGTCTATCAC GTAAAGGAAA ATGGCGACCT TGAGCGTATT CCTTCTAAAG TTGAAAATGG 3480

GCAAGTTGTT TTTAAAACGA ACCACTTCAG TTTGTTTGCG ATTAAGACAC TTTCTAAGGA 3540

TCAAAATGTT ACTCCACCGA AGCAGACTAA ACCTTCTACC CAAGGCAGTC AAGTAGAGAT 3600

TGCAGAGAGT CAAACTGGAA AATTCCAGAG TAAAGCAGCT AATCATAAAG CACTGGCTAC 3660

TGGAAATGAA ACAGTGGCAA AAGGAAATCC TACATCAACA ACGGAAAAGA AATTGCCATA 3720

TACAGGAGTG GCATCTAATC TAGTTCTTGA AATTATGGGT CTCCTTGGTT TGATTGGAAC 3780

TTCATTCATC GCAATGAAAA GAAGAAAATC ATGATTCAGT TTTTTAAAAA TATCCACTTT 3840

CGATATCTAG CATTTGATTG GTTATCTGTG GATGATTCTA AAGATGTTAC CTATCGTTGG 3900

TATGTAACAA TTATAAGTCA TTTCATATAA AAGAGGCTCT TTGTCAACTG TAGTTGGTTG 3960

AAACAAGGCT ACAAACTAGA AAGGACGCAT TTTGTCCTTT CTTTTTGATG TTGAGGGCAA 4020

TGAAAATACG CTTTTTGAAG TTTTCAAAAT TCCGAAAACT AAAGATATTG TATTTGAAAA 4080

GTTTAATGAG ATGATTAGTT GCTTCCAATT TTGCGTTGGA GTAGGTTTAC TGAAGGACGT 4140

TGACGATATT CTCTTTGCTT TTGAGAATGA TTTTAAAGAT AGTCTGAAAA AGAGGATGAA 4200

(2) INFORMATION FOR SEQ ID Nθ:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 984 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( i) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Ser Glu Leu Val Lys Asp Asp ser Val Lys Thr Thr Glu Val Ala Ala 1 5 10 15

Lys Pro Tyr Pro ser Met Ala Gin Thr Asp Gin Gly Asn Asn Ser ser 20 25 30

Ser Ser Glu Leu Glu Thr Thr Lys Met Glu ie Pro Thr Thr Asp lie 35 40 45

Lys Lys Ala Val Glu Pro Val Glu Lys Thr Ala Gly Glu Thr ser Ala 50 55 60

Thr Asp Thr Gly Lys Arg Glu Lys Gin Leu Gin Gin Trp Lys Asn Asn 65 70 75 80

Leu Lys Asn Asp Val Asp Asn Thr lie Leu Ser His Glu Gin Lys Asn 85 90 95

Glu Phe Lys Thr Lys lie Asp Glu Thr Asn Asp Ser Asp Ala Leu Leu 100 105 110

Glu Leu Glu Asn Gin Phe Asn Glu Thr Asn Arg Leu Leu His lie Lys 115 120 125

Gin His Glu Glu Val Glu Lys Asp Lys Lys Ala Lys Gin Gin Lys Thr 130 135 140

Leu Lys Gin Ser Asp Thr Lys Val Asp Leu Ser Asn lie Asp Lys Glu 145 150 155 160

Leu Asn His Gin Lys ser Gin Val Glu Lys Met Ala Glu Gin Lys Gly 165 170 175 lie Thr Asn Glu Asp Lys Asp Ser Met Leu Lys Lys lie Glu Asp ie 180 185 190

Arg Lys Gin Ala Gin Gin Ala Asp Lys Lys Glu Asp Ala Glu Val Lys 195 200 205

Val Gin Leu Glu Glu Glu Ala His ser Lys Leu Lys Gin Val Val Glu 210 215 220

Asp Phe Arg Lys Lys Phe Lys Thr Ser Glu Gin Val Thr Pro Lys Lys 225 230 235 240

Arg Val Lys Arg Asp Leu Ala Ala Asn Glu Asn Asn Gin Gin Lys lie 245 250 255

Glu Leu Thr Val Ser Pro Glu Asn ie Thr Val Tyr Glu Gly Glu Asp 260 265 270

Val Lys Phe Thr Val Thr Ala Lys Ser Asp Ser Lys Thr Thr Leu Asp 275 280 285

Phe Ser Asp Leu Leu Thr Lys Tyr Asn Pro Ser Val Ser Asp Arg lie 290 295 300

ser Thr Asn Tyr Lys Thr Asn Thr Asp Asn His Lys He Ala Glu He 305 310 315 320

Thr He Lys Asn Leu Lys Leu Asn Glu Ser Gin Thr Val Thr Leu Lys 325 330 335

Ala Lys Asp Asp Ser Gly Asn Val Val Glu Lys Thr Phe Thr He Thr 340 345 350

Val Gin Lys Lys Glu Glu Lys Gin Val Pro Lys Thr Pro Glu Gin Lys 355 360 365

Asp Ser Lys Thr Glu Glu Lys Val Pro Gin Glu Pro Lys Ser Asn Asp 370 375 380

Lys Asn Gin Leu Gin Glu Leu He Lys ser Ala Gin Gin Glu Leu Glu 385 390 395 400

Lys Leu Glu Lys Ala He Lys Glu Leu Met Glu Gin Pro Glu He Pro 405 410 415

Ser Asn Pro Glu Tyr Gly He Gin Lys ser He Trp Glu Ser Gin Lys 420 425 430

Glu Pro He Gin Glu Ala He Thr ser Phe Lys Lys He He Gly Asp 435 440 445

Ser ser Ser Lys Tyr Tyr Thr Glu His Tyr Phe Asn Lys Tyr Lys Ser 450 455 460

Asp Phe Met Asn Tyr Gin Leu His Ala Gin Met Glu Met Leu Thr Arg 465 470 475 480

Lys Val Val Gin Tyr Met Asn Lys Tyr Pro Asp Asn Ala Glu He Lys 485 490 495

Lys He Phe Glu Ser Asp Met Lys Arg Thr Lys Glu Asp Asn Tyr Gly 500 505 510

Ser Leu Glu Asn Asp Ala Leu Lys Gly Tyr Phe Glu Lys Tyr Phe Leu 515 520 525

Thr Pro Phe Asn Lys He Lys Gin He val Asp Asp Leu Asp Lys Lys 530 535 540

Val Glu Gin Asp Gin Pro Ala Pro He Pro Glu Asn Ser Glu Met Asp 545 550 555 560

Gin Ala Lys Glu Lys Ala Lys He Ala Val Ser Lys Tyr Met Ser Lys 565 570 575

Val Leu Asp Gly Val His Gin His Leu Gin Lys Lys Asn Asn Ser Lys 580 585 590 lie Val Asp Leu Phe Lys Glu Leu Glu Ala He Lys Gin Gin Thr He 595 600 605

Phe Asp He Asp Asn Ala Lys Thr Glu Val Glu He Asp Asn Leu Val 610 615 620

His Asp Ala Phe Ser Lys Met Asn Ala Thr Val Ala Lys Phe Gin Lys 625 630 635 640

Gly Leu Glu Thr Asn Thr Pro Glu Thr Pro Asp Thr Pro Lys He Pro 645 650 655

Glu Leu Pro Gin Ala Pro Asp Thr Pro Gin Ala Pro Asp Thr Pro His 660 665 670

Val Pro Glu Ser Pro Lys Ala Pro Glu Ala Pro Arg Val Pro Glu Ser 675 680 685

Pro Lys Thr Pro Glu Ala Pro His Val Pro Glu ser Pro Lys Ala Pro 690 695 700

Glu Ala Pro Arg Val Pro Glu Ser Pro Lys Thr Pro Glu Ala Pro His 705 710 715 720

Val Pro Glu Ser Pro Lys Thr Pro Glu Ala Pro Lys He Pro Glu Pro 725 730 735

Pro Lys Thr Pro Asp Val Pro Lys Leu Pro Asp val Pro Lys Leu Pro 740 745 750

Asp Val Pro Lys Leu Pro Asp Ala Pro Lys Leu Pro Asp Gly Leu Asn 755 760 765

Lys Val Gly Gin Ala Val Phe Thr ser Thr Asp Gly Asn Thr Lys Val 770 775 780

Thr Val Val Phe Asp Lys Pro Thr Asp Ala Asp Lys Leu His Leu Lys 785 790 795 800

Glu Val Thr Thr Lys Glu Leu Ala Asp Lys He Ala His Lys Thr Gly 805 810 815

Gly Gly Thr Val Arg Val Phe Asp Leu Ser Leu Ser Lys Gly Gly Lys 820 825 830

Glu Thr His Val Asn Gly Glu Arg Thr Val Arg Leu Ala Leu Gly Gin 835 840 845

Thr Gly ser Asp Val His Val Tyr His Val Lys Glu Asn Gly Asp Leu 850 855 860

Glu Arg He Pro Ser Lys val Glu Asn Gly Gin val Val Phe Lys Thr 865 870 875 880

Asn His Phe Ser Leu Phe Ala He Lys Thr Leu ser Lys Asp Gin Asn 885 890 895

Val Thr Pro Pro Lys Gin Thr Lys Pro ser Thr Gin Gly ser Gin Val 900 905 910

Glu He Ala Glu Ser Gin Thr Gly Lys Phe Gin ser Lys Ala Ala Asn 915 920 925

His Lys Ala Leu Ala Thr Gly Asn Glu Thr Val Ala Lys Gly Asn Pro 930 935 940

Thr Ser Thr Thr Glu Lys Lys Leu Pro Tyr Thr Gly Val Ala ser Asn 945 950 955 960

Leu Val Leu Glu He Met Gly Leu Leu Gly Leu lie Gly Thr ser Phe 965 970 975

He Ala Met Lys Arg Arg Lys Ser 980

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GCGGATCCGC TTATGTGACA TTCATC 26

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GCGTCGACAA CCTTTACTTC GGCATC 26

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GCGTCGACCT AGAAGAGGAA GCTCAT 26

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCGGATCCAT CAAATGCTAG ATATCG 26

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 932 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTTATGTACA TTCATCTTTA TTTTTCCTGT CTATGCGGTT ATTCTTTATC AAAGAATAGC 60

AGAGGAAGAA AAATTATTGC AGGAAGTTAT TATTCCGAAT GGAAGAATGA AAGGTTAAAA 120

ATAATATACC CAATTTAATA TGCAGTTCAT ATTGGAAGGG TATACTGTAG ATAAATAAAA 180

TATTGGGGAT ATCGATATGT TTAAATCTAA TTATGAAAGA AAAATGCGTT ATTCCATTCG 240

TAAATTTAGT GTAGGAGTAG CTAGTGTAGC GGTAGCTAGT TTATTCATGG GAAGCGTTGC 300

TCATGCAAGT GAGCTTGTAA AGGACGATAG TGTGAAGACT ACCGAGGTTG CAGCTAAGCC 360

CTATCCAAGT ATGGCTCAAA CAGATCAAGG AAATAATTCA TCATCCTCGG AACTTGAGAC 420

AACAAAGATG GAAATTCCTA CAACAGACAT AAAAAAAGCT GTTGAACCGG TCGAGAAAAC 480

AGCTGGGGAA ACATCTGCCA CTGATACTGG AAAACGAGAG AAACAATTAC AACAATGGAA 540

AAATAATCTA AAAAATGATG TGGATAACAC AATTCTATCT CATGAACAGA AAAATGAGTT 600

TAAAACAAAA ATTGATGAAA CAAATGATTC TGATGCATTA TTAGAATTAG AAAATCAATT 660

TAACGAAACT AATAGACTGT TACACATCAA ACAACATGAA GAAGTTGAGA AAGATAAGAA 720

AGCTAAGCAA CAGAAAACTC TGAAACAGTC AGATACGAAA GTAGATCTAA GCAATATTGA 780

CAAAGAGCTT AATCATCAAA AAAGTCAAGT TGAAAAAATG GCAGAGCAAA AGGGAATCAC 840

AAATGAAGAT AAAGATCTAT GCTGAAAAAA ATCGAAGATA TTCGTAAACA AGCTCAACAA 900

GCAGATAAAA AAGAGATGCC GAAGTAAAGG TT 932

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3730 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genoris)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

AAAAATTATT GCAGGAAGTT ATTATTCCGA ATGGAAGAAT GAAAGGTTAA AAATAATATA 60

CCCAATTTAA TATGCAGTTC ATATTGGAAG GGTATACTGT AGATAAATAA AATATTGGAG 120

GATATCGATA TGTTTAAATC TAATTATGAA AGAAAAATGC GTTATTCCAT TCGTAAATTT 180

AGTGTAGGAG TAGCTAGTGT AGCGGTACGT AGTTTGTTCA TGGGAAGCGT TGCTCATGCA 240

AGTGAGCTTG TAAAGGACGA TAGTGTGAAG ACTACCGAGG TTGCAGCTAA GCCCTATCCA 300

AGTATGGCTC AAACAGATCA AGGAAATAAT TCATCATCCT CGGAACTTGA GACAACAAAG 360

ATGGAAATTC CTACAACAGA CATAAAAAAA GCTGTTGAAC CGGTCGAGAA AACAGCTGGG 420

GAAACATCTG CCACTGATAC TGGAAAACGA GAGAAACAAT TACAACAATG GAAAAATAAT 480

CTAAAAAATG ATGTGGATAA CACAATTCTA TCTCATGAAC AGAAAAATGA GTTTAAAACA 540

AAAATTGATG AAACAAATGA TTCTGATGCA TTATTAGAAT TAGAAAATCA ATTTAACGAA 600

ACTAATAGAC TGTTACACAT CAAACAACAT GAAGAAGTTG AGAAAGATAA GAAAGCTAAG 660

CAACAGAAAA CTCTGAAACA GTCAGATACG AAAGTAGATC TAAGCAATAT TGACAAAGAG 720

CTTAATCATC AAAAAAGTCA AGTTGAAAAA ATGGCAGAGC AAAAGGGAAT CACAAATGAA 780

GATAAAGATT CTATGCTGAA AAAAATCGAA GATATTCGTA AACAAGCTCA ACAAGCAGAT 840

AAAAAAGAAG ATGCCGAAGT AAAGGTTCGT GAAGAACTAG GTAAACTCTT TAGTTCAACT 900

AAAGCTGGTC TGGATCAAGA AATTCAAGAG CATGTGAAGA AAGAAACGAG TAGTGAGGAA 960

AATACTCAGA AAGTTGATGA ACACTATGCT AATAGCCTTC AGAACCTTGC TCAAAAATCT 1020

CTTGAAGAAC TAGATAAGGC AACTACCAAT GAACAAGCTA CACAAGTTAA AAATCAATTC 1080

TTAGAAAACG CTCAAAAGCT CAAAGAAATA CAACCTCTTA TCAAAGAAAC GAATGTGAAA 1140

TTGTATAAGG CTATGAGTGA GAGCTTGGAG CAGGTTGAGA AGGAATTAAA ACATAATTCG 1200

GAAGCTAATT TAGAAGATTT GGTTGCGAAA TCTAAAGAAA TCGTAAGAGA ATACGAAGGA 1260

AAACTTAATC AATCTAAAAA TCTTCCAGAA TTAAAGCAAC TAGAAGAGGA AGCTCATTCG 1320

AAGTTGAAAC AAGTTGTGGA GGATTTTAGA AAAAAATTTA AAACGTCAGA GCAAGTGACA 1380

CCAAAAAAAC GTGTCAAACG AGATTTAGCT GCTAATGAAA ATAATCAACA AAAGATTGAG 1440

TTAACAGTTT CACCAGAGAA TATCACTGTA TATGAAGGTG AAGACGTGAA ATTTACAGTC 1500

ACAGCTAAAA GTGATTCGAA GACGACGTTG GACTTCAGTG ATCTTTTAAC AAAATATAAT 1560

CCGTCTGTAT CAGATAGAAT TAGTACAAAT TATAAGACTA ACACGGATAA TCATAAGATT 1620

GCCGAAATCA CTATCAAGAA TTTGAAGCTA AATGAAAGTC AAACAGTGAC TCTAAAAGCT 1680

AAAGATGATT CTGGCAATGT AGTTGAAAAA ACATTCACTA TTACAGTGCA AAAGAAAGAG 1740

GAGAAACAAG CAAGAACCAA AATCAAATGA CAAGAATCAA TTACAAGAGT TGATTAAATC 1800

AGCTCAACAA GAACTGGAAA AGTTAGAAAA AGCAATAAAA GAATTAATGG AGCAACCAGA 1860

GATTCCATCC AATCCAGAGT ATGGTATTCA AAAATCTATT TGGGAGTCAC AAAAAGAGCC 1920

TATCCAGGAA GCCATAACAA GTTTTAAGAA GATTATTGGT GATTCATCTT CAAAATACTA 1980

CACAGAGCAC TATTTTAACA AATATAAATC TGATTTTATG AATTATCAAC TTCATGCACA 2040

AATGGAGATG CTGACTAGAA AAGTGGTTCA GTATATGAAC AAATATCCTG ATAATGCAGA 2100

AATTAAAAAG ATATTTGAGT CAGATATGAA GAGAACGAAA GAAGATAATT ACGGAAGTTT 2160

AGAAAATGAT GCTTTGAAAG GCTATTTTGA GAAATATTTC CTTACACCAT TTAATAAAAT 2220

TAAGCAGATT GTAGATGATT TGGATAAAAA AGTAGAACAA GATCAGCCAG CACCAATTCC 2280

GGAAAATTCA GAAATGGATC AGGCTAAGGA AAAGGCTAAG ATTGCTGTAT CGAAGTATAT 2340

GAGTAAGGTT TTAGATGGAG TTCATCAACA TCTGCAGAAG AAAAATCACA GTAAAATTGT 2400

TGATCTTTTT AAGGAACTTG AAGCGATTAA ACAACAAACT ATTTTTGATA TTGACAATGC 2460

AAAGACTGAA GTAGAGATTG ATAACTTAGT ACACGATGCA TTCTCAAAAA TGAATGCTAC 2520

TGTTGCTAAA TTTCAAAAAG GTCTAGAGAC AAATACGCCA GAAACTCCAG ATACACCGAA 2580

GATTCCAGAG CTACCTCAAG CCCCAGATAC ACCGCAGGCT CCAGACACAC CGCATGTTCC 2640

GGAATCACCA AAGGCCCCAG AAGCACCGCG TGTTCCGGAA TCACCAAAGA CTCCAGAAGC 2700

ACCGCATGTT CCGGAATCAC CAAAGACTCC AGAAGCACCA AAGATTCCGG AACCCCCTAA 2760

GACTCCAGAC GTCCCTAAGC TTCCAGACGT CCCTAAGCTT CCAGATGCAC CGAAGTTACC 2820

AGATGGGTTA AATAAAGTTG GACAAGCAGT ATTTACATCA ACTGATGGAA ATACTAAGGT 2880

TACGGTTGTA TTTGATAAAC CTACAGATGC TGATAAGTTA CATCTCAAGG AAGTAACGAC 2940

GAAAGAGTTG GCTGATAAAA TTGCTCATAA AACAGGAGGA GGAACAGTTC GTGTGTTTGA 3000

CTTATCTCTT TCTAAAGGAG GCAAGGAAAC ACATGTCAAT GGAGAACGAA CTGTTCGGCT 3060

CGCGCTTGGG CAGACTGGCT CAGATGTTCA CGTCTATCAC GTAAAGGAAA ATGGCGACCT 3120

TGAGCGTATT CCTTCTAAAG TTGAAAATGG GCAAGTTGTT TTTAAAACGA ACCACTTCAG 3180

TTTGTTTGCG ATTAAGACAC TTTCTAAGGA TCAAAATGTT ACTCCACCGA AGCAGACTAA 3240

ACCTTCTACC CAAGGCAGTC AAGTAGAGAT TGCAGAGAGT CAAACTGGAA AATTCCAGAG 3300

TAAAGCAGCT AATCATAAAG CACTGGCTAC TGGAAATGAA ACAGTGGCAA AAGGAAATCC 3360

TACATCAACA ACGGAAAAGA AATTGCCATA TACAGGAGTG GCATCTAATC TAGTTCTTGA 3420

AATTATGGGT CTCCTTGGTT TGATTGGAAC TTCATTCATC GCAATGAAAA GAAGAAAATC 3480

ATGATTCAGT TTTTTAAAAA TATCCACTTT CGATATCTAG CATTTGATTG GTTATCTGTG 3540

GATGATTCTA AAGATGTTAC CTATCGTTGG TATGTAACAA TTATAAGTCA TTTCATATAA 3600

AAGAGGCTCT TTGTCAACTG TAGTTGGTTG AAACAACGTA CAAACTAGAA AGGACGCATT 3660

TTGTCCTTTC TTTTTGATGT TGAGGGCAAT GAAAATACGC TTTTTGAAGT TTTCAAAATT 3720

CCGAAAACTA 3730