AU2002304016B2 - Outer surface proteins, their genes, and their use - Google Patents

Description

Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE

SPECIFICATION

DIVISIONAL

PATENT

APPLICANT:

Invention Title: Microscience Limited OUTER SURFACE PROTEINS, THEIR GENES, AND THEIR USE The following statement is a full description of this invention, including the best method of performing it known to me: PACommonWord97\1 1501-12000\1 1822mic\20021 127.doc WO 00/37490 PCT/GB99/04376 OUTER SURFACE PROTEINS, THEIR GENES, AND THEIR USE Field of the Invention This invention relates to the identification of outer surface proteins, their genes, and their use. More particularly, it relates to their use in therapy, for immunisation and in screening for drugs.

Background to the Invention Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is the causative agent of various conditions. In particular, GBS causes: Early onset neonatal infection.

This infection usually begins in utero and causes severe septicaemia and pneumonia in infants, which is lethal if untreated and even with treatment is associated with a 10-20% mortality rate.

Late onset neonatal infection.

This infection occurs in the period shortly after birth until about 3 months of age. It causes a septicaemia, which is complicated by meningitis in 90% of cases. Other focal infections also occur including osteomyelitis, septic arthritis, abscesses and -endopthalmitis.

Adult infections.

These appear to be increasingly common and occur most frequently in women who have just delivered a baby, the elderly and the immunocompromised. They are characterised by septicaemia and focal infections including osteomyelitis, septic arthritis, abscesses and endopthalmitis.

Urinary tract infections.

GBS is a cause of urinary tract infections and in pregnancy accounts for about 10% of all infections.

Veterinary infections.

GBS causes chronic mastitis in cows. This, in turn, leads to reduced milk production and is therefore of considerable economic importance.

WO 00/37490 PCT/G B99/04376 2 GBS infections can be treated with antibiotics.

However, immunisation is preferable. It is therefore desirable to develop an immunogen that could be used in a therapeutically-effective vaccine.

Summary of the Invention The present invention is based on the identification of a series of genes in GBS, and also related organisms, the products of which may be associated with the outer surface of the organism. and may therefore be useful as targets for immunotherapy.

According to one aspect of the invention, a peptide is encoded by an operon including any of the genes identified herein as MS4, MS10, MS11, MS14 and MS16, obtainable from Group B Streptococcus, or a homologue or functional fragment thereof. Such a peptide is suitable for therapeutic use, e.g. when isolated.

The term "functional fragments" is used herein to define a part of the gene or peptide which retains the activity of the whole gene or peptide. For example, a functional fragment of the peptide may be used as an antigenic determinant, useful in a vaccine or in the production of antibodies.

A gene fragment may be used to encode the active peptide. Alternatively, the gene fragment may have utility in gene therapy, targetting the wild-type gene in vivo to exert a therapeutic effect.

A peptide according to the present invention may comprise any of the amino acid sequences identified herein as SEQ ID NOS. 2, 4, 6, 8, 10 and 12, or a functional fragment thereof.

Because of the extracellular or cell surface location, the peptides of the present invention may be suitable candidates for the production of therapeutically-effective vaccines against GBS. The term "therapeutically-effective" is intended to include the prophylactic effect of vaccines.

For example, a vaccine may comprise a peptide according to WO 00/37490 PCT/GB99/04376 3 the invention, or the means for its expression, for the treatment of infection.

This vaccine may be administered to females either prior to or during pregnancy to protect mother and neonate against infection by GBS.

According to another aspect of the invention, the peptides or genes may be used for screening potential antimicrobial drugs or for the detection of virulence.

A further aspect of this invention is the use of any of the products identified herein, for the treatment or prevention of a condition associated with infection by a Group B Streptococcal strain.

Although the protein has been described for use in the treatment of patients, veterinary uses of the products of the invention are also considered to be within the scope of the present invention. In particular, the peptides or the vaccines may be used in the treatment of chronic mastitis, especially in cows.

Description of the Invention The present invention is described with reference to Group B Streptococcal strain M732. However, all the GBS strains and many other bacterial strains are likely to -include related peptides or proteins having amino acid sequence homology with the peptide of M732. Organisms likely to contain the peptides include, but are not limited to, S. pneumoniae, S. pyogenes, S. suis, S. milleri, Group C and Group G Streptococci and Enterococci. Vaccines to each of these may be developed in the same way as described for GBS.

Preferably, the peptides that may be useful for the production of vaccines have greater than 40% sequence similarity with the peptides identified herein. More preferably, the peptides have greater than 60% sequence similarity. Most preferably, the peptides have greater than 80% sequence similarity, e.g. 95% similarity.

Having characterised a gene according to the invention, it is possible to use the gene sequence to WO 00/37490 PCT/GB99/04376 4 establish homologies in other microorganisms. In this way it is possible to determine whether other microorganisms have similar outer surface products. Sequence homologies may be established by searching in existing databases, e.g.

EKBL or Genbank.

Peptides or proteins according to the invention may be purified and isolated by methods known in the art. In particular, having identified the gene sequence, it will be possible to use recombinant techniques to express the genes in a suitable host. Active fragments and homologues can be identified and may be useful in therapy. For example, the peptides or their active fragments may be used as antigenic determinants in a vaccine, to elicit an immune response.

They may also be used in the preparation of antibodies, for passive immunisation, or diagnostic applications. Suitable antibodies include monoclonal antibodies, or fragments thereof, including single chain fv fragments. Methods for the preparation of antibodies will be apparent to those skilled in the art.

The preparation of vaccines based on attenuated microorganisms is known to those skilled in the art.

Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g. alum, as necessary or desired, and used in therapy, to provide effective immunisation against Group B Streptococci or other related microorganisms. The preparation of vaccine formulations will be apparent to the skilled person.

More generally, and as is well known to those skilled in the art, a suitable amount of an active component of the invention can be selected, for therapeutic use, as can suitable carriers or excipients, and routes of administration. These factors will be chosen or determined according to known criteria such as the nature/severity of the condition to be treated, the type or health of the subject etc.

The products of the present invention were identified as follows: WO 00/37490 PCT/GB99/04376 Todd-Hewitt broth was inoculated with GBS and allowed to grow overnight at 37°C. The cells were harvested by centrifugation and washed with Phosphate Buffered Saline (PBS). The cells were resuspended in an osmotic buffer Sucrose, 20mM Tris-HCl pH 7.0, 10mM MgCl 2 containing protease inhibitors (1 mM PMSF, 10 AM Iodoeacetic Acid, 10 mM 1,10-Phenanthroline, 1 M Pepstatin A) and Mutanolysin at a final concentration of 4 Units per microlitre. This was incubated (shaking) at 37 0 C for 2 hours.

Cells and debris were removed first by high speed centrifugation, then ultra-centrifugation for 1 hour. The resultant supernatant containing cell wall proteins was concentrated under pressure using an ultrafiltration device (10,000 molecular weight cut-off).

The sample was dialysed against ultra high quality water and lyophilised. After resuspension in loading buffer, the proteins were separated by preparative 2- Dimensional-Gel Electrophoresis. Following electrophoresis an individual spot was chosen for study. The spot was subjected to in-gel tryptic digestion. The resulting peptides were extracted from the gel and purified using microbore RP-HPLC. Fractions were collected every seconds and a portion of these consistent with the regions of UV absorbance were analysed by Delayed Extraction-Matrix Assisted Laser Desorption-Time of Flight Mass Spectrometry (DE-MALDI-TOF-MS). Peptides not observed in a blank preparation were then subjected to sequencing using Nanospray-MS/MS Using this peptide sequence information, degenerate oligonucleotides were designed to be used in a polymerase chain reaction (PCR) to amplify the DNA segment lying between the peptide sequences identified.

PCR amplification resulted in the production of several polynucleotide fragments, each of which was cloned into the pCR 2.1-TOPO vector (Invitrogen BV, Netherlands) according to manufacturers protocol.

WO 00/37490 PCT/GB99/04376 6 The DNA fragment in each plasmid was identified by sequencing and then used to obtain the full-length gene sequence, as follows.

Using the identified DNA fragment, oligonucleotide primers were designed for genomic DNA sequencing. These primers were designed so as to sequence in an -outward' direction from the obtained sequence. Once read, the sequence obtained was checked to see if the 5' and 3' termini of the gene had.been reached. The presence of these features was identified by checking against homologous sequences, and for the 5' end the presence of an AUG start codon (or accepted alternative) preceded by a Shine-Dalgarno consensus sequence, and for the 3' end, the presence of a translation termination (Stop) codon.

Upon identification of the full-length gene, primers were designed for amplification of full-length product from GBS genomic DNA. Primers used included restriction enzyme recognition sites (NcoI at the 5'end and EcoOl09I at the 3' end) to allow subsequent cloning of the product into the Lactococcal expression system used.

PCR was carried out using the primers, and the products cloned into a pCR 2.1 cloning vector (In Vitrogen). Following confirmation of the presence of the cloned fragment, the DNA was excised using the restriction enzymes NcoI and EcoOl09I.

The vector into which this fragment was inserted was a modified version of pNZ8048 (Kuipers, O. P. et al. (1998) J. Biotech 64: 15-21). This vector, harbouring a lactococcal origin of replication, a chloramphenicol resistance marker, an inducible nisin promoter and a multicloning site was altered by the replacement of the multicloning site with two 10X His tags, flanked on the most end with an NcoI site, split in the middle with a multicloning site (including an Eco0109I site), and a Stop (termination) codon at the 3'end of the His tags.

The gene of interest was inserted so that a 10X His tag was in the 3' position relative to the coding region.

WO 00/'37490 PCT/GB99/04376 7 Following transformation of the recombinant plasmid into L.lactis (strain NZ9000 Kuipers, 0. P. et al. (1998) supra), a 400 ml liquid culture was set up and translation of the protein was induced by the addition of nisin to the culture. After a 2 hour incubation, the cells were harvested and lysed by bead beating. The resultant lysate was cleared by centrifugation, then passed over a metal affinity (Talon, Clonetech) column. The column was washed repeatedly before bound proteins were eluted with Imidazole.

To identify fractions containing the His-tagged recombinant protein, an aliquot from each fraction was analysed by SDS-PAGE, Western blotted and probed with anti- His antibodies.

The recombinant protein obtained was then used to immunise New Zealand white rabbits, with pre-immune sera being harvested prior to immunisation. Following a boost, the rabbits were sacrificed and sera collected. This sera was used in Western blots, ELISA and animal protection models.

Using the sera obtained from the animal studies, immunosorption studies were carried out.

Group B Streptococcus was grown in 20ml Todd Hewitt broth (THB) for 8 hours, harvested and resuspended in PBS. 50Ai aliquots of this were used to coat wells in a 96 well plate (Nunc Immuno-Sorb). This was left at 4°C overnight to allow for absorbance of the bacteria onto the plate. Plates were washed twice with PBS, then blocked with 3%BSA in PBS for lhr at 37"C. Plates were again washed. Serial 10 fold dilutions of the sera were made in PBS and 50Al of these dilutions were added to the wells of the plate, in duplicate. The plate was covered and incubated for 1 hr at 37cC. The plate was washed, then 50g1 anti-rabbit alkaline phosphatase conjugated secondary antibody at a concentration of 1:5000 was added to each well. Following incubation at 37 0 C for an hour, the plate was washed again. 50gl substrate (PNPP) was added to each WO 00/37490 PCT/GB99/04376 8 well, and the reaction allowed to proceed for 30min before the absorbance was read at 405 nm.

Animal protection studies were also carried out to test the effectiveness of protection on the immunised rabbits.

GBS M732 was grown up in THB until mid-log phase was reached approximately 5 hours. Cells were counted in a counting chamber, and bacteria were diluted to give a concentration of 2x10 bacteria per ml in pre-immune or test sera. 501 of this was injected via the intraperitoneal route into 0-1 day old mice. The mice were observed for survival over 48 hours.

The following Examples illustrate the invention.

Example 1 A first plasmid was termed MS4. The cloned DNA fragment was sequenced and the nucleotide and deduced amino acid sequence (SEQ ID NO. 1 and 2) was used to search protein databases.

Homologues to the GBS MS4 gene product can be identified in Clostridium perfingens, Haemophilus influenzae, Neisseria flavescens and Thermatoga maritima.

In all cases the homologues are the genes for Ornithine Carbamoyltransferase (OCT). In eukaryotic systems this enzyme catalyses the second step in the _Urea cycle, the conversion of ornithine to citrulline, a reaction requiring carbomyl phosphate. In prokaryotes, ODC is one of the three enzymes involved in Arginine Deaminase activity a system which protects bacteria from acid damage. In particular, ODC is responsible for the conversion of citrulline to ornithine and carbamoyl phosphate (the opposite role to that in eukaryotes) (Casiano-Colon, A and Marquis, R. E. 1988. Appl. Environ. Microbiol. 54: 1318- 1324, Cunin, R. et al. 1986. Microbiol. Rev. 50: 314-352).

Animal protection studies were carried out as described above. The results are as follows: WO 00/37490 PCT/GB99/04376 9 Treatment pups pups surviving at time (hrs) 24 48 PBS 15 6 0 Pre-Immune 41 18 1 Test 41 33 14 Example 2 A second plasmid was termed MS11. The nucleotide and deduced amino acid sequence (SEQ ID NOS. 3 and 4) were used to search protein databases.

Homologues to the GBS MS11 gene product can be identified in Lactobacillus delbrueckii, Thermotoga maritima, Clostridium acetobulylicum, Bacillus megaterium, Triticum aestivium and Synechocystis PCC6803.

In all cases the homologues are the genes for the protein Phosphoglycerate Kinase (PGK). PGK is a major enzyme in the glycolytic pathway, being involved in the conversion of Glyceraldehyde-3-phosphate to Phosphoenolpyruvate. In particular, it is involved in the catalysis of the reaction between Glycerate-1,3-diphosphate and 3-Phospho-Glycerate, releasing a phosphate in the forward reaction.

Example 3 A third plasmid was termed pMS16. The 5' and 3' cloned DNA fragments were sequenced and the nucleotide and deduced amino acid sequences for each are shown as SEQ ID NOS. 5 and 6 for the 5' fragment and SEQ ID NOS. 7 and 8 for the 3' fragment.

Homologues to the GBS MS16 gene product can be identified in Bacillus stearothermophilus, Bacillus subtilis and Mycoplasma genitalium.

In all cases the homologues are the genes for the protein Glucose-6-Phosphate Isomerase (GPI).

The enzyme Glucose-6-Phospate Isomerase catalyses the reaction between Glucose-6-phosphate and Fructose-6- Phosphate in both glycolysis (G6P to F6P) and WO 00/37490 PCT/GB99/04376 gluconeogenesis (F6P to G6P). Mutations in the gpi gene have been shown to confer purine analogue sensitivity to organisms.

Example 4 A fourth plasmid was termed pMS14. The cloned DNA fragment was sequenced and the nucleotide and deduced amino acid sequence (SEQ ID NOS. 9 and 10) was used to search protein databases.

Homologues to the GBS MS14 gene product can be identified in Bacillus subtilis, Bacillus stearothermophilus, Mus musculus, Bos taurus and Zea mays.

In all cases the homologues are the genes for the protein Purine Nucleoside Phosphatase (PNP). The function of this enzyme is to cleave the nucleosides guanosine or inosine to their respective basis and sugar-1-phosphate molecules in the presence of orthophosphate.

Example A fifth plasmid was termed pMS10. The cloned DNA fragment was sequenced. The nucleotide and deduced amino acid sequence (SEQ ID NOS. 11 and 12) was used to search protein databases.

Homologues to the GBS MS10 gene product can be identified in Streptococcus mutans, Nicotiana plumb, Pisum sativum and Zea mays. In all cases the homologues are the genes for the protein Nonphosphorylating, NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase (NPGAP-3-DH).

NPGAP-3-DH has been reported as being an important means of generating NADPH for biosynthetic reactions in S. mutans (as opposed to NAD-specific GAP-3-DH which satisfies the requirements of the glycolytic pathway) (Boyd, D.A., Cvitkovitch, D. G. and Hamilton, I. R 1995 J. Bacteriol.

177: 2622-2727).

WO 00/37490 WO 0037490PCT/GB99/04376 SEQUENCE LISTING <110> microscience Limited <120> OUTER SURFACE PROTEINS, THEIR GENES, AND THEIR USE <130> REP05969W0 <140> <141> <160> 12 <1'70> Patentln Ver. 2.1 <210> <211> <212> <213> <220> <221> <222> 1 1014

DNA

group B streptococcus

COS,

<400> 1 atg aca Met Thr caa gta ttt Gin Val Phe caa gga cgt agt Gin Gly Arg Ser ita gca gaa aaa Leu Ala C-lu Lys gat ttt Asp Phe tct cgt gag Ser Arg Glu gaa Glu ttt gaa tat ctt Phe Glu Tyr Leu att gat Ile Asp 25 ttt tca gct Phe Ser Ala cat tta aaa His Leu Lys ggt aaa aat Gly Lys Asn gac ctt Asp 1.eu att gct Ile Ala aaa Lys aaa cgt ggt gtt Lys Arg Giy Val cct Pro 40 cat cat tat ctt His His Tyr Leu gaa Glu ctc tta ttt gaa Leu Leu Phe Giu aca tct act cgt Thr Ser Thr Akrg act Th r cgc qca gcc ttt Arg Ala Ala Phe 144 192 240 a ca Thr act gca gca att Thr A la ALa Ile gac Asp 70 cta ggc gct cat Leu Gly Ala His ccg Pro 75 gaa tac ctt ggt Glu Tyr Leu Gly gca Al a aat gat att caa Asn Asp Ile Gin ctt Leu ggt aaa aaa gaa Gly Lys Lys Giu tca Ser 90 aca gaa gat Thr Giu Asp act gct aag Thr Ala Lys 288 WO 00/37490 WO 0037490PCT/GB99/04376 gtt tta gga Val Leu Gly cgt Arg 100 atg ttt gat ggt Met Phe Asp Gly att Ile 105 gaa ttc cgt ggt Glu Phe Arg Gly ttt agc caa Phe Ser Gin 110 gtc tgg aat Vai Trp Asn aga atg Arg Met ggt tta Giy Leu 130 gtt Vai 115 gaa gag ctt gct Giu Giu Leu Ala gaa Giu 120 ttt tct 99a gta Phe Ser Gly Vai cct Pro 125 aca gat gaa tgg Thr Asp Giu Trp, cca aca caa atg Pro Thr Gln Met gct gac tac ctt Ala Asp Tyr Leu 384 432 480 528 act Thr 145 atc aaa gaa aac Ile Lays Giu Asn ggt aaa, ctt gaa.

Gly Lys Leu Glu g gt Gi y 155 att act ctt gtt Ile Thr Leu Vai tgt ggt gac gga cgt Cys Gly Asp Gly Arg 165 aac aat gtt gc Asn Asn Val Al a aac As n 170 tcg ctt tta gtg Ser Leu Leu Val gct ggg A-1a Giy 175 act ttg atg Thr Leu Met ccw gct gaa Xaa Al a Glu 195 ggg Gi y 180 gtc aat gia. cac Val Asn Val H-is ttt tct cca aaa Phe Ser Pro Lys gaa. ctt tty Glu Leu Phe 190 aaa gaa tct Lys Giu Ser 576 624 gag att gtt aaa Giu Ile Val Lys ttg Leu 200 gct gaa gga tat Al a Giu Gly Tyr gc Al a 205 ggg gct Gly Ala 210 cac gtt ctc gtt His Val Leu Val act Thr 215 gat aat gta gac Asp Asn Val Asp gaa Gi u 220 gct gta, aag gga Ala Val. Lys Gly gca Al a 225 gac gtc ttt tac Asp Val Phe Tyr act Thr 230 gat gtc tgg gta Asp Val Trp Vai tcg Ser 235 atg gga gaa gaa Met Gly Giu Glu ga t Asp 240 672 '72 0 '768 816 aag ttc aaa gaa Lys Phe Lys Giu cgc Arg 245 gtt gaa ctt ctt Val Glu Leu Leu caa Gin 250 cca tat caa gta Pro Tyr Gin Val aac atg Asn Met 255 cac tgc His Cys gaa ctg att Giu Leu Ile tta. oct gca Leu Pro Al1a 275 aaa Lys 260 aaa gct aat aat Lys Ala Asn A-sn gat Asp 265 aat cti atc ttc A-sn Leu Ile Phe tta Le u 270 ttc cat gat aca Phe His Asp Thr aat Asn 280 acc gtt tat ggc Thr Vai Tyr Giy aaa Lys 285 gac gtc gct Asp Val Ala PCT/GB99/043 7 6 WO 00/37490 gaa aaa Glu Lys 290 ttt ggg gtc aag Phe Gly Val Lys gaa atg gaa Glu Met Glu 295 gtt act gat gaa Val Thr Asp Glu 300 gtc tto cgt Val Phe Arg agc Ser 305 aaa tat got cgt Lys Tyr Ala Arg cat His 310 ttc gao caa got Phe Asp Gin Ala gaa aat cgt atg Glu Asn Arg Met 315 aat ctt tto att Asn Leu Phe Ile caC His 960 1008 att aaa gct gta Ile Lys Ala Val atg Met 325 got gca aco ctt Ala Ala Thr Leu gga Giy 330 cca aaa Pro Lys 335 1014 gtt taa Val <210> 2 <211> 337 <212> PRT <2i3> group B streptococcus <400> 2 Met Thr 1 Gin Vai Phe Gin Giy Arg ser Phe Leu Ala Giu Lys Asp Phe Ser Arg Glu Glu Phe Giu Tyr Leu Asp Phe Ser Ala His Leu Lys Gly Lys Asn Asp Leu Lys Lys Arg Gly Val Pro 40 His His Tyr Leu Glu Ile Ala Leu Leu Phe Glu Thr Ser Thr Arg Thr krg Ala Ala Phe Thr Thr Ala Ala Ile Asp 70 Leu Giy Ala His Pro 75 Glu Tyr Leu Gly Ala Asn Asp Ile Gin Leu Giy Lys Lys Glu Ser 90 Thr Giu Asp Thr Ala Lys Vai Leu Gly Arg 100 Met Phe Asp Giy Ile Glu Phe Arg Gly Phe Ser Gin Arg Met Val Glu Giu Leu Ala 115 Glu 120 Phe Ser Giy Vai Pro 125 Val Trp Asn Gly Leu Thr Asp Giu Trp His Pro Thr Gin Met Leu Ala Asp Tyr Leu WO 00/37490 PCT/G B99/04376 Thr 145 Ile Lys Glu Asn Gly Lys Leu Glu Gly le Thr Leu Val 155 Cys Gly Asp Gly Asn Asn Val Ala Asn 170 Ser Leu Leu Val Ala Gly 175 Thr Leu Met Xaa Ala Glu 195 Giy 180 Val As Val His Ile 185 Phe Ser Pro Lys Glu Leu Phe 190 Lys Giu Ser Glu Ile Val Lys Ala Glu Gay Tyr Gly Ala 210 His Val Leu Val Asp Asr Val Asp Glu 220 Ala Val Lys Gly Ala 225 Asp Val Phe Tyr Asp Val Trp Val Ser 235 Met Gly Giu Glu Asp 240 Lys Phe Lys Glu Arg 245 Val Giu Leu Leu Gin 250 Pro Tyr Gin Val Asn Met 255 Glu Leu Ile Leu Pro Ala 275 Lys 260 Lys Ala Asn Asn Asp 265 Asn Leu Ile Phe Leu His Cys 270 Asp Val Ala Phe His Asp Thr Asr 280 Thr Val Tyr Gly Lys 285 Glu Ser 305 Lys 290 Phe Gly Vai Lys Glu 295 Met Glu Val Thr Asp 300 Glu Val Phe Arg Lys Tyr Ala Arg Phe Asp Gin Ala Glu 315 Asn Arg Met His Thr 320 Ile Lys Ala Val Met 325 Ala Ala Thr Leu Gly 330 Asn Leu Phe Ile Pro Lys 335 <210> 3 <211> 1197 <212> DNA <213> group B streptococcus <220> WO 00/37490 WO 001.7490PCT/GB99/04376 <221> CDS <222> <400> 3 atg gct met Ala 1 aaa ttg act Lys Leu Thr 5 gtt aaa gac gtt Val Lys A-sp Val ttg aag gta aaa aaa gtc Leu Lys Val Lys Lys Val.

ctc gtt cgt Leu Val A-rg aac gac aac Asn A sp Asfl gtt Val gac tit aat gtg Asp Phe Asn Val cct Pro 25 ttg aaa gac Leu Lys ASP cgt atc act gcg Arg Ile Thr Ala g ct Al1 a 40 ctt cca aca atc Leu Pro Thr Ile ggc gtt aic act Gly Val Ile Thr aag tat atc atc Lys Tyr ile Ile gga cgt gtt aaa Gly Arg Val Lys gaa caa Giu Gin ggt ggt cgt gct Gly Giy Arg Ala atc Ile 55 cic ttc ict cac Leu Phe Ser His ctt Leu gaa Glu gaa gct gac aaa Giu Ala Asp Lys gga aaa ica ctt Gly Lys Ser Leu gca Al a '7 5 CCg gta gct gCt Pro Va]. Al1a Al1a gati Asp 192 240 288 tta gct gct aaa Leu Ala Ala Lys ctt Leu ggt caa gat gt Gly Gin A-sp Val.

gia Val 90 ttc cca ggt gtt Phe Pro Gly Val act cgt Thr Arg ggt gca Gly Ala aaa tia Lys Leu 100 gaa gaa gca atc Giu Giu Ala Ile aat As n 105 gct1 ttg gaa gat Al1a Leu Glu Asp gga caa gt Gly Gin Val 110 aag aaa gaa Lys L.ys Gi'u ctt tig gtt Leu Leu Val 115 gaa aac act cgt Glu Asn Thr Arg ttt Ph e 120 gaa gat gtt gac Giu Asp Val. ASP ggi Gi y 125 tct aag Ser Lys 130 aat gac gaa gaa Asn Asp Glu Glu ct Leu 135 ggt aaa tac tgg gct ica cit gga gai Gly Lys Tyr Trp Ala Ser Leu Giy Asp 140 gga Gi y 145 atc tt git aac Ile Phe Val. Asn gat Asp 150 gca ttt ggt aca Ala Phe Gly Thr gca Al a 155 cac cgi gct cat His Arg Al1a Pis gca Al1 a 160 tca aac gta ggt Ser Asn Vai Gly att Ile 165 tca gca aac gtt Ser Al1a Asn Vai aaa gci gta gct Lys Ala Val Ala ggt tic Gly Phe 175 WO 00/37490 WO 00/74 90PCT/GB99/04376 ctt -ctt Leu Leu gas cgc Giu Arg att ggt Ile Gly 210 gaa sac Giu Asn 180 gaa att gct tac Giu Ile Ala Tyr atc Ile 185 caa gsa gca gtt Gin Giu Ala Vai gas act cca Giu Thr Pro 190 tct gat aag Ser Asp Lys cca Pro 195 ttc gta. gct att Phe Val Ala Ile ggt ggc tcs ass Gly Giy Ser Lys gtt Vai 205 57 6 624 67 2 gtt atc gas sac Val Ile Giu Asn ctt gas ass. gct Leu Giu Lys Ala gat Asp 220 ass, gtt ctt atc Lys Val Leu Ile ggt ggt ggt Giy Gly Giy 225 atg act tac Met Thr Tyr 230 aca ttc tac asa Thr Phe Tyr Lys gct Ala 235 caa ggt atc gas Gin Gly Ile Giu ggt sac tcs ctt Giy Asn Ser Leu gta Val 245 gsa gsa gac ass Giu Giu Asp Lys tt g Leu 250 gat gtt gct ass Asp Val Ala Lys gac ctc Asp Leu 255 ctt gas, ass Leu Giu Lys gca. aac gca Al a Asn Al1a 275 tca Ser 260 sac ggt ass. ttg Asn Gly Lys Leu atc Ile 265 tig cca, gtt gac Leu Pro Val Asp tcs asa ga Ser Lys Giu 27 0 gsa ggt ga Giu Giy Giu ttt gct ggt tat Phe Ala Giy Tyr act Thr 280 gas. gtt cgc gac Giu Val Arg Asp act Thr 285 gca gtt Ala Val 290 tcs, gas ggg ttc Ser Giu Giy Phe ctt Leu 295 ggt ctt gac sic Giy Leu Asp Ile ggt Gi y 300 cct ass. tca. atc Pro Lys Ser Ile gct Ala 305 as ttt gst gas Lys Phe Asp Giu gca Ala, 310 cit act ggt gct Leu Thr Gly Al1a 555.

Lys 315 aca. gtt gta tgg Thr Vai Vai Trp sac As n 320 912 960 1008 ggs. cct atg ggt Giy Pro Met Giy gt c Vsi 325 itt gsa sac cct ?he Giu Asn Pro ga c Asp 330 tic caa. gct ggt Phe Gin Al1a Giy sca atc Thr Ile 335 ggt gta atg Giy Val Met ggt ggt ggt Gly Giy Giy 355 gct atc gtt ass Als ale Val Lys cca agc gtt Pro Giy Vai aaas tca atc aic Lys Ser Ile Ile 350 ggt cgt gct gac Giy Arg Ala Asp 365 1056 1104 gat tcs gca. gca Asp Ser Ala Ala gct Ala 360 gct atc sac. ct Ala Ile Asn Leu WO 00/37490 aaa ttC tca Lys Phe Ser 370 PCT/GB99/04376 tgg Trp atc tct act lie Ser Thr 375 ggt ggt 9ga gca Gly Gly Gly Ala atg gaa ttg ctc Met Giu Leu Leu 1152 gaa ggt aaa gia tta cca Glu Gly Lys Val Leu Pro 385 390 ggt ttg gca gca Gly Leu Ala Ala ttg Leu 395 act gaa aaa taa Thr Giu Lys 1197 <210> 4 <211> 398 <212> PRT <213> group B streptococcus <400> 4 Met Ala 1 Lys Leu Thr 5 Val Lys Asp Val Asp 10 Leu Lys Val Lys Lys Val Ile Thr 1eu Val Arg Asn Asp Asn Val Asp Phe Asn Val Pro 25 Leu Lys Asp Gly Val Arg Ile Thr Ala Leu Pro Thr Ile Tyr Ile Ile Glu Gin Gly Gly Arg Ala Ile Leu Phe Ser His Gly Arg Val Lys Glu Ala Asp Lys Gly Lys Ser Leu Al a Pro Val Ala Ala Asp Leu Ala Ala Lys Leu Gly Gin Asp Val Val 90 Phe Pro Giy Val Thr Arg Gly Ala Lys Leu Leu Vai 115 Leu 100 Giu Giu Ala Ile As n 105 Ala Leu Giu Asp Gly Gin Val 110 Lys Lys Glu Glu Asn Thr Arg Phe 120 Glu Asp Val Asp G1y 125 Ser Lys 130 Asn Asp Giu Glu Leu 135 Gly Lys Tyr Trp Al a 140 Ser Leu Giy Asp Giy 145 Ile Phe Val Asn Asp 150 Ala Phe Gly Thr Ala 155 His Arg Ala His Al a 160 Ser Asn Val Gly Ile 165 Ser Ala Asn Val Glu 170 Lys Ala Val Ala Gly Phe 175 WO 00/37490 Leu Leu Glu PCT/GB99/04376 Asn 180 Giu Ile Ala Tyr Ile Gin Giu Ala Vai Giu Thr Pro 185 190 Glu Arg Pro 195 Phe Val Ala Ile Gly Giy Ser Lys Val 205 Ser Asp Lys Ile Giy 210 Val Ile Glu Asn Leu 215 Leu Giu Lys Ala Asp Lys Val Leu 220 Gin Gly Ile Glu Ile Ile 240 Gly 225 Gly Giy Met Thr Thr Phe Tyr Lys Gly Asn Ser Leu Val 245 Giu Giu Asp Lys Leu 250 Asp Val Ala Lys Asp Leu 255 2eu Giu Lys Ala Asn Ala 275 Ser 260 Asn Giy Lys Leu Leu Pro Val Asp Ser Lys Glu 270 Glu Gly Glu Phe Ala Giy Tyr Glu Val Arg Asp Thr 285 Ala Val 290 Ser Giu Giy Phe Leu 295 Giy Leu Asp le Gly 300 Pro Lys Ser Ile Ala 305 Lys Phe Asp Glu Leu Thr Giy Ala Thr Val Val Trp Gly Pro Met Gly Val 325 Phe Glu Asn Pro Asp 330 Phe Gin Ala Giy Thr Ile 335 Gly Val Met Gly Gly Giy 355 Asp 340 Ala Ile Val Lys Pro Giy Vai Lys Ser Ile Ile 350 Arg Ala Asp Asp Ser Ala Ala Ala 360 Ala Ile Asn Leu Gly 365 Lys Phe 370 Ser Trp Ile Ser Thr 375 Gly Giy Gly Ala Ser 380 Met Glu Leu Leu Gly Lys Vai Leu Pro 390 Gly Leu Ala Ala Leu Thr Giu Lys 395 <210> <211> <212> <213> 516

DNA

group B streptococcus WO 00/37490 WO 0037490PCTIG B99/04376 <220> <221> CDS <222> (516) <400> atg aca Met Thr

I

cat att. aca ttt gac tia ttc His Ile Thr Phe Asp Leu Phe 5 gtc ttg ggt caa Val Leu Gly Gln itt gta Phe Val ggc gaa cat Gly Giu His gct ttc ctt.

Ala Phe Leu gag Gi u ita gac tat cia Leu Asp Tyr Leu cca Pro 25 cta caa gta agt Pro Gin Val Ser gca gca gat Al a Al a As p ttc gga tgg Leu Gly Trp cgt caa ggg act Arg Gin Giy Thr gigt.

Gi y 40 cci gga ica gat Pro Gly Ser Asp tt Phe atg gaa Met Giu cci cca gaa aat Pro Pro Giu Asn gat aaa gaa gaa Asp Lys Giu Giu itt ict cgc ati Phe Ser Arg Ile gia tc gtg gt Val Leu Val Val caa Gin att Ile aaa Lys gtt gct gaa aag Ala Ala Giu Lys ati Ile '70 aaa ica gat agc Lys Ser Asp Ser gaa Giu 75 ggt ati ggt ggt Giy Ilie Gly Giy tcg Ser tat cit ggt gta Tyr Leu Giy Ala aaa Lys 90 gca gta att gat Ala Ala Ile Asp tit tig Phe Leu aat aat cat Asn Asn His tag att ctt Gin Ile Leu 115 tt Phe 100 gci aat tig caa Ala Asn Leu Gin act Thr 105 gca gaa gaa cgi Ala Giu Giu Arg aaa gcg cci Lys Ala Pro 110 cit gcc gat Leu Ala Asp tat gci gga aat Tyr Ala Giy Asn tct Ser 120 ati tca tct. act Ile Ser Ser Thr tat Tyr 125 tta gti Leu Val 130 gaa iac gic caa Giu Tyr Vai Gin aaa gaa ttc ta Lvs Giu Phe Ser gia Val 140 aai gic ati tca Asn Val Ile Ser 432 480 aaa Lys 145 ica ggt aca aca 5cr Gly Thr Thr act Th r 150 gaa tta gcg ati Giu Pro Al1a Ile gci.

Al1 a 155 ttc cgi gia iii Phe Arg Vai Phe aaa Lys 160 gaa cit cia git aaa aag tat cgg ica aga aga agt 516 Giu Leu Leu Val Lys Lys Tyr Arg Ser Arg Arg Ser WO 00/37490 WO 0037490PCT/GB99/04376 <210> 6 <211> 172 <212> PRT <213> group B streptococcus <400> 6 Met Thr 1 His Ile Thr 5 Phe Asp Leu Phe Lys 10 Val Leu Gly Gin Phe Val Gly Glu His Ala Phe Leu Gi u Leu Asp Tyr Leu Pro 25 Pro Gin Val Ser Ala Ala Asp Leu Gly Trp Arg Gin Giy Thr G1 y 40 Pro Gly Ser Asp Phe Met Giu Pro Pro Glu Asn Tyr Asp Lys Giu Glu Ser Arg Ile Gin Lys Ala Ala Giu Lys Lys Ser Asp Ser Val Leu Val Val Ile so Gly Ile Gly Gly Ser Tyr Leu Giy Ala Ala Ala Ile Asp Phe Leu Asn Asn His Gin Ile Leu 115 Phe 100 Ala Asri Leu Gin Thr 105 Ala Giu Giu Arg Lys Ala Pro 110 Tyr Leu Ala Asp 125 Tyr Ala Giy Asn Ser 120 Ile Ser Ser Thr Leu Val 130 Giu Tyr Vai Gin Asp 135 Lys Giu Phe Ser Val Asn Val Ile Ser 140 Ser Giy Thr Thr Thr 150 Glu Pro Ala Ile Al a 155 Phe Arg Val. Phe Lys 160 Giu Leu Leu Vai Lys 165 Lys Tyr Arg Ser Arg Arg Ser 170 <210> 7 <211> 318 <212> DNA <213> group B streptococcus PCT/GB99/04376 WO 00/37490 <220> <221> CDS <222> (318) <400> 7 ait aac cga aga lie Asn Arg Arg 1 aga tgg ict tgg tta tct tca aga aaa Arg Trp Ser Trp Leu Ser Ser Arg Lys 10 gat gta Asp Val gat tit gtt Asp Phe Val gat ggt ggg Asp Gly Gly aat Asn aaa aaa gca aca Lys Lys Ala Thr gat ggt A sp Gly 25 gtg ctt ctt Val Leu Leu gct cat aca Ala His Thr caa gaC gct Gin Asp Ala gtt cca aat atg Val Pro Asn Met ttt Ph e 40 gta acg ctt cct Val Thr Leu Pro aca Thr tac act Tyr Thr ctt ggt tac act Leu Gly Tyr Thr att Ile 55 tac ttc ttt gag Tyr Phe Phe Giu tta Leu gca att ggc ctt Ala Ile Gly Leu 192 240 ggt tat ctt aac Gly Tyr Leu Asn tca Ser gta aat cca ttt Val Asn Pro Phe ga t Asp 75 caa ccg ggg gta Gin Pro Gly Val gaa Giu gca tat aaa cgt aat atg ttc gca ttt Ala Tyr Lys Arg Asn Met Phe Ala Phe ggt Gi y 90 aaa cct gga ttc Lys Pro Gly Phe gaa gag Giu Giu 288 ctt age get Leu Ser Ala <210> 8 <211> 105 <212> PRT <213> group <400> 8 Ile Asn Arg 1 Asp Phe Val ga a Glu 100 ttg aat gca cgt Leu Asn Ala Arg ctt taa Leu 105 B streptococcus Arg Phe Arg Trp 5 Asn Lys Lys Ala Ser Trp Leu Ser Ser Arg Lys 10 Asp Val Thr Asp 25 Gly Val 1,eu Leu Ala His Thr Asp Gly Gly Val AspGlyGlyVaiPro Asn Met Phe Val Thr Leu Pro Thr Gin Asp Ala WO 00/37490 WO 0037490PCT/GB99/04376 Ala Ile Gly Leu Tyr Thr Leu Gly Tyr Thr Ile Tyr Phe Phe Giu 55 Leu Ser Gly Tyr Leu Asri Ser '70 Val. Asn Pro Phe Gin Pro Gly Vai Giu Ala Tyr Lys Arg Asn Met Phe Ala Phe Gi y 90 Lys Pro Gly Phe Glu Glu Leu Ser Ala Gl.u 100 Leu Asn Ala Arg <210> 9 <211> 804 <212> DNA <213> group B streptococcus <220> <221> CDS <222> (804) <400> 9 aig aca Met Thr I-1 tta tta gaa Leu Leu Giu 5 aaa att. aat gag Lys Ile Asn Glu act Th r 10 aga. gac ttt ttg Arg Asp Phe Leu caa gca Gin Ala.

aaa ggc gic Lys Gly Val.

gga gaa ttg Gly Giu Leu a ca Thr gca cca gaa ttt.

Ala Pro Gi1u Phe ggy Xaa 25 ctt att tta ggc Leu Ile Leu Gly tct ggt tta.

Ser Gly Leu gat tat gca Asp Tyr Ala gct. gaa gaa atc Ala Glu Giu Ile gaa Gi u 40 aat cct ati gtt Asn pro Ile Val gt g Val gac atc Asp Ile ccrn aat tgg gga Xaa Asn Trp Gly cag Gin 55 tca aca gta gtt Ser Thr Val Val ggt Gi y cat gct gga. aaa His Ala Gly Lys 192 240 tt Phe agt gia tgg gat Ser Va). Trp Asp tca ggc cgt aag Ser Gly Arg Lys gia Val.

'75 tta gcg ctt caa Leu Al1a Leu Gin ggt Gi y cgt ttt cat ttt tay gaa ggw aat aca atg9 gaa gtc gtt act ttc cca Arg Phe His Phe Tyr Giu Xaa Asn Thr Met Giu Val Val. Thr Phe Pro 288 PCT/GB99/04376 WO 00/37490 gta cgt atc Val Arg Ile gca gcg ggt Ala Ala Gly 115 atg Met 100 aga gca ttg gct Arg Ala Leu Ala tgc Cys 105 cac agt gtg ctt His 5cr Val Leu gig act aai Val Thr Asn 110 ctg atc aaa Leu Ile Lys ggg att gga tac Gly Ile Gly Tyr gga Gi y 120 cca gga act tta Pro Giy Thr Leu atg Met 125 gac cac Asp His 130 atc aat atg ati Ile Asn Met Ilie ggg Gi y 135 act aac cci cic Thr Asn Pro Leu ata Ile 140 ggt gag aac ct G1iy Giu Asn Leu gaa Giu 145 gaa iii gga cca Giu Phe Gly Pro cgt Arg 150 tic cca gac aig Phe Pro Asp Met icg 5cr 155 gat gct tay aca Asp Ala Tyr Thr 432 480 528 aca tat cga caa Thr Tyr Arg Gin gct cac caa at Ala His Gin Ile gct Al. a 1,70 gaa aac gat atc Giu Asn Asp Ile aaa ctc Lys Leu 175 gaa gaa ggt Giu Giu Gly gca gaa ati Ala Giu Ile 195 gig Val 180 tac tig ggt gta Tyr Leu Giy Val tca 5cr 185 gga ccc act tat Gly Pro Thr Tyr gaa aca cct Giu Thr Pro 190 gt~a ggt atg Vai Gly Met cgt gca tic caa Arg Al1a Phe Gin a ca Thr 200 atg ggc gca caa Met Gly Ala Gin g cg Al. a 205 tcc acg Ser Thr 210 gtt cca gag gig Val Pro Giu Vai aic Ile 215 gtt gca gct cac Val Al.a Al a His ica Ser 220 ggg ctt aaa gig Gly Leu Lys Val gga att ica gca Giy Ile Ser Ala att le 230 act aac cit gcc Thr As n Leu Ala gct Al1 a 235 ggc tic caa ica Giy Phe Gin ser gag Glu 240 672 '72 0 '768 ctc aat cat gag Leu Asn His Giu gag Giu 245 gtc gtt gaa gt Val Val Giu Val act Thr 250 cag cgi att aaa Gin Arg Ile Lys gaa gat Giu Asp 255 ttc aag gga Phe Lys Gly ggt aaa ica tta Giy Lys 5cr Leu gtt Vai 265 gct gaa ctc Al1a Giu Leu <210> WO 0037490PCT/GB99/04376 WO 00/37490 Leu Asn His Glu Giu Val Val Giu 245 Val Thr 250 Gin Arg Ile Lys Giu Asp 255 Phe Lys Gly Leu 260 Gly Lys Ser Leu Val Aia Giu Leu 265 <210> <211> <212> <213> <220> <221> <222> 11 1428

DN~A

group B streptococcus

CDS

(1)..(1428) <400> 11 ttg aca aaa gaa tat Leu Thr Lys Giu Tyr 1 5 caa aat tat gtc Gin Asn Tyr Vai aat Asn 10 ggC gaa tgg aaa Giy Giu Trp Lys tca tct Ser Ser gtt aat cag Val Asn Gin att 1.1e gag att ttg tca Giu Ile Leu Ser cca Pro 25 att gat gat tot Ile Asp Asp Ser tca ttg gga Ser Leu Giy atg aaa gcg Met Lys Ala ttc gtg oca Phe Val Pro gcg atg act oga gaa gaa gtt gat cat Ala Met Thr Arg Giu Giu Val ASP His 40 goct Al a ggt cgt Gly Arg gag gct tta oca Giu Ala Leu Pro got Al1 a 55 tgg gct got tta Trp Ala Ala Leu aca Thr gta tat gaa cgt Val Tyr Glu Arg gca Al a oaa tac ctt cat GIn Tyr Leu His aaa Lys '70 gcc goa gac at Ala Ala Asp Ile at Ile 75 gaa cgt gat aaa Giu Ara Asp Lys gaa Giu 192 240 288 gaa att gct aot Glu Ile Ala Thr gt Val tta gca aaa gaa Leu Ala Lys Giu at Ile 90 tot aaa got tac Ser Lys Ala Tyr aat got As n Ala toa gta act Ser Val Thr gag Giu 100 gtt gta agg aca Val Val Arg Thr got Al1 a 105 gat Ott att cgt Asp Leu Ile Arg tat goa goa Tyr Ala Ala 110 PCTIGB99/04376 WO 00/37490 <211> 268 <212> PRT <213> group <400> 10 Met Thr Leu 1 Lys Gly Val Gly Giu Leu B streptococcus Leu Giu Lys lie Asn Giu Thr Arg Asp Phe Leu Gin Ala Thr Ala Pro Giu Phe Leu Ile Leu Gly Ser Gly Leu Asp Tyr Ala Ala Glu Giu Ile Asn Pro Ile Val Asp Ile Xaa Asn Trp Gly Ser Thr Val Val Gly His Ala Giy Lys Ser Vai Trp Asp Leu Ser Giy Arg Lys Leu Ala Leu Gin Giy Pro Arg Phe His Phe Tyr Giu Xaa Asn Thr Met 90 Glu Vai Vai Thr Phe Val Arg Ile Ala Ala Giy 115 Met 100 Arg Ala Leu Ala His Ser Val Leu Vai 110 Thr Asn Gly Ile Gly Tyr Giy 120 Pro Gly Thr Leu Met 125 Leu Ile Lys Asp His 130 Ile Asn Met Ile Gly 135 Thr Asn Pro 1eu Ile 140 Gly Giu Asn Leu Glu Phe Giy Pro Arg 150 Phe Pro Asp Met Ser 155 Asp Ala Tyr Thr Ala 160 Thr Tyr Arg Gin Lys 165 Ala His Gin Ile Al a 170 Glu Asn Asp Ile Lys Leu 175 Glu Giu Gly Ala Giu Ile 195 Val 180 Tyr Leu Giy Val Gly Pro Thr Tyr Arg Ala Phe Gln Thr 200 Met Giy Ala Gin Ala 205 Ser Gly 220 Glu Thr Pro 190 Val Gly Met Leu Lys Val Ser Thr 210 Val Pro Giu Val Ile 215 Val Ala Ala His Leu Gly Ile Ser Ala Ile Thr Asn Leu Ala Ala Giy Phe Gin Ser Glu PCT/GB99/043 76 WO 00/37490 gaa gaa gga G2.u Giu Gly 115 att cgt tta tea lie Arg Leu Ser act Thr 120 tca get. gac gaa Ser Al1a Asp Glu ggt Gi y 125 gga aaa atg Gly Lys Met gat get.

Asp Ala 130 tea aea ggt cat Ser Thr Giy His aag Lys 135 ttg get gtt. att Leu Al1a Val Ile egt Arg 140 egt eaa eea gta Arg Gin Pro Val ggt G1 y 145 ate gtt tta gea Ile Vai Leu Ala gea ect tat aat Al a Pro Tyr Asn ect. gtt aae etc Pro Vai Asn Leu 432 480 528 gga tea aaa att, Giy Ser Lys Ile geg Al1 a 165 eca get eta att Pro Leu Ile ggt, Gi y 1 7 0 gga aac gtt gtg Gly As n Val Val atg ttt Met Phe 1,75 aaa cea eea Lys Pro Pro ttt gca gaa.

Phe Al a Giu 195 aca Thr 180 caa ggt tea, gtc GIn Gly Ser Val.

tea Ser 185 gga. ett. gtt tta.

Gly Leu Vai Leu gea aaa get Al a Lys Ala 190 att. aca gga Ile Thr Gly gea ggt ctt eca Ala Giy Leu Pro ga 200 ggt gtc ttt. aat Gly Vai Phe Asn act Thr 205 ege ggt Arg Giy 210 tet gag att gga Ser Giu Ile Gly gat tac Asp Tyr 215 att gtt gag Ile Val Glu eat His 220 gaa gaa gtt aat Giu Giu Val Asn att, aac ttt aca lie Asn Phe Thr gga Gi y 230 tea aeg cca gtt Ser Thr Pro Vai eaa egt att, ggt Gin Arg Ile Giy aag Lys 240 672 720 768 ttg gea, gga atg Leu Ala Gly Met ggt ate gte ita.

Gly Ile Vai Leu 260 gtt gea ggt get Val Ala Giy Al1a 275 egt Arg 245 eea att, atg ctt Pro Ile Met Leu gag Giu 250 ttg gge ggt aag Leu Gly Giy Lys gat gca Asp Al a 255 get. gat get gac Al a Asp Al1a Asp c tt.

Leu 265 gat aac get get Asp Asn Al a Al1a aaa eaa ate Lys GIn Ile 2,70 gca att aag Ala le Lys 816 864 tat gat tae Tyr Asp Ty tct.

Ser 280 gga eaa ege tgt Gly Gin Ar-g Cys aeg Thr 285 e gt Arg gtg Val 290 ctt gte gtt gaa Leu Vai Val Giu gaa Gi u 295 gtt. gew gat gaa Val Xaa Asp Glu ttg Leu 300 gea gaa aaa ata Al1a Glu Lys Ile 912 PCT/GB99/043 7 6 WO 00/37490 tct Ser 305 gaa aat gta gca Glu Asn Val Ala tta tca gta ggt gat cca tit gat aat gca Leu Ser Val Gly Asp Pro Phe Asp Asn A.1a 320 acg gtg aca ccg Thr Val Thr Pro gtt Val 325 att gat gat aat Ile Asp Asp Asn tca gct gac ttt att gaa agc Ser Ala Asp Phe Ile Giu Ser 330 335 1008 tta gta gia Leu Val Val ga t A-sp 340 gca cgt caa aaa Ala Arg Gin Lys gag aaa gaa ttg A-1a Lys Giu Leu aai gaa ttt Asn Giu Phe 350 cat gtt act His Val Thr 1056 aaa cgt Lys Arg tta cat Leu Asp 3*70 gat As p 355 ggt cgt cta tta Gly Arg Leu Leu act Thr 360 cca gga ttg tt Pro Gly Leu Phe gat Asp 365 aig aaa cia gct Met Lys Leu Al1a tg g Trp 375 gaa gag cct ttt G1u Giu Pro Phe gga Gi y 380 cca att ctc eca Pro Ile Leu Pro 1104 1152 1200 1248 at Ile 385 ati cgt gtc aag Ile Arg Val Lys gat Asp 390 gca gaa gaa gci Ala Giu Glu Ala gt Val 395 gct ati gcc aac A1a Ile Al1a Asn aaa Lys 400 tct gat i~tt. gga Ser Asp Phe Gly tta Leu 405 caa tca ica gic Gin Ser Ser Vai ttt Phe 410 aca cgt. gat ttc Thr Arg Asp Phe caa aaa Gin Lys 415 gca ttt gat Ala Phe Asp aia Ile 420 gca aai aaa att Al a Asn Lys Leu gaa Giu 425 gtt ggt aca gtt Val. Gly Thr Vai cac att aac His le Asn 430 gga ctc aaa Gly Leu Lys 1296 aai aag Asn Lys gga tat Gly Ser 450 act Thr 435 gga cgt ggt ccw Gly Arg Gly Xaa gat Asp 440 aai. itc cca ttc A-sn Phe Pro Phe tta Leu 445 1344 ggt gca ggt gtt caa ggt aic aga Giy Al1a Gly Val Gin Giy Ile Arg 455 tat ica att. gaa gca atg Tyr Ser Ile Giu Ala Met 460 1392 1428 a ca Thr 465 aat gia aaa tag Asn Val Lys Ser gtt cia gat ai~g Val Leu Asp Met aaa tag Lys 475 <210> 12 <211> 475 <212> PRT PCT/GB99/04376 WO 00/37490 <213> group B streptococcus <400> 12 Leu Thr Lys 1 Val As Gin Phe Vai Pro Giv Arg Glu Gin Asn Tyr Val Asn Gly Glu Trp Lys Ser Ser Glu Tyr Ile Glu Ile Leu Ser Ai a Pro 25 Glu Ile Asp Asp Ser Met Thr Arg Glu 40 Trp Vai Asp His Ala Val Ser Leu Gly Met Lys Ala Tyr Gu Arg Ala Leu Pro Ala Ala Leu Ala Gin Thr Glu Tyr Leu His Ala Asp Ile Glu Ile 75 Ser Arg Asp Lys Glu Ile Ala Thr Val Val Ala Lys Glu Ile 90 Asp Lys Ala Tyr Asn Ala Ser Val Thr Glu Glu Gly 115 Asp Ala Ser Glu 100 Ile Vai Arg Thr Ala 105 Ser Leu Ile Arg Tyr Ala Ala 110 Gly Lys Met Arg Leu Ser Thr 120 Leu Ala Asp Glu Gly Thr Gly His 130 Glv Ile Lys 135 Ala Ala Val Ile Arg 140 Pro Arg Gin Pro Val Val Leu AIa 145 Gly Ile 150 Pro Pro Tyr Asn Tyr 155 Giy Val Asn Leu Ser 160 Ser Lys Ile Ala 165 Gin Ala Leu Ile Gly 170 Giy Asn Val Val Met Phe 175 Lys Pro Pro Phe Ala Glu 195 Arg Gly Ser 210 Thr 180 Ala Gly Ser Val Ser 185 Giy Leu Vai Leu Gly Leu Pro Al a 200 Tyr Val Phe Asn Thr 205 Glu Ala Lys Al a 190 Ile Thr Gly Glu Val Asn Glu Ile Gly Asp 215 Ile Val Glu His 220 Phe 225 Ile Asn Phe Thr Giy 230 Ser Thr Pro Val Gly 235 Gin Arg Ile Gly Lys 240 PCT/GB99/04376 WO 00/37490 Leu Ala Gly Met Arg Pro Ile Met Leu 245 Glu Leu 250 Gly Gly Lys Asp Ala 255 Gly Ile Val Val Ala Gly 275 Leu 260 Ala Asp Ala Asp Leu 265 Asp Asn Ala Ala Lys Gin Ile 270 Ala Ile Lys Ala Tyr Asp Tyr Ser 280 Gly Gin Arg Cys Thr 285 Arg Val 290 Leu Val Val GlU Val Xaa Asp Glu Leu 300 Ala Giu Lys Ile Ser 305 Glu Asn Val Ala Leu Ser Val Gly Asp 315 Pro Phe Asp Asn Al a 320 Thr Val Thr Pro Val 325 Ile Asp Asp Asn Ser 330 Ala Asp Phe, Ile Glu Ser 335 Leu Val Val Lys Arg Asp 355 Asp 340 Ala Arg Gin Lys Giy 345 Ala Lys Giu Leu Asn Giu Phe 350 His Val Thr Gly Arg Leu Leu Thr 360 Pro Gly Leu Phe Asp 365 Leu Asp 370 Met Lys Leu Ala Trp 375 Glu Giu Pro Phe Gly 380 Pro Ile Leu Pro Ile Arg Val Lys Asp 390 Ala Giu Giu Ala Ala Ile Ala Asn Lys 400 Ser Asp Phe Gly Gin Ser Ser Vai Phe 410 Thr Arg Asp Phe Gin Lys 415 Ala Phe Asp Asn Lys Thr 435 Ile 420 Ala Asn Lys Leu Val Gly Thr Vai His le Asn 430 Gly Arg Gly Xaa Asn Phe Pro Phe Leu Gly Leu Lys 445 Gly Ser 450 Gly Ala Gly Val Gin 455 Gly Ile Arg Tyr Ser Ile Giu Ala Met 460 Asn Vai Lys Ser Ile 470 Val Leu Asp Met Lys 475

Claims (11)

1. A Ornithine Carbamoyl Transferase peptide obtained from Group B streptococcus, or a fragment thereof that is capable of eliciting an immune response, when used in therapy.

2. A peptide according to claim 1, comprising the amino acid sequence identified herein as SEQ ID NO. 2.

3. A polynucleotide encoding a peptide according to claim 1 or claim 2, for therapeutic use.

4. A host transformed to express a peptide according to claim 1 or claim 2. A vaccine comprising a peptide according to claim 1 or claim 2, or the means for its expression.

6. Use of the peptide according to claims 1 or 2, the polynucleotide according to claim 3, or the host according to claim 4, for screening potential drugs or for the detection of virulence.

7. Use of the peptide according to claims 1 or 2, the polynucleotide according to claim 3, or the host according to claim 4, for the manufacture of a medicament for use in the treatment or prevention of a condition associated with bacterial infection.

8. Use according to claim 7, wherein the infection is a Group B streptococcal infection.

9. Use according to claim 7 or claim 8, wherein the infection is a focal infection. Use according to claim 7 or claim 8, wherein the infection is a urinary tract infection. 12

11. An antibody raised against a peptide according to claim 1 or claim 2.

12. Use according to claim 7 or claim 8, wherein the medicament treats or prevents infection in a neonate. WO 00/37490 WO 0037490PCT/GB99/04376 SEQUENCE LISTING <110> microscience Limited <120> OUTER SURFACE PROTEINS, THEIR GENES, AND THEIR USE <130> REP05969W0 <140> <141> <160> 12 <1'70> Patentln Ver. 2.1 <210> <211> <212> <213> <220> <221> <222> 1 1014 DNA group B streptococcus COS, <400> 1 atg aca Met Thr caa gta ttt Gin Val Phe caa gga cgt agt Gin Gly Arg Ser ita gca gaa aaa Leu Ala C-lu Lys gat ttt Asp Phe tct cgt gag Ser Arg Glu gaa Glu ttt gaa tat ctt Phe Glu Tyr Leu att gat Ile Asp 25 ttt tca gct Phe Ser Ala cat tta aaa His Leu Lys ggt aaa aat Gly Lys Asn gac ctt Asp 1.eu att gct Ile Ala aaa Lys aaa cgt ggt gtt Lys Arg Giy Val cct Pro 40 cat cat tat ctt His His Tyr Leu gaa Glu ctc tta ttt gaa Leu Leu Phe Giu aca tct act cgt Thr Ser Thr Akrg act Th r cgc qca gcc ttt Arg Ala Ala Phe 144 192 240 a ca Thr act gca gca att Thr A la ALa Ile gac Asp 70 cta ggc gct cat Leu Gly Ala His ccg Pro 75 gaa tac ctt ggt Glu Tyr Leu Gly gca Al a aat gat att caa Asn Asp Ile Gin ctt Leu ggt aaa aaa gaa Gly Lys Lys Giu tca Ser 90 aca gaa gat Thr Giu Asp act gct aag Thr Ala Lys 288 WO 00/37490 WO 0037490PCT/GB99/04376 gtt tta gga Val Leu Gly cgt Arg 100 atg ttt gat ggt Met Phe Asp Gly att Ile 105 gaa ttc cgt ggt Glu Phe Arg Gly ttt agc caa Phe Ser Gin 110 gtc tgg aat Vai Trp Asn aga atg Arg Met ggt tta Giy Leu 130 gtt Vai 115 gaa gag ctt gct Giu Giu Leu Ala gaa Giu 120 ttt tct 99a gta Phe Ser Gly Vai cct Pro 125 aca gat gaa tgg Thr Asp Giu Trp, cca aca caa atg Pro Thr Gln Met gct gac tac ctt Ala Asp Tyr Leu 384 432 480 528 act Thr 145 atc aaa gaa aac Ile Lays Giu Asn ggt aaa, ctt gaa. Gly Lys Leu Glu g gt Gi y 155 att act ctt gtt Ile Thr Leu Vai tgt ggt gac gga cgt Cys Gly Asp Gly Arg 165 aac aat gtt gc Asn Asn Val Al a aac As n 170 tcg ctt tta gtg Ser Leu Leu Val gct ggg A-1a Giy 175 act ttg atg Thr Leu Met ccw gct gaa Xaa Al a Glu 195 ggg Gi y 180 gtc aat gia. cac Val Asn Val H-is ttt tct cca aaa Phe Ser Pro Lys gaa. ctt tty Glu Leu Phe 190 aaa gaa tct Lys Giu Ser 576 624 gag att gtt aaa Giu Ile Val Lys ttg Leu 200 gct gaa gga tat Al a Giu Gly Tyr gc Al a 205 ggg gct Gly Ala 210 cac gtt ctc gtt His Val Leu Val act Thr 215 gat aat gta gac Asp Asn Val Asp gaa Gi u 220 gct gta, aag gga Ala Val. Lys Gly gca Al a 225 gac gtc ttt tac Asp Val Phe Tyr act Thr 230 gat gtc tgg gta Asp Val Trp Vai tcg Ser 235 atg gga gaa gaa Met Gly Giu Glu ga t Asp 240 672 '72 0 '768 816 aag ttc aaa gaa Lys Phe Lys Giu cgc Arg 245 gtt gaa ctt ctt Val Glu Leu Leu caa Gin 250 cca tat caa gta Pro Tyr Gin Val aac atg Asn Met 255 cac tgc His Cys gaa ctg att Giu Leu Ile tta. oct gca Leu Pro Al1a 275 aaa Lys 260 aaa gct aat aat Lys Ala Asn A-sn gat Asp 265 aat cti atc ttc A-sn Leu Ile Phe tta Le u 270 ttc cat gat aca Phe His Asp Thr aat Asn 280 acc gtt tat ggc Thr Vai Tyr Giy aaa Lys 285 gac gtc gct Asp Val Ala PCT/GB99/043 7 6 WO 00/37490 gaa aaa Glu Lys 290 ttt ggg gtc aag Phe Gly Val Lys gaa atg gaa Glu Met Glu 295 gtt act gat gaa Val Thr Asp Glu 300 gtc tto cgt Val Phe Arg agc Ser 305 aaa tat got cgt Lys Tyr Ala Arg cat His 310 ttc gao caa got Phe Asp Gin Ala gaa aat cgt atg Glu Asn Arg Met 315 aat ctt tto att Asn Leu Phe Ile caC His 960 1008 att aaa gct gta Ile Lys Ala Val atg Met 325 got gca aco ctt Ala Ala Thr Leu gga Giy 330 cca aaa Pro Lys 335 1014 gtt taa Val <210> 2 <211> 337 <212> PRT <2i3> group B streptococcus <400> 2 Met Thr 1 Gin Vai Phe Gin Giy Arg ser Phe Leu Ala Giu Lys Asp Phe Ser Arg Glu Glu Phe Giu Tyr Leu Asp Phe Ser Ala His Leu Lys Gly Lys Asn Asp Leu Lys Lys Arg Gly Val Pro 40 His His Tyr Leu Glu Ile Ala Leu Leu Phe Glu Thr Ser Thr Arg Thr krg Ala Ala Phe Thr Thr Ala Ala Ile Asp 70 Leu Giy Ala His Pro 75 Glu Tyr Leu Gly Ala Asn Asp Ile Gin Leu Giy Lys Lys Glu Ser 90 Thr Giu Asp Thr Ala Lys Vai Leu Gly Arg 100 Met Phe Asp Giy Ile Glu Phe Arg Gly Phe Ser Gin Arg Met Val Glu Giu Leu Ala 115 Glu 120 Phe Ser Giy Vai Pro 125 Val Trp Asn Gly Leu Thr Asp Giu Trp His Pro Thr Gin Met Leu Ala Asp Tyr Leu WO 00/37490 PCT/G B99/04376 Thr 145 Ile Lys Glu Asn Gly Lys Leu Glu Gly le Thr Leu Val 155 Cys Gly Asp Gly Asn Asn Val Ala Asn 170 Ser Leu Leu Val Ala Gly 175 Thr Leu Met Xaa Ala Glu 195 Giy 180 Val As Val His Ile 185 Phe Ser Pro Lys Glu Leu Phe 190 Lys Giu Ser Glu Ile Val Lys Ala Glu Gay Tyr Gly Ala 210 His Val Leu Val Asp Asr Val Asp Glu 220 Ala Val Lys Gly Ala 225 Asp Val Phe Tyr Asp Val Trp Val Ser 235 Met Gly Giu Glu Asp 240 Lys Phe Lys Glu Arg 245 Val Giu Leu Leu Gin 250 Pro Tyr Gin Val Asn Met 255 Glu Leu Ile Leu Pro Ala 275 Lys 260 Lys Ala Asn Asn Asp 265 Asn Leu Ile Phe Leu His Cys 270 Asp Val Ala Phe His Asp Thr Asr 280 Thr Val Tyr Gly Lys 285 Glu Ser 305 Lys 290 Phe Gly Vai Lys Glu 295 Met Glu Val Thr Asp 300 Glu Val Phe Arg Lys Tyr Ala Arg Phe Asp Gin Ala Glu 315 Asn Arg Met His Thr 320 Ile Lys Ala Val Met 325 Ala Ala Thr Leu Gly 330 Asn Leu Phe Ile Pro Lys 335 <210> 3 <211> 1197 <212> DNA <213> group B streptococcus <220> WO 00/37490 WO 001.7490PCT/GB99/04376 <221> CDS <222> <400> 3 atg gct met Ala 1 aaa ttg act Lys Leu Thr 5 gtt aaa gac gtt Val Lys A-sp Val ttg aag gta aaa aaa gtc Leu Lys Val Lys Lys Val. ctc gtt cgt Leu Val A-rg aac gac aac Asn A sp Asfl gtt Val gac tit aat gtg Asp Phe Asn Val cct Pro 25 ttg aaa gac Leu Lys ASP cgt atc act gcg Arg Ile Thr Ala g ct Al1 a 40 ctt cca aca atc Leu Pro Thr Ile ggc gtt aic act Gly Val Ile Thr aag tat atc atc Lys Tyr ile Ile gga cgt gtt aaa Gly Arg Val Lys gaa caa Giu Gin ggt ggt cgt gct Gly Giy Arg Ala atc Ile 55 cic ttc ict cac Leu Phe Ser His ctt Leu gaa Glu gaa gct gac aaa Giu Ala Asp Lys gga aaa ica ctt Gly Lys Ser Leu gca Al a '7 5 CCg gta gct gCt Pro Va]. Al1a Al1a gati Asp 192 240 288 tta gct gct aaa Leu Ala Ala Lys ctt Leu ggt caa gat gt Gly Gin A-sp Val. gia Val 90 ttc cca ggt gtt Phe Pro Gly Val act cgt Thr Arg ggt gca Gly Ala aaa tia Lys Leu 100 gaa gaa gca atc Giu Giu Ala Ile aat As n 105 gct1 ttg gaa gat Al1a Leu Glu Asp gga caa gt Gly Gin Val 110 aag aaa gaa Lys L.ys Gi'u ctt tig gtt Leu Leu Val 115 gaa aac act cgt Glu Asn Thr Arg ttt Ph e 120 gaa gat gtt gac Giu Asp Val. ASP ggi Gi y 125 tct aag Ser Lys 130 aat gac gaa gaa Asn Asp Glu Glu ct Leu 135 ggt aaa tac tgg gct ica cit gga gai Gly Lys Tyr Trp Ala Ser Leu Giy Asp 140 gga Gi y 145 atc tt git aac Ile Phe Val. Asn gat Asp 150 gca ttt ggt aca Ala Phe Gly Thr gca Al a 155 cac cgi gct cat His Arg Al1a Pis gca Al1 a 160 tca aac gta ggt Ser Asn Vai Gly att Ile 165 tca gca aac gtt Ser Al1a Asn Vai aaa gci gta gct Lys Ala Val Ala ggt tic Gly Phe 175 WO 00/37490 WO 00/74 90PCT/GB99/04376 ctt -ctt Leu Leu gas cgc Giu Arg att ggt Ile Gly 210 gaa sac Giu Asn 180 gaa att gct tac Giu Ile Ala Tyr atc Ile 185 caa gsa gca gtt Gin Giu Ala Vai gas act cca Giu Thr Pro 190 tct gat aag Ser Asp Lys cca Pro 195 ttc gta. gct att Phe Val Ala Ile ggt ggc tcs ass Gly Giy Ser Lys gtt Vai 205 57 6 624 67 2 gtt atc gas sac Val Ile Giu Asn ctt gas ass. gct Leu Giu Lys Ala gat Asp 220 ass, gtt ctt atc Lys Val Leu Ile ggt ggt ggt Giy Gly Giy 225 atg act tac Met Thr Tyr 230 aca ttc tac asa Thr Phe Tyr Lys gct Ala 235 caa ggt atc gas Gin Gly Ile Giu ggt sac tcs ctt Giy Asn Ser Leu gta Val 245 gsa gsa gac ass Giu Giu Asp Lys tt g Leu 250 gat gtt gct ass Asp Val Ala Lys gac ctc Asp Leu 255 ctt gas, ass Leu Giu Lys gca. aac gca Al a Asn Al1a 275 tca Ser 260 sac ggt ass. ttg Asn Gly Lys Leu atc Ile 265 tig cca, gtt gac Leu Pro Val Asp tcs asa ga Ser Lys Giu 27 0 gsa ggt ga Giu Giy Giu ttt gct ggt tat Phe Ala Giy Tyr act Thr 280 gas. gtt cgc gac Giu Val Arg Asp act Thr 285 gca gtt Ala Val 290 tcs, gas ggg ttc Ser Giu Giy Phe ctt Leu 295 ggt ctt gac sic Giy Leu Asp Ile ggt Gi y 300 cct ass. tca. atc Pro Lys Ser Ile gct Ala 305 as ttt gst gas Lys Phe Asp Giu gca Ala, 310 cit act ggt gct Leu Thr Gly Al1a

555. Lys 315 aca. gtt gta tgg Thr Vai Vai Trp sac As n 320 912 960 1008 ggs. cct atg ggt Giy Pro Met Giy gt c Vsi 325 itt gsa sac cct ?he Giu Asn Pro ga c Asp 330 tic caa. gct ggt Phe Gin Al1a Giy sca atc Thr Ile 335 ggt gta atg Giy Val Met ggt ggt ggt Gly Giy Giy 355 gct atc gtt ass Als ale Val Lys cca agc gtt Pro Giy Vai aaas tca atc aic Lys Ser Ile Ile 350 ggt cgt gct gac Giy Arg Ala Asp 365 1056 1104 gat tcs gca. gca Asp Ser Ala Ala gct Ala 360 gct atc sac. ct Ala Ile Asn Leu WO 00/37490 aaa ttC tca Lys Phe Ser 370 PCT/GB99/04376 tgg Trp atc tct act lie Ser Thr 375 ggt ggt 9ga gca Gly Gly Gly Ala atg gaa ttg ctc Met Giu Leu Leu 1152 gaa ggt aaa gia tta cca Glu Gly Lys Val Leu Pro 385 390 ggt ttg gca gca Gly Leu Ala Ala ttg Leu 395 act gaa aaa taa Thr Giu Lys 1197 <210> 4 <211> 398 <212> PRT <213> group B streptococcus <400> 4 Met Ala 1 Lys Leu Thr 5 Val Lys Asp Val Asp 10 Leu Lys Val Lys Lys Val Ile Thr 1eu Val Arg Asn Asp Asn Val Asp Phe Asn Val Pro 25 Leu Lys Asp Gly Val Arg Ile Thr Ala Leu Pro Thr Ile Tyr Ile Ile Glu Gin Gly Gly Arg Ala Ile Leu Phe Ser His Gly Arg Val Lys Glu Ala Asp Lys Gly Lys Ser Leu Al a Pro Val Ala Ala Asp Leu Ala Ala Lys Leu Gly Gin Asp Val Val 90 Phe Pro Giy Val Thr Arg Gly Ala Lys Leu Leu Vai 115 Leu 100 Giu Giu Ala Ile As n 105 Ala Leu Giu Asp Gly Gin Val 110 Lys Lys Glu Glu Asn Thr Arg Phe 120 Glu Asp Val Asp G1y 125 Ser Lys 130 Asn Asp Giu Glu Leu 135 Gly Lys Tyr Trp Al a 140 Ser Leu Giy Asp Giy 145 Ile Phe Val Asn Asp 150 Ala Phe Gly Thr Ala 155 His Arg Ala His Al a 160 Ser Asn Val Gly Ile 165 Ser Ala Asn Val Glu 170 Lys Ala Val Ala Gly Phe 175 WO 00/37490 Leu Leu Glu PCT/GB99/04376 Asn 180 Giu Ile Ala Tyr Ile Gin Giu Ala Vai Giu Thr Pro 185 190 Glu Arg Pro 195 Phe Val Ala Ile Gly Giy Ser Lys Val 205 Ser Asp Lys Ile Giy 210 Val Ile Glu Asn Leu 215 Leu Giu Lys Ala Asp Lys Val Leu 220 Gin Gly Ile Glu Ile Ile 240 Gly 225 Gly Giy Met Thr Thr Phe Tyr Lys Gly Asn Ser Leu Val 245 Giu Giu Asp Lys Leu 250 Asp Val Ala Lys Asp Leu 255 2eu Giu Lys Ala Asn Ala 275 Ser 260 Asn Giy Lys Leu Leu Pro Val Asp Ser Lys Glu 270 Glu Gly Glu Phe Ala Giy Tyr Glu Val Arg Asp Thr 285 Ala Val 290 Ser Giu Giy Phe Leu 295 Giy Leu Asp le Gly 300 Pro Lys Ser Ile Ala 305 Lys Phe Asp Glu Leu Thr Giy Ala Thr Val Val Trp Gly Pro Met Gly Val 325 Phe Glu Asn Pro Asp 330 Phe Gin Ala Giy Thr Ile 335 Gly Val Met Gly Gly Giy 355 Asp 340 Ala Ile Val Lys Pro Giy Vai Lys Ser Ile Ile 350 Arg Ala Asp Asp Ser Ala Ala Ala 360 Ala Ile Asn Leu Gly 365 Lys Phe 370 Ser Trp Ile Ser Thr 375 Gly Giy Gly Ala Ser 380 Met Glu Leu Leu Gly Lys Vai Leu Pro 390 Gly Leu Ala Ala Leu Thr Giu Lys 395 <210> <211> <212> <213> 516 DNA group B streptococcus WO 00/37490 WO 0037490PCTIG B99/04376 <220> <221> CDS <222> (516) <400> atg aca Met Thr I cat att. aca ttt gac tia ttc His Ile Thr Phe Asp Leu Phe 5 gtc ttg ggt caa Val Leu Gly Gln itt gta Phe Val ggc gaa cat Gly Giu His gct ttc ctt. Ala Phe Leu gag Gi u ita gac tat cia Leu Asp Tyr Leu cca Pro 25 cta caa gta agt Pro Gin Val Ser gca gca gat Al a Al a As p ttc gga tgg Leu Gly Trp cgt caa ggg act Arg Gin Giy Thr gigt. Gi y 40 cci gga ica gat Pro Gly Ser Asp tt Phe atg gaa Met Giu cci cca gaa aat Pro Pro Giu Asn gat aaa gaa gaa Asp Lys Giu Giu itt ict cgc ati Phe Ser Arg Ile gia tc gtg gt Val Leu Val Val caa Gin att Ile aaa Lys gtt gct gaa aag Ala Ala Giu Lys ati Ile '70 aaa ica gat agc Lys Ser Asp Ser gaa Giu 75 ggt ati ggt ggt Giy Ilie Gly Giy tcg Ser tat cit ggt gta Tyr Leu Giy Ala aaa Lys 90 gca gta att gat Ala Ala Ile Asp tit tig Phe Leu aat aat cat Asn Asn His tag att ctt Gin Ile Leu 115 tt Phe 100 gci aat tig caa Ala Asn Leu Gin act Thr 105 gca gaa gaa cgi Ala Giu Giu Arg aaa gcg cci Lys Ala Pro 110 cit gcc gat Leu Ala Asp tat gci gga aat Tyr Ala Giy Asn tct Ser 120 ati tca tct. act Ile Ser Ser Thr tat Tyr 125 tta gti Leu Val 130 gaa iac gic caa Giu Tyr Vai Gin aaa gaa ttc ta Lvs Giu Phe Ser gia Val 140 aai gic ati tca Asn Val Ile Ser 432 480 aaa Lys 145 ica ggt aca aca 5cr Gly Thr Thr act Th r 150 gaa tta gcg ati Giu Pro Al1a Ile gci. Al1 a 155 ttc cgi gia iii Phe Arg Vai Phe aaa Lys 160 gaa cit cia git aaa aag tat cgg ica aga aga agt 516 Giu Leu Leu Val Lys Lys Tyr Arg Ser Arg Arg Ser WO 00/37490 WO 0037490PCT/GB99/04376 <210> 6 <211> 172 <212> PRT <213> group B streptococcus <400> 6 Met Thr 1 His Ile Thr 5 Phe Asp Leu Phe Lys 10 Val Leu Gly Gin Phe Val Gly Glu His Ala Phe Leu Gi u Leu Asp Tyr Leu Pro 25 Pro Gin Val Ser Ala Ala Asp Leu Gly Trp Arg Gin Giy Thr G1 y 40 Pro Gly Ser Asp Phe Met Giu Pro Pro Glu Asn Tyr Asp Lys Giu Glu Ser Arg Ile Gin Lys Ala Ala Giu Lys Lys Ser Asp Ser Val Leu Val Val Ile so Gly Ile Gly Gly Ser Tyr Leu Giy Ala Ala Ala Ile Asp Phe Leu Asn Asn His Gin Ile Leu 115 Phe 100 Ala Asri Leu Gin Thr 105 Ala Giu Giu Arg Lys Ala Pro 110 Tyr Leu Ala Asp 125 Tyr Ala Giy Asn Ser 120 Ile Ser Ser Thr Leu Val 130 Giu Tyr Vai Gin Asp 135 Lys Giu Phe Ser Val Asn Val Ile Ser 140 Ser Giy Thr Thr Thr 150 Glu Pro Ala Ile Al a 155 Phe Arg Val. Phe Lys 160 Giu Leu Leu Vai Lys 165 Lys Tyr Arg Ser Arg Arg Ser 170 <210> 7 <211> 318 <212> DNA <213> group B streptococcus PCT/GB99/04376 WO 00/37490 <220> <221> CDS <222> (318) <400> 7 ait aac cga aga lie Asn Arg Arg 1 aga tgg ict tgg tta tct tca aga aaa Arg Trp Ser Trp Leu Ser Ser Arg Lys 10 gat gta Asp Val gat tit gtt Asp Phe Val gat ggt ggg Asp Gly Gly aat Asn aaa aaa gca aca Lys Lys Ala Thr gat ggt A sp Gly 25 gtg ctt ctt Val Leu Leu gct cat aca Ala His Thr caa gaC gct Gin Asp Ala gtt cca aat atg Val Pro Asn Met ttt Ph e 40 gta acg ctt cct Val Thr Leu Pro aca Thr tac act Tyr Thr ctt ggt tac act Leu Gly Tyr Thr att Ile 55 tac ttc ttt gag Tyr Phe Phe Giu tta Leu gca att ggc ctt Ala Ile Gly Leu 192 240 ggt tat ctt aac Gly Tyr Leu Asn tca Ser gta aat cca ttt Val Asn Pro Phe ga t Asp 75 caa ccg ggg gta Gin Pro Gly Val gaa Giu gca tat aaa cgt aat atg ttc gca ttt Ala Tyr Lys Arg Asn Met Phe Ala Phe ggt Gi y 90 aaa cct gga ttc Lys Pro Gly Phe gaa gag Giu Giu 288 ctt age get Leu Ser Ala <210> 8 <211> 105 <212> PRT <213> group <400> 8 Ile Asn Arg 1 Asp Phe Val ga a Glu 100 ttg aat gca cgt Leu Asn Ala Arg ctt taa Leu 105 B streptococcus Arg Phe Arg Trp 5 Asn Lys Lys Ala Ser Trp Leu Ser Ser Arg Lys 10 Asp Val Thr Asp 25 Gly Val 1,eu Leu Ala His Thr Asp Gly Gly Val AspGlyGlyVaiPro Asn Met Phe Val Thr Leu Pro Thr Gin Asp Ala WO 00/37490 WO 0037490PCT/GB99/04376 Ala Ile Gly Leu Tyr Thr Leu Gly Tyr Thr Ile Tyr Phe Phe Giu 55 Leu Ser Gly Tyr Leu Asri Ser '70 Val. Asn Pro Phe Gin Pro Gly Vai Giu Ala Tyr Lys Arg Asn Met Phe Ala Phe Gi y 90 Lys Pro Gly Phe Glu Glu Leu Ser Ala Gl.u 100 Leu Asn Ala Arg <210> 9 <211> 804 <212> DNA <213> group B streptococcus <220> <221> CDS <222> (804) <400> 9 aig aca Met Thr I-1 tta tta gaa Leu Leu Giu 5 aaa att. aat gag Lys Ile Asn Glu act Th r 10 aga. gac ttt ttg Arg Asp Phe Leu caa gca Gin Ala. aaa ggc gic Lys Gly Val. gga gaa ttg Gly Giu Leu a ca Thr gca cca gaa ttt. Ala Pro Gi1u Phe ggy Xaa 25 ctt att tta ggc Leu Ile Leu Gly tct ggt tta. Ser Gly Leu gat tat gca Asp Tyr Ala gct. gaa gaa atc Ala Glu Giu Ile gaa Gi u 40 aat cct ati gtt Asn pro Ile Val gt g Val gac atc Asp Ile ccrn aat tgg gga Xaa Asn Trp Gly cag Gin 55 tca aca gta gtt Ser Thr Val Val ggt Gi y cat gct gga. aaa His Ala Gly Lys 192 240 tt Phe agt gia tgg gat Ser Va). Trp Asp tca ggc cgt aag Ser Gly Arg Lys gia Val. '75 tta gcg ctt caa Leu Al1a Leu Gin ggt Gi y cgt ttt cat ttt tay gaa ggw aat aca atg9 gaa gtc gtt act ttc cca Arg Phe His Phe Tyr Giu Xaa Asn Thr Met Giu Val Val. Thr Phe Pro 288 PCT/GB99/04376 WO 00/37490 gta cgt atc Val Arg Ile gca gcg ggt Ala Ala Gly 115 atg Met 100 aga gca ttg gct Arg Ala Leu Ala tgc Cys 105 cac agt gtg ctt His 5cr Val Leu gig act aai Val Thr Asn 110 ctg atc aaa Leu Ile Lys ggg att gga tac Gly Ile Gly Tyr gga Gi y 120 cca gga act tta Pro Giy Thr Leu atg Met 125 gac cac Asp His 130 atc aat atg ati Ile Asn Met Ilie ggg Gi y 135 act aac cci cic Thr Asn Pro Leu ata Ile 140 ggt gag aac ct G1iy Giu Asn Leu gaa Giu 145 gaa iii gga cca Giu Phe Gly Pro cgt Arg 150 tic cca gac aig Phe Pro Asp Met icg 5cr 155 gat gct tay aca Asp Ala Tyr Thr 432 480 528 aca tat cga caa Thr Tyr Arg Gin gct cac caa at Ala His Gin Ile gct Al. a 1,70 gaa aac gat atc Giu Asn Asp Ile aaa ctc Lys Leu 175 gaa gaa ggt Giu Giu Gly gca gaa ati Ala Giu Ile 195 gig Val 180 tac tig ggt gta Tyr Leu Giy Val tca 5cr 185 gga ccc act tat Gly Pro Thr Tyr gaa aca cct Giu Thr Pro 190 gt~a ggt atg Vai Gly Met cgt gca tic caa Arg Al1a Phe Gin a ca Thr 200 atg ggc gca caa Met Gly Ala Gin g cg Al. a 205 tcc acg Ser Thr 210 gtt cca gag gig Val Pro Giu Vai aic Ile 215 gtt gca gct cac Val Al.a Al a His ica Ser 220 ggg ctt aaa gig Gly Leu Lys Val gga att ica gca Giy Ile Ser Ala att le 230 act aac cit gcc Thr As n Leu Ala gct Al1 a 235 ggc tic caa ica Giy Phe Gin ser gag Glu 240 672 '72 0 '768 ctc aat cat gag Leu Asn His Giu gag Giu 245 gtc gtt gaa gt Val Val Giu Val act Thr 250 cag cgi att aaa Gin Arg Ile Lys gaa gat Giu Asp 255 ttc aag gga Phe Lys Gly ggt aaa ica tta Giy Lys 5cr Leu gtt Vai 265 gct gaa ctc Al1a Giu Leu <210> WO 0037490PCT/GB99/04376 WO 00/37490 Leu Asn His Glu Giu Val Val Giu 245 Val Thr 250 Gin Arg Ile Lys Giu Asp 255 Phe Lys Gly Leu 260 Gly Lys Ser Leu Val Aia Giu Leu 265 <210> <211> <212> <213> <220> <221> <222> 11 1428 DN~A group B streptococcus CDS (1)..(1428) <400> 11 ttg aca aaa gaa tat Leu Thr Lys Giu Tyr 1 5 caa aat tat gtc Gin Asn Tyr Vai aat Asn 10 ggC gaa tgg aaa Giy Giu Trp Lys tca tct Ser Ser gtt aat cag Val Asn Gin att 1.1e gag att ttg tca Giu Ile Leu Ser cca Pro 25 att gat gat tot Ile Asp Asp Ser tca ttg gga Ser Leu Giy atg aaa gcg Met Lys Ala ttc gtg oca Phe Val Pro gcg atg act oga gaa gaa gtt gat cat Ala Met Thr Arg Giu Giu Val ASP His 40 goct Al a ggt cgt Gly Arg gag gct tta oca Giu Ala Leu Pro got Al1 a 55 tgg gct got tta Trp Ala Ala Leu aca Thr gta tat gaa cgt Val Tyr Glu Arg gca Al a oaa tac ctt cat GIn Tyr Leu His aaa Lys '70 gcc goa gac at Ala Ala Asp Ile at Ile 75 gaa cgt gat aaa Giu Ara Asp Lys gaa Giu 192 240 288 gaa att gct aot Glu Ile Ala Thr gt Val tta gca aaa gaa Leu Ala Lys Giu at Ile 90 tot aaa got tac Ser Lys Ala Tyr aat got As n Ala toa gta act Ser Val Thr gag Giu 100 gtt gta agg aca Val Val Arg Thr got Al1 a 105 gat Ott att cgt Asp Leu Ile Arg tat goa goa Tyr Ala Ala 110 PCTIGB99/04376 WO 00/37490 <211> 268 <212> PRT <213> group <400> 10 Met Thr Leu 1 Lys Gly Val Gly Giu Leu B streptococcus Leu Giu Lys lie Asn Giu Thr Arg Asp Phe Leu Gin Ala Thr Ala Pro Giu Phe Leu Ile Leu Gly Ser Gly Leu Asp Tyr Ala Ala Glu Giu Ile Asn Pro Ile Val Asp Ile Xaa Asn Trp Gly Ser Thr Val Val Gly His Ala Giy Lys Ser Vai Trp Asp Leu Ser Giy Arg Lys Leu Ala Leu Gin Giy Pro Arg Phe His Phe Tyr Giu Xaa Asn Thr Met 90 Glu Vai Vai Thr Phe Val Arg Ile Ala Ala Giy 115 Met 100 Arg Ala Leu Ala His Ser Val Leu Vai 110 Thr Asn Gly Ile Gly Tyr Giy 120 Pro Gly Thr Leu Met 125 Leu Ile Lys Asp His 130 Ile Asn Met Ile Gly 135 Thr Asn Pro 1eu Ile 140 Gly Giu Asn Leu Glu Phe Giy Pro Arg 150 Phe Pro Asp Met Ser 155 Asp Ala Tyr Thr Ala 160 Thr Tyr Arg Gin Lys 165 Ala His Gin Ile Al a 170 Glu Asn Asp Ile Lys Leu 175 Glu Giu Gly Ala Giu Ile 195 Val 180 Tyr Leu Giy Val Gly Pro Thr Tyr Arg Ala Phe Gln Thr 200 Met Giy Ala Gin Ala 205 Ser Gly 220 Glu Thr Pro 190 Val Gly Met Leu Lys Val Ser Thr 210 Val Pro Giu Val Ile 215 Val Ala Ala His Leu Gly Ile Ser Ala Ile Thr Asn Leu Ala Ala Giy Phe Gin Ser Glu PCT/GB99/043 76 WO 00/37490 gaa gaa gga G2.u Giu Gly 115 att cgt tta tea lie Arg Leu Ser act Thr 120 tca get. gac gaa Ser Al1a Asp Glu ggt Gi y 125 gga aaa atg Gly Lys Met gat get. Asp Ala 130 tea aea ggt cat Ser Thr Giy His aag Lys 135 ttg get gtt. att Leu Al1a Val Ile egt Arg 140 egt eaa eea gta Arg Gin Pro Val ggt G1 y 145 ate gtt tta gea Ile Vai Leu Ala gea ect tat aat Al a Pro Tyr Asn ect. gtt aae etc Pro Vai Asn Leu 432 480 528 gga tea aaa att, Giy Ser Lys Ile geg Al1 a 165 eca get eta att Pro Leu Ile ggt, Gi y 1 7 0 gga aac gtt gtg Gly As n Val Val atg ttt Met Phe 1,75 aaa cea eea Lys Pro Pro ttt gca gaa. Phe Al a Giu 195 aca Thr 180 caa ggt tea, gtc GIn Gly Ser Val. tea Ser 185 gga. ett. gtt tta. Gly Leu Vai Leu gea aaa get Al a Lys Ala 190 att. aca gga Ile Thr Gly gea ggt ctt eca Ala Giy Leu Pro ga 200 ggt gtc ttt. aat Gly Vai Phe Asn act Thr 205 ege ggt Arg Giy 210 tet gag att gga Ser Giu Ile Gly gat tac Asp Tyr 215 att gtt gag Ile Val Glu eat His 220 gaa gaa gtt aat Giu Giu Val Asn att, aac ttt aca lie Asn Phe Thr gga Gi y 230 tea aeg cca gtt Ser Thr Pro Vai eaa egt att, ggt Gin Arg Ile Giy aag Lys 240 672 720 768 ttg gea, gga atg Leu Ala Gly Met ggt ate gte ita. Gly Ile Vai Leu 260 gtt gea ggt get Val Ala Giy Al1a 275 egt Arg 245 eea att, atg ctt Pro Ile Met Leu gag Giu 250 ttg gge ggt aag Leu Gly Giy Lys gat gca Asp Al a 255 get. gat get gac Al a Asp Al1a Asp c tt. Leu 265 gat aac get get Asp Asn Al a Al1a aaa eaa ate Lys GIn Ile 2,70 gca att aag Ala le Lys 816 864 tat gat tae Tyr Asp Ty tct. Ser 280 gga eaa ege tgt Gly Gin Ar-g Cys aeg Thr 285 e gt Arg gtg Val 290 ctt gte gtt gaa Leu Vai Val Giu gaa Gi u 295 gtt. gew gat gaa Val Xaa Asp Glu ttg Leu 300 gea gaa aaa ata Al1a Glu Lys Ile 912 PCT/GB99/043 7 6 WO 00/37490 tct Ser 305 gaa aat gta gca Glu Asn Val Ala tta tca gta ggt gat cca tit gat aat gca Leu Ser Val Gly Asp Pro Phe Asp Asn A.1a 320 acg gtg aca ccg Thr Val Thr Pro gtt Val 325 att gat gat aat Ile Asp Asp Asn tca gct gac ttt att gaa agc Ser Ala Asp Phe Ile Giu Ser 330 335 1008 tta gta gia Leu Val Val ga t A-sp 340 gca cgt caa aaa Ala Arg Gin Lys gag aaa gaa ttg A-1a Lys Giu Leu aai gaa ttt Asn Giu Phe 350 cat gtt act His Val Thr 1056 aaa cgt Lys Arg tta cat Leu Asp 3*70 gat As p 355 ggt cgt cta tta Gly Arg Leu Leu act Thr 360 cca gga ttg tt Pro Gly Leu Phe gat Asp 365 aig aaa cia gct Met Lys Leu Al1a tg g Trp 375 gaa gag cct ttt G1u Giu Pro Phe gga Gi y 380 cca att ctc eca Pro Ile Leu Pro 1104 1152 1200 1248 at Ile 385 ati cgt gtc aag Ile Arg Val Lys gat Asp 390 gca gaa gaa gci Ala Giu Glu Ala gt Val 395 gct ati gcc aac A1a Ile Al1a Asn aaa Lys 400 tct gat i~tt. gga Ser Asp Phe Gly tta Leu 405 caa tca ica gic Gin Ser Ser Vai ttt Phe 410 aca cgt. gat ttc Thr Arg Asp Phe caa aaa Gin Lys 415 gca ttt gat Ala Phe Asp aia Ile 420 gca aai aaa att Al a Asn Lys Leu gaa Giu 425 gtt ggt aca gtt Val. Gly Thr Vai cac att aac His le Asn 430 gga ctc aaa Gly Leu Lys 1296 aai aag Asn Lys gga tat Gly Ser 450 act Thr 435 gga cgt ggt ccw Gly Arg Gly Xaa gat Asp 440 aai. itc cca ttc A-sn Phe Pro Phe tta Leu 445 1344 ggt gca ggt gtt caa ggt aic aga Giy Al1a Gly Val Gin Giy Ile Arg 455 tat ica att. gaa gca atg Tyr Ser Ile Giu Ala Met 460 1392 1428 a ca Thr 465 aat gia aaa tag Asn Val Lys Ser gtt cia gat ai~g Val Leu Asp Met aaa tag Lys 475 <210> 12 <211> 475 <212> PRT PCT/GB99/04376 WO 00/37490 <213> group B streptococcus <400> 12 Leu Thr Lys 1 Val As Gin Phe Vai Pro Giv Arg Glu Gin Asn Tyr Val Asn Gly Glu Trp Lys Ser Ser Glu Tyr Ile Glu Ile Leu Ser Ai a Pro 25 Glu Ile Asp Asp Ser Met Thr Arg Glu 40 Trp Vai Asp His Ala Val Ser Leu Gly Met Lys Ala Tyr Gu Arg Ala Leu Pro Ala Ala Leu Ala Gin Thr Glu Tyr Leu His Ala Asp Ile Glu Ile 75 Ser Arg Asp Lys Glu Ile Ala Thr Val Val Ala Lys Glu Ile 90 Asp Lys Ala Tyr Asn Ala Ser Val Thr Glu Glu Gly 115 Asp Ala Ser Glu 100 Ile Vai Arg Thr Ala 105 Ser Leu Ile Arg Tyr Ala Ala 110 Gly Lys Met Arg Leu Ser Thr 120 Leu Ala Asp Glu Gly Thr Gly His 130 Glv Ile Lys 135 Ala Ala Val Ile Arg 140 Pro Arg Gin Pro Val Val Leu AIa 145 Gly Ile 150 Pro Pro Tyr Asn Tyr 155 Giy Val Asn Leu Ser 160 Ser Lys Ile Ala 165 Gin Ala Leu Ile Gly 170 Giy Asn Val Val Met Phe 175 Lys Pro Pro Phe Ala Glu 195 Arg Gly Ser 210 Thr 180 Ala Gly Ser Val Ser 185 Giy Leu Vai Leu Gly Leu Pro Al a 200 Tyr Val Phe Asn Thr 205 Glu Ala Lys Al a 190 Ile Thr Gly Glu Val Asn Glu Ile Gly Asp 215 Ile Val Glu His 220 Phe 225 Ile Asn Phe Thr Giy 230 Ser Thr Pro Val Gly 235 Gin Arg Ile Gly Lys 240 PCT/GB99/04376 WO 00/37490 Leu Ala Gly Met Arg Pro Ile Met Leu 245 Glu Leu 250 Gly Gly Lys Asp Ala 255 Gly Ile Val Val Ala Gly 275 Leu 260 Ala Asp Ala Asp Leu 265 Asp Asn Ala Ala Lys Gin Ile 270 Ala Ile Lys Ala Tyr Asp Tyr Ser 280 Gly Gin Arg Cys Thr 285 Arg Val 290 Leu Val Val GlU Val Xaa Asp Glu Leu 300 Ala Giu Lys Ile Ser 305 Glu Asn Val Ala Leu Ser Val Gly Asp 315 Pro Phe Asp Asn Al a 320 Thr Val Thr Pro Val 325 Ile Asp Asp Asn Ser 330 Ala Asp Phe, Ile Glu Ser 335 Leu Val Val Lys Arg Asp 355 Asp 340 Ala Arg Gin Lys Giy 345 Ala Lys Giu Leu Asn Giu Phe 350 His Val Thr Gly Arg Leu Leu Thr 360 Pro Gly Leu Phe Asp 365 Leu Asp 370 Met Lys Leu Ala Trp 375 Glu Giu Pro Phe Gly 380 Pro Ile Leu Pro Ile Arg Val Lys Asp 390 Ala Giu Giu Ala Ala Ile Ala Asn Lys 400 Ser Asp Phe Gly Gin Ser Ser Vai Phe 410 Thr Arg Asp Phe Gin Lys 415 Ala Phe Asp Asn Lys Thr 435 Ile 420 Ala Asn Lys Leu Val Gly Thr Vai His le Asn 430 Gly Arg Gly Xaa Asn Phe Pro Phe Leu Gly Leu Lys 445 Gly Ser 450 Gly Ala Gly Val Gin 455 Gly Ile Arg Tyr Ser Ile Giu Ala Met 460 Asn Vai Lys Ser Ile 470 Val Leu Asp Met Lys 475