WO2001073037A2 - R5 protein, a cell-surface protective antigen of group b streptococci - Google Patents

Abstract

The invention relates to R5 protein, a cell-surface protective antigen of group B streptococci, a DNA-Sequence coding for R5 protein and compositions comprising R5 protein useful for vaccination against infection with group B streptococci.

Description

R5 protein, a new cell-surface protective antigen of group B streptococci

The invention relates to R5 protein, a new cell-surface pro- tective antigen of group B streptococci, a DNA-Sequence coding for R5 protein and compositions comprising R5 protein useful for vaccination against infection with group B streptococci.

A list of the referenced literature with detailed bibliographic data is given at the end of this description.

Introduction

Group B streptococci (GBS) have emerged as an important cause of infection in neonates characterized by a high mortality rate even in developed countries (Baker and Edwards, 1995). In the United States alone more than 15,000 cases and 1,300 deaths due to GBS occur each year (Zangwill et al., 1992). The problem of GBS infection of neonates lies in the fast and dramatic course of infection that can only be treated inadequately with antibiotics (Hall et al., 1976). Since many women of child-bearing age have vaginal GBS colonization, they are tested during pregnancy for carriage of GBS (Pass et al., 1979). Besides affecting neonates, GBS cause a number of maternal peripartum diseases and are responsible for serious illness in non-pregnant adults (Farley et al., 1993). Necro- tizing fasciitis and toxic shock like syndrome due to GBS has recently been reported in adults (Gardam et al., 1998).

GBS strains comprise nine serotypes based on the presence of specific capsular polysaccharides. Of these serotypes la, lb, II and III have been more prevalent. Approximately 40% of the isolates from cases with invasive GBS disease express la po- lysaccharide and 27% express type III polysaccharide (Lin et al., 1998). Because of the increasing antibiotic resistance and the restriction of antibiotic therapy during pregnancy (Pearlman et al., 1998), it is desirable to develop alterna- tives to antibiotic therapy. Clinical studies have shown that newborns whose mothers had high titers of anti-GBS antibodies were seldom infected (Baker and Kasper, 1976) An attractive alternative to classic antibiotic therapy could be vaccination of women of child-bearing age to protect newborns against GBS infection (Baker et al., 1988). Since GBS capsule plays an important role in the virulence, attempts have been made to develop a vaccine based on capsular polysaccharides. However, these vaccination studies were unsuccessful because of antigenic variation and the low immunogenicity of capsule po- lysaccharides. A potential solution to this problem can be the use of glycocojugates as candidate vaccines (Wessels et al. 1998). Because of suboptimal immunogenicity of capsule-based vaccines, interest has shifted towards the surface proteins antigens of GBS as vaccine candidates or carrier proteins for specific GBS polysaccharides. These antigens include α and β antigen of the c protein complex (Jerlstrδm et al., 1991; Ma- doff et al., 1991), an α-like protein (Kvam et al., 1993), the R proteins (Flores and Ferrieri, 1989) and protein Rib (Stalhammar-Carlemalm et al., 1993). R proteins are cell surface proteins of GBS that are resistant towards certain proteases (Flores and Ferrieri, 1989). In animal models c protein antigens were protective (Valtonen et al., 1986). The c protein antigens are expressed in 90% of types la and lb isolates and by 50% of type II invasive isolates, however, they are rarely expressed by type III GBS (Ferrieri and Flores, 1997).

Rib protein also confers protective immunity in mice (Stalhammar-Carlemalm et at., 1993). Other R proteins are also biologically important and a correlation between low levels of maternal antibodies to R proteins and neonatal septicaemia has been reported (Linden et al., 1983). Four species of R protein, R1-R4, have been identified (Flores and Ferrieri, 1989,1996; Wilkinson, 1972). This invention describes a new species of R-protein designated R5 that is expressed by a number of clinically relevant GBS serotypes and is protective antigen in a mouse model.

In one aspect the invention relates to a DNA sequence compri- sing a DNA sequence selected from the group consisting of:

(a) the following DNA sequence: 10 20 30 40 50 60 70 -: SO

I234557a90 1234557390 12315.7330 liHi»» 12145S7330 133-1557330 12HSS7»3 12:i:i7.3: 123t5S73?0

AAACTCΓ TT TAAΓΓACCΓT TATATTCGAT AOAACTATCC AATCTOTTTT ATC AΛACTA CCACGT ΓΓA C7AT 7A?TT TTTΛATTTAT CTTCAATGAG CX-A CTCΓΓT ACTTTTATGT AAA-TΓCAAAA TATTCTTAVA τtτ-/Αi Aτ TTT AΛAΓTT

ACGCTITrAT UTILCTTCT TETrfAAATG TAAAAACTCT TACACGAAAC ACCAACATAT TATCTTTCGT CAA7A7AAT7 ΓTCAAAAAGG 270

M P R 0 ϊ N P E K G

TrrAAArrr. TCCATTCCTA AATTTTCCCT TOCAATAGCT TCACTTC T TACCATCATΓ ATTATTCATT TTA-— CA C TACTTGCACA 350

L N F S I R K F S V C I A S V A I C S C. L p r L ? Q V L A D TCAJOACAA T AG GTTACAT CTCCAACAA T CAACACCT CTAACCA Aλ ACAT AAA CTTGGTCAAT CC7; :A TT CΛλCTCCTAC 45

E T T S V T S A T T P T C V T T T D A N L V N P ^*N N S T P T TTCCACTAA ACCACTCCAA CTACCAC CA ACCAAGTAAT TTOAGTAATA CTTCACAAAT CATTAACCCT CCAA'- :TA.O CA CAACATC S»O

S T N R S A T S T Q C S N L S N T S E I I K p A 7 L A λ T S ACCAACAACT CATAATGTAG CCCCATCAGT AC.AJC_W-.AOG ACC7AT C A CTACTCCCGA TTCCACGTTA CW;:^T^T AΓOCTCΛCAG 630

P T T D H V A P S V D K R T Y A 7 S C D W T L Q N ? Y A D S TCTTCCAAAT AAAAATATTΓ CT CAACTCT TCC CATCAA TCATTTAAAA CTCCTCAAΛC AACCCTCCTT CCTC - ΓA A TCAACCCT 7-Ό

V R N K N T S P S V R H Z S P K S A E T T V V P. K 0 N S T V 7ΛAAOTGACT CCCACAATTA CTCCTGTAGA AGCGAATSAT CAAGCTT TC CTATTTTCAC AAATGCTCGT AT ΛCR^O AATATAAACC 810

K V T A T I T P V E C H D E C S C I L T N C C N Q Ξ E Y K A AA AT ACAA ATCTTTGTTC CAAATGTACA TCCTCCAλλλ ATACCTCCT7 TACCrrGTCTA TACλ AACCA CGA- TArr AACCACGTAC 9C0

T S E M P V C N V D P A K I P A L G V Y T Q p G ?. T E C G S TAAACTATCT CATAAGCTCA ATTTTAATGG GAACGCC CA TCAA TATTT TAACATT AA ATTTCACAAC GO..T7ACAC ACCCTΛTTAT 930

K L S D K N P N G K A P S T I L T L K F D K A V 7 D ? I 1 TGAXTTATCT CGTGT GGA CTAATCCACG TCTATCATTT ACλCAλΛCTC TCAP3GAAAA TOGTAACATT GTTi_AAAAAT TTCλCTATGC 1030

D S G V G G N A R L S F T E T V M E N G * _ V E X P D Y A ACCTCCTTCA TATAATTCAA CGTTCTTTGA TGCAATAACT C ACCTATAA CCCTTGAAλλ AG AAGTTCA GGAGT-TAATT TAACAGTTAC 1170

R C S Y N S T F F D C I T P C I S L E K A S S G V . L T V T AGCGAΛTACG CTTGΛTGTAA CAGACAACAλ CAC TTTAAT CA3T AST7G TTAATCCATC TGACGAAGAC TTTTT AAT GTCCAGATAG 1250

A N T V D V T D K N T P N E S V V N P S D E D P V .N G P D R AACCCCTCAT c GT AG cccGAACACG CTCAATCCGT TTAλλAGGAA CCTTTACAGA CG TTCATTT AAA77A7ATC A CAACCTGT 13 SO

T P D A V P A C T C S I P. L K C 7 F T D A S P K Y H Q A V TCCATCTACA CCATTTTCTA AACAAAAATA TCATACACCA CATCGATATT A AATASTAT TGCCAATGTT CCΛr AA T. TA3TTAAACC 1440

P S T A F S K E K Y H T G D C Y Y N S I A N V R P T V K P ASATAG ATT AArCGT TAA ATAAGCACCC AAGTGATGTT ATTGATT TA AAATT CGA TAATAATGAT ATTrπ-AACG ATGACTTACT 1530

D S I N G L N K H A S D V I D Y K I S D N N D I S _Ϊ. D D L L

T GT TλTcr CTΓTCG XAC AAAATCCCCG TCGATCAGTT GTΓCTAAATT ATATTGATAC AGAACGCAAT AττλT τrA CTGAATACAA IS 20

R S V R L U N P R C S V V V N Y I D T E G N I I C T E Y K ACATACCACC GATGCAATTC CΛ3GAA ACA TTACAATACG GCACAATCAT CAC ACAC T TAATTCAGA GCCACAJTTG AC GTCCTTC 1710

D T T D A I P G T H Y N T A E S S G D L N S D A T V E R P S CΛCTλTTλ T AAACA CGTA-AGGTGTATCA TTΓAOTACCA CAAAATATTA CAtTTG CAiTr TCGTAAGGTT

CTACCTTCG ^' 1»30

T I T D G K V Y D L V A E M I T V P C R v N 3 0 C T L A TACAAATCCG AGCTCATTTA ATTATCCTA TGATCCAOCA T TO ACAAG TTCCAGAAX; TA TAAGTCA CTAArrTAT- T7ATTAGCAT 1330 N G S S P >τ ' Y G T D λ λ S A E V A E G T K S V I Y V I S I

TAAA AAGAA TCAAAcSϊjA. ATGTTCATGC CCGCTATGTT ATTTTACGCA CAGAGACTCA CrrTCCAACT G rA-VACAC TAAATCAGA 1930

K Q E S K G N V H A R Y V I L G T Ξ T E L A S A K T K S E ACCrCCAATT CATCAGGCAT ATTCACATAA ACCACCTCCλ ACTCTT AAA AAGATOGTAA GTTGTATGAA TTT-TA AT_J T GTGATAA 2070

A P T D E A Y S D K A P A T L E K D C K Y E F V H V R D M TAAGCCCCAT CCTCCAOCAC ATGGTAACGT GACTCAACAA CUTCAOACCA TTACATATGA ATATGTTCAA CTT TT.4AC CTCCTGTCCT - 2150

K G D A P A D C K V T E Q D Q T I T Y E Y V E V P K C R V V TGTACATTAT GTTATCGAGG GTACTGCTA AAAACCTAAA GATACTTATC T C TACCXC AACACCTTAT ATTT- ATA AAGAAGGTCA 2250

V D Y V I E G T A T K P K D T Y V D T P T λ Y I R 0 S E C Q ACCTATTC T TATAATA AC CTCAGAATCA TTCTCACAAA CCATTGTTA TTGACAAAOA TCCAATAAAA AT 7A3 TTTCAATTCA 2340

A I P Y N T A E N D S E K P L L L Ω Z D C 7. K Y Ϊ L V S I 0 AL-V.GCGTCA CCACCGGACA AACGAACA T TCCACAAGCG GAACAACATC TTGT 7AT A ATAT GTAAA CT GTλ ACO TT CAACTGT 2430

E G S A A E K G T L R E C E Q H V V Y Q Y R K V Ξ V P S V TAAAG TEH AATGTT ATC CCCGCTATCT TATTTTAC G ACλ AGA TC ACCTTGCAAG TCCTAAAACA CTT AA7 A AACCTCCAAT 2520

K V C N V H λ R Y V I L G T Ξ T E L A S A R T V S S E A P I TCATGACG A TATT AGATA AAGCACCTCC AACT TTCAA AA.- AT33TA AOrrGTArGA ATTTCTACAT

A7AACCC CA 2S10

D E A Y S O K A P A T L E K. D C K L Y E P V H X S K G D TCCTC AGCA GATCGTAArr:

ACATCACACC ATTA ATATC AATATAACCT TAAOAAAGAT

CTGTA CAA 2700

A P A D G K V T E Q D Q T I T Y E K L K K D D A 3 A V G H TCTAGT ATT AACTATCTK; A GAAACACC TAATCTTATC AAA.\AA CAA TAC ACATA A ACXAATCA A-S^CTT CCA CA CλrATCλ 2730

V V I H Y D E T C N V I K K P I L D T H E S T P Y D TA CA A2A7 TATAΛAT C CTGAAATTAλ ATTTA-XT CT AA ATTTA7A A.\~ CTTTC TCCAAAAA λTO-^ΛΛT- AAΓΓ CGAAA 2330

T T D Y K P A E I F N C K I K L V S A X T f. .'i Z F G K CCTCA T AA C3 A AA λO AA3T A TTA TOTG AT CA CAAT AQTAA AAT TA CT A AAACGAA AX -" A rTT TATTC AAO 2370

V T E C T T E V T Y V Ϊ P. E S V X S T L P K 2 T O I S I P S C AAT A^AC T CCTAArr TTATATCTA CCAAArTACA CAAAATAC^C CTAATAAACG A TATTAA T T-^,?— O. ATC AACCAA 306-

E S E S ? K F 1 S T Q T T E N R ? N K G V L 7 S 5 S S ? T N TAAAT r TT C T AArr CAC CACGA AACTAATAy. TT T CA^ T TTrOGCAT TA TCTACTA C AΛ1V.ACA3 CTACTTTGOC 3150

K S V L P T T C E E S » R I L G V V C I T L V A 7 7 A L A A CTA ACT TTAAAA T-A GTAAAAATTA AAArT rTCT CA-7TC .7 T ACCCAA7A.: CL ΓTAΓΓATA AGA _{■ ■} ^• _{■ ■} ^■ _■ ^• : CTTGATAATC 32 :

A S S L K R R K N .

A A7CATACA VT'ITJATCAA CTATTTGTTA TCG7A77TT C CT77AX7 C OT AATTA TA CTA7AT TA.\A-V'.:- A CTTT TTACCA 3333

GT TA AAAA AAGT AT AT TTCATCC TT TTTTTO AAT TTA7 ATTT7 AAAAAC -AA TAAAATA7TA 77. . :.--: 7 341- or its complementary strand,

(b) DNA sequences which hybridise under stringent conditions to regions of a DNA sequence according to (a) encoding pro- teins or to fragments, for example having a degree of homolo- gy of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, of said DNA sequence,

(c) DNA sequences which hybridise to DNA sequences according to (a) and/or (b) because of a degeneration of the genetic code,

(d) allelic variants and mutants of DNA sequences according to (a) to (c) in which one or more nucleotide residues, for example 1 to 300, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 10 or 1 to 5 residues, are deleted and/or substituted and/or inserted and/or one or more nucleotide segments are inverted, wherein said allelic variants and mutants encode isofunctional expression pro- ducts.

In a further aspect the invention relates to recombinant expression vectors comprising DNA sequences as defined above. An example for such a recombinant expression vector is that deposited under EMBL accession number AJ133114.

In another aspect the invention relates to procaryotic or eucaryotic cells transformed or transfected with DNA sequences or recombinant expression vectors as defined above or ha- ving a DNA sequence as defined above stably integrated into the chromosomal DNA. An example for such cells are Escheri- chia coli cells. In yet another aspect the invention relates to the expression products or partial expression products of DNA sequences or of recombinant expression vectors as defined above.

In still another aspect the invention relates to synthetic proteins or polypeptides having the amino acid sequence of an expression product or partial expression product as defined above .

Moreover, the invention relates to a process for the preparation of expression products or partial expression products as defined above comprising the steps of cultivating cells as defined above in a suitable culture medium and isolating the expression products or partial expression products from the cells and/or the culture medium.

In addition, the invention relates to a protein or polypepti- de comprising an amino acid sequence selected from the group consisting of:

(a) the following amino acid sequence:

10 20 30 40 50 60 70 ~1 90^*

1234557390 1234567a90 1234557390 1234557390 1234557330 1234567990 12)4557-30 I2i;:57no 1234567330

AAATTCT TT TAATTACCTT TATATTCGAT ACAAGTATCC AATCTGTTTT ATGTAAACTA CCACCTCTTA C7A7 TATTT TT AATTTAT 90

CTTGAATCAG CCACCTCΓTΓ ACTTTTATCT AΛACTCAAAΛ TATTCTTAVA

TΓTCAAATTT

130 ACCCTΓΓTAT TTrrrcTTG TΓTTTAAATG TAAAAACTGT TA AGGAAAG AGGAACATAT TATXTΓΓTCGT CAATA7AATT TTCAAAAAGO 270

H P R Q t P E K G

TTTAAAΓΠT TCCATTCGTA AATTTTCCGT TCCAATAG T TCACTTCCTA TACCATCATT ATTATTCATT TTA—CAOC TACTTCCACA 350 L N P S I R K P S V C I A S V A I G Ξ L L P I L P Q V L A D

TGACACAA T AGTCTTACAT CTCCAACAA TCCAACACCT GTAACCA AA CACATGCAAA CΓTCCTCAAT CC7AA-ΛATT CAACTCCTAC 450

E T T S V T S A T T P T G V T T T D A N L V N P U N S T P T TTCCACTAAT ACCAGTGCAA CTACCACTCA AGGAAGTAAT TTCAGTAATA CTTCAGAAAT CATTAACCCT GCAA TTT C CΛCCAACATC SLO

S T N R S A T S T Q G S N L S N T S E I I K P A 7 L A A T S AC AACAACT GATAATGTAG CCCCAT AGT AGACAAGAGG ACCTATCCT CTACRRCCCCA TTCGACCTTA CAAAAT CAT ATOCTCACAG 630

P T T D N V A P S V D K R T Y A T S G D W T L Q N P Y A D S TCTTCGAAAT AAAAATATTT CTCCAACTGT TCCTCATGAA T ATTTAAAA GTO TGAAAC AA CCTCCTT CC7CA-CATA ACTCAACCGT 720

V R N K N I S P S V R H E S P K S A E T T V V R K D H S T V TAAAGTGACR GCGACAATTA CTCCTCTAGA AGCGAATGAT CAAGGTTCΠ. CTATTTTGAC AAATGGTCGT AA7 A07CAC- AA7ATAAAGC 8L0 V T A T I T P V E G N D E G S C I L T N C C N Q S E Y K A AACATCAGAA ATCTTTGT G GAAATGTACA TCCTGCAΛΛΛ ATACCTGCTT TAOGTGTCTA TACACAA CA CGACC7ACTC A CCAGGTAG 900

T S E N P V G N V D P A K I P A L G V Y T Q P G R T E C G S TAAA TATCT CATAAGCTCA ATTTTAATGG C-AACCCCCCA TCAA TATTT TAACATTGAA ATTTCACΛAC CCA-TTACAG A CCTATTAT 930

K L S D K L N P N G K A P S T I L T L K. F D K A V T D ? I 1 TGAΓTTATCΓ ∞TCTTCCAG CTAATCCACG TCTAT ATTT ACACAAACTG TCATGGAAAA TGCTAAGATT CTTGAAAAAT TΓCACTATGC 103

D L S G V G C N A R L S F T E T V M E N C K I V E K F D Y A

ACC CCTT A TATAATTCAA TGGAATAACT CCACCTATAA GCCTTCAΛAA AGCAAOTTCA TAACAGTTAC 1170

R C S Y N S T P F D G I T P G I S L E K A S S G V M L T V T AGCGAATACG CTTGATGTAA CAGACAAGAA CACCTTTAAT Λ-TCAGTTC TTAATCCATC TGACGAAGAC TTT-TCAATO CTCCAGATAG 1250

A N T V D V T D K N T P N E S V V N P S D E D F V N C P D R AACCCCTGAT CCACTCCCAG CC-3GAACAGG CTCAATCCGT TTλλλASGΛA CCTTTACAGA CCCTTCATTT AAATTA7ATC ATCAAGCTGT 1350

T P D A V P A G T G S I P. L K G 7 F T D A S F K L Y H Q A V TCCATCTACA CCATTTTCTA AAGAAAAATA TCATACAGGA GATCGATATT ATAATACTAT TGCGAATCTT V -AACTC TAGTTAAACC 1440

P S T A F S K E K Y H T G D G Y Y N S I A N V R ? T V V K P AGATAGTATT AATCGTTTλλ ATAAGCACCC AAGTGATGTT ATTCATTATA AAATTTCCCA TAATAATCAT ATTTCCAACC ATCACTTACT 1530

D S I N C L N K H A S D V I D Y K I S D N N D I S K D D L L TCGTTTATCr CTTCGC IAC AΛAΛTCCGCG TC-3ATCAGTT GTTCTAAATT ATATTGATAC AGAAGGCAAT ATTATTGGTA CTGAATACAA 1520

R L S V R L Q N P R C S V V V N Y I D T E G N I I G T E Y K AGATACCACC GATC AATTC CAGGAACACA TTACAATACG CiCAGAATCAT CAGGAGACCT TλλrrCACAC CCCA-λOTTC AGCSICCTTC 1710

D T T D A I P G T H Y N T A E S S G D L N S D A T V E R P S CACTATTACT AAACACCCTA.-AGGTGTATGA TTTAGTACCA GAAAATATTA CACTGCCACT TCGTAAGGTT AATT OGATG CTACGTTGC ^' 1300

T I T K D G K V Y D L V A E M I T V P V C R V N S D G T L A TACAAATCCC AGCTCATTTA ATTATGGTAC TGATGCAGCA TCTCCAGAAC TTCCACAACG TACTAACTCA GTAATTTATC TTATTAGCAT 1330

T N G S S F N Y C T D A A S A E V A E G T K S V I Y V I S I TAAACAACAA TCAAAGfϊ-3A ATGTTCATGC CCGCTATCPrT ATTTTAGCGA CAGACJ.CTCA C TTCCAAGT GCTAAAλCλG TTAAATCACA 1930

K Q E S K G N V H A R Y V I L G T B T B L A S A K T V K S E ACCrCCAATT GATCAGGCAT ATTCACATAA ACCACCTCCA AACATCCTAλ CTTGTATGAA TTT TACATC 20 0

A P I D E A Y S D K A P A T L E K D G K L Y E F V H V R D N TAAGCCCGAT CCTCCACCAG ATGGTAAGCT CACTCAACAA GATCACΛCCA TTACATATCA ATATCTTGAA GTT 7AACG CTTCGTGTGGT - 2150

K G D A P A D C K V T E Q D Q T I T Y E Y V E V ? K G R V V TCTAGATTAT GTTATCGAGG GTACTGCTAC AAAACCTAAA GATACTTATG TCCATACCCC AACACCTTAT ATTCOTCATA AACAACCTCA 2250

V D Y V I E G T A T K P K D T Y V D T P T A Y I S D K E G Q AGCTATTCCT TATAATACAC CTGAGAATCA TTCTCACAAA CCATTCITAC TTCACAAAGA TGCAATAAAA TATiAA TAC 2340

A I P Y N T A E N D S E K P L L L D K D C t K Y S V S I Q ACACCCGTCA GCACCGGΛCA AACCIAACACT TCCACAAGGG GAACAACATG TTGTCTATCA ATATCGTAAA CTCC7A ACC TTCCAACTCT 2130

E G S A A E K G T L R E C E Q H V V Y y Y R K V V ϊ V P S V TAAλGT Ξ3 AATGTTCATG CCCGCTATGT TATTTTACCG ACAJGJiSACTC ACCTTCCAAC TCCTAAAACA GTTAAATCAO AAJ3CTCCAAT 2520

K V G N V H λ R Y V I L G T E T E L A S A R T X S E A P I TGATC-ACGCA TATTCAGAT AAGCACCTGC AACTCTTCAA AAACATCG ATTTCTACAT A7AACCCCCA 2510

D E A Y S D K A P A T L E K D G K L Y E P V H V X D N K G D TCCTCCAGCA GATCGTAA03 ^CTCAACA AGATCAGACC ATTACATATC AATATAAGCT TAACAAAGAT CAC7CA A G CTCTAOCGAA 27C0

A P A D G V T E Q D Q T I T Y E Y K L K K D D A D A V G H TGTAGTCATT AλCTATGTCσ ACGAAACACG TAATCTTATC AAAAAA CAA TACTACATAC ACACGAATCA A.-^X77 CCA CArCATATCA 2730

V V I N Y V D E T C N V I K K P I L D T H E S K V G 7 P Y D TACCACAGAT TATAAATTCG CTGAAATTAA ATTTA.ATCCT AACATTTATλ AACTCCTTTC TCCAAAAACT ATC A-AT AATTCGGAAA 2330

T T D Y K F A E I K F N G K I Y K L V S A K T M 0 .'I E F G K C-T A TGAA GGCACAACAG

TGTG7ATCCA CAATCAGTAA AATCTACCCT ACC-AAACCAA AC C-7 A777 CTλrrCCAAG 2970

V T E C T T E V T Y V Y P. E S V K S T L P K E T 0 I S I P S CCAAT AGAG TCrt TAAGT TTATATCTAC CCAAACTλCA GA A7AGAC C7 ATAAACC AGTATTAA T T A7 7AAAA ATCCAACCAλ 3 5 C

E S E S P K F 1 S T Q T 7 E N R P H K C V L T S S K N P T H TAAATCrCTT CTTC AACTA CAC CAGGA λAGTλλTAGA ATTCTCCCAG TTGTTGCGAT TACTCTA 7A C A.^-V. AO CTACTTTGCC 31S0

K S V L P T T G E E S H R I L G V V G I T L V A 7 7 A T L A ACCTA CACT TTAAAAC5AC GTAAAAATTA AAAtTCTTCT CACTCC^TCT CACCCAATA.: COTTATTATA A :;^■;^■,- ,-;-,^■ CTTGATAATC 3243

A S S L K R R K N . ACATCATACA TTTT ATύλA CrTATTTGTTA TCGTATCTTT GCCTT TA^T CACTCAA7TA TACCTA7AT 7AAA^.\ CCTCTTACGA 3333

C TACAAAA AACT AT AT r.TiTCCΛAT TTλTCATTTT AAAAACCGAA TAAAATA7TA T . .v:O CT 341C (b) allelic variants and mutants of the amino acid sequence according to (a) in which one or more amino acid residues, for example 1 to 100, 1 to 80, 1 to 50, 1 to 30, 1 to 15, 1 to 10 or 1 to 5 residues, are deleted and/or substituted and/or inserted and/or one or more amino acid segments are inverted, wherein said allelic variants and mutants are iso- functional,

(c) an amino acid sequence having fused to its C-terminus and/or N-terminus an amino acid sequence, wherein the resulting fusion protein or fusion polypeptide is isofunctional and/or the additional C-terminal or N-terminal amino acid sequences may be easily eliminated under physiological conditions .

Further, the invention relates to polyclonal or monoclonal antibodies specifically directed against expression products or partial expression products as defined above or against synthetic proteins or polypeptides as defined above or against proteins or polypeptides as defined above and to hy- bridoma cells producing monoclonal antibodies as defined above.

The above polyclonal or monoclonal antibodies may be detec- tably labeled. For example, the label may be selected from one or more members of the group consisting of radioactive, coloured or fluorescent groups, groups for immobilisation to a solid phase and groups for an indirect or direct reaction, particularly by means of enzyme-conjugated secondary antibo- dies, the biotin/avidin(streptavidin) system or the collodial gold system. In yet another aspect the invention relates to the use of DNA sequences as defined above or expression products or partial expression products as defined above or synthetic proteins or polypeptides as defined above or proteins or poly- peptides as defined above or polyclonal or monoclonal antibodies as defined above in an assay for diagnosting an infection with group B streptococci. Each of these compounds may be part of a kit for use in such assay.

In still another aspect the invention relates to compositions comprising DNA sequences as defined above or expression products or partial expression products as defined above or synthetic proteins or polypeptides as defined above or proteins or polypeptides as defined above as an antigen for prophylac- tic or therapeutic vaccination (i.e. active immunization) against an infection with group B streptococci. Each of these compounds for use as an antigen may be incorporated into, for example, liposomes, ISCOMs (immunostimulating complexes) or microparticles. For increasing the immunogenicity and/or ef- ficacy of the antigen composition the expression products or partial expression products as defined above, the synthetic proteins or polypeptides as defined above or the proteins or polypeptides as defined above may be conjugated with a carbohydrate moiety.

Each of the above antigen compositions may be used, e.g., for subcutaneous or mucosal, e.g. intranasal, administration.

Further, the invention relates to compositions comprising po- lyclonal or monoclonal antibodies as defined above for immu- notherapy (i.e. by passive immunization) of an infection with group B streptococci. In the following the invention is explained in more detail, however, without limiting its scope.

Purification of cell-surface R proteins from S. agalactiae Compton R

The purified product obtained after alkaline extraction and FPLC anion exchange chromatography gave four bands of 125 kDa, 120 kDa, 115 kDa and 110 kDa in silver stained SDS-PAGE gel. Western blot analysis indicated all bands were immunore- active against polyclonal reference R-antiserum with the 125 kDa and 120 KDa bands reacting most prominently. Alkaline extraction in the presence of protease inhibitors or further purification by gel filtration did not change the gel pat- tern. These purified proteins were used to raise a polyclonal antiserum for screening of the gene library.

The attached Figures show:

Figure 1: The nucleotide and deduced amino acid sequence of the gene encoding R5 protein. Putative -10 and —35 regions are boxed, the Shine Dalgarno region is underlined. Within the R5 coding region, the initial codons of the two repeats are boxed and the regions coding for the LPXTG membrane an- chor consensus sequence as well as the charged C-terminal tail are underlined.

Figure 2: A. The recombinant expression of GST-tagged R5 in E. coli XLl-Blue MRF' and purification by glutathione-agarose affinity chromatography. Coomassie stained gel (lanes 1-3) showing E. coli whole cell extract prior to induction (la- nel), E. coli whole cell extract after induction (lane2), and purified recombinant R5 fusion protein (lane 3). Western blot of the above antigens reacted with monospecific anti-R5 rabbit serum (lanes 4-6).

B. A Western blot analysis of GBS strains using monospecific R5 antiserum. Purified recombinant R5 (lane 1), Compton R (lane 2), 71-735 (serotype III/R1) (lane 3), H4A-0126 (type Ia/Rl, R5) (lane 4), 76-043 (type III/R4) (lane 5), E. coli XLl-Blue expressing R5 (lane 6), E. coli XLl-Blue control (lane 7 ) .

Figure 3: An immunoelectron microscopy of S. agalactiae Compton R using monospecific R5 antiserum. Immunogold particles are indicated by an arrow. Bar represents 100 nm.

Figure 4: Panel A: The identification of the R proteins of S. agalactiae Compton R by immunodiffusion in agarose slides. Center well: HCl extract of the GBS Compton R strain. Well 1: antiserum to the R3, R4 and R5 proteins of Compton R; wells 2 and 5: antiserum to the R3 and R5 proteins of Compton R; wells 3 and 6: antiserum to the recombinant R5 protein (anti- R5); and well 4: antiserum to the R3 protein from Compton R. Panel B: Effect of enzyme treatment (1 hr/37°C) on the immuno- precipitin reactions of the R3 and R5 proteins from the HCl extract of S. agalactiae Compton R. Center well: antiserum to the R3 and R5 proteins of Compton R. Well 1: untreated HCl extract; well 2 : pH 4 buffer control; well 3: treated with 0.2% pepsin (pH 4); well 4: treated with 5% trypsin (pH 8); welt 5: treated with 0.2% trypsin; and well 6 : pH 8 buffer control. Figure 5: Vaccination with the R5 protein triggers the elici- tation of protective responses. (A) R5-specific serum antibodies in mice after subcutaneous (R5-Alum) or intranasal (R5- CTB) immunization. Results are expressed as the geometric me- an end point titer (GMT); the SEM is indicated by vertical lines. (B) Survival times of vaccinated and non-vaccinated mice after challenge with group B streptococci. Animals were challenged with an inoculum corresponding to the LD₉₀ of either Compton R (CR) or B176 strain, respectively, at day 37 af- ter the primary immunization, and mortality was daily recorded.

Cloning and nucleotide sequence analysis of the gene encoding R5 protein

A lambda Zap gene library of S. agalactiae Compton R chromosomal DNA was screened using polyclonal antiserum raised against purified cell-surface R proteins isolated from the homologous strain. One clone which expressed a 125 kDa appa- rent molecular weight protein reactive against the antiserum (result not shown) was in vivo-excised and the plasmid, designated pSE3. The 5.0 kb DNA insert of the plasmid was subjected to DNA sequence analysis which revealed a single open reading frame of 2937 bp encoding a predicted 105 kDa protein (Fig. 1). A putative Shine Dalgarno sequence ( 5 ' -GAGGAAG-3 ' ) was detected 5 bp upstream of the ATG start codon. Both -35 (5'-TTGGAT-3' ) and -10 ( 5 ' -TATAAT-3 ' ) consensus sequences at least typical for E. coli were detected 94 and 66 bp upstream of the open reading frame, respectively. A putative 39 amino acid signal sequence was detected at the amino-terminus of the putative protein which shared DNA similarity with other Gram-positive cell-surface protein signal sequences (von Hei- jne, 1985). The carboxy-terminus of the protein contained a membrane anchor LPXTG sequence typical of membrane-anchored surface proteins of many streptococci and other Gram-positive bacteria (Hollingshead et al., 1986). The carboxy-terminus of the protein also contained two identical 76 amino acid repeats separated by a 101 amino acid spacer. After searching the EMBL database, the protein was found to share limited DNA similarity only with the protein encoded by the Streptococcus suis mrp gene of unknown function (Smith et al., 1992). The R protein analysed in this study which was present in cell- surface extracts of S. agalactiae Compton R was immunologi- cally distinct from Rl, R3 and R4 (see below) and thus designated R5. The S. agalactiae gene encoding R5 was designated sar5. (EMBL accession number AJ133114).

Expression and purification of GST-tagged R5

Using the 5 ' -BamHI and 3 ' -Smal primers the sar5 gene was PCR amplified without the amino-terminal signal sequence and car- boxy-terminal membrane-anchor and ligated into the relevant enzyme restriction sites of the expression vector pGEX-2T (Pharmacia) to form pSE4. Induction of E. coli XLl-Blue MRF' (pSE4) resulted in the expression of GST-tagged R5 of 158 kDa apparent molecular weight, which was subsequently purified using glutathione-agarose affinity chromatography (Figure 2 A, lanes 1-3). The purified recombinant R5 protein was used to raise monospecific rabbit polyclonal antiserum which recognized the degraded R5 protein of 125 kDa apparent molecular weight in S. agalactiae Compton R whole cell extracts, the 32 kDa apparent molecular weight GST in E. coli XLl-Blue MRF' (pGEX-2T) whole cell extracts and the 158 kDa degraded recombinant R5 fusion protein in E. coli XLl-Blue MRF' (pSE4) who- le cell extracts (Figure 2A, lanes 4-6). A commercially acquired anti-GST monoclonal antibody reacted against GST and GST-tagged R5, but not against native R5 in S. agalactiae Compton R whole cell extracts (data not shown).

Western blot analysis using anti-R5 serum

Western blot analysis using R5 antiserum revealed that R5 antigen produced by S. agalactiae Compton R, partially purified Compton R surface proteins and purified recombinant GST- tagged R5 are degraded following sample preparation, resulting in multiple bands being detected (Figure 2B, lanes 1, 2,4, 6). However, lack of reactivity of the R5 antiserum against E. coli XLl-Blue MRF' confirms the lack of cross- reactive antibodies and the purity of the GST-tagged R5 preparation used to prepare the polyclonal antiserum (Figure 2B, lane 1 ) .

The R5 antiserum was also used to detect R5 in a number of different group B streptococcal serotypes. Lack of reactivity of the antiserum with S. agalactiae 71-735 (serotype III/R1, lane 3) and 76-043 (serotype III/R4, lane 5) revealed that R5 was not immunologically related to Rl and R4. S. agalactiae H4A-0126 (serotype la/Rl) on the other hand did express a protein of higher apparent molecular weight which was both reactive with the R5 antiserum and produced a degradation profile similar to Compton R, suggesting that H4A-0126 also expressed R5, that R5 antigen displayed size variation, and that R5 was expressed in more than one serotype of S. agalac- tiae (Figure 2B). Further proof of the immunological distinction between Rl, R3, R4 and R5 was the fact that the antise- rum to R3 and R5 from Compton R did not react with proteins from strains 71-735 or 76-043 (Fig. 2B)

Cell-surface expression of R5 in S. agalactiae Compton R

To confirm and characterize the location of R5 protein on the surface of S. agalactiae cells, immunoelectron microscopy was conducted by using monospecific polyclonal anti-R5 antiserum. Immunogold particles were found to be evenly distributed on the cell surface (Figure 3) of the parental S. agalactiae strain Compton R indicating that R5 protein is homogeneously expressed on the cellular surface. Anti-GST antibodies as well as preimmune serum were used as controls and did not reveal positive reactions (data not shown) .

Ouchterlony double diffusion (DD) analysis of S. agalactiae Compton R proteins with different antisera

Identification of the cloned protein as R5 was based on DD studies of the Compton R strain with reference antisera. Figure 4, panel A, shows that the HCl extract of Compton R strain (center well) tested with University of Minnesota (UM) antiserum to Compton R (well 1), gave three separate precipi- tin bands with the diffuse R4 band closest to the antiserum well, the dense R3 band in the middle, and the lighter R5 band very close to the R3 band and farthest away from the antiserum well. Previous work with anti-Compton R sera from Prague and UM placed in adjacent wells showed the same three precipitin bands, but the R5 band was lighter and closer to R3 with the Prague antiserum than the UM antiserum (data not shown). When the antiCompton R was absorbed to remove anti-R4 antibodies (wells 2 and 5), only the R3 and R5 bands were seen. When the serum was absorbed to remove anti-R4 and anti- R5 antibodies (well 4), only R3 was left. R5 antiserum produced with the recombinant protein (wells 3 and 6) gave one precipitin band, and this band gave a reaction of identity with the R5 band of the antiserum to Compton R produced with whole bacterial cells (wells 1, 2, and 5). In addition, when antiserum to R5 protein was placed in the center well, a precipitin reaction of identity was observed with the purified recombinant protein and the HCl extract of the Compton R strain, while the extracts of strains 71-735 (III/Rl) and 76- 043 (III/R4) did not react with this antiserum (data not shown) .

Enzyme susceptibility studies of the R5 from S. agalactiae Compton R

To compare the enzyme susceptibility of R5 to that of R3, the HCl extract of Compton R was treated with trypsin or pepsin and then compared by DD to buffer-treated controls. Figure 4, panel B, shows the R3 (inside, denser) and R5 (outer, lighter) precipitin bands of the untreated extract (well 1) when the antiserum to R3 and R5 was placed in the center well. Treatment of the extract with 0.2% trypsin (well 5) eliminated R5 but did not affect the R3 reaction, while 5% trypsin greatly diminished the R3 reaction (well 4). By contrast, only the R5 precipitin band remained after treatment with 0.2% pepsin at pH 4 (well 3), and neither the R3 or R5 reaction was seen after treatment with 0.5% pepsin at pH 2, the optimum and conventional pH for pepsin activity (not shown). Buf- fer only at pH 8, pH 4 or pH 2 (not shown) did not affect either the R3 or the R5 precipitin band. Vaccination with R5 protein protects mice against challenge with virulent strains of group B streptococci

For immunization studies His-tagged R5 was used to avoid the modulatory effects of GST. The gene encoding for the whole R5 protein was cloned in an expression vector (pQE30) and his- tagged protein purified by affinity chromatography. Immunization of mice by the subcutaneous route with recombinant R5 protein triggered the elicitation of very efficient antigen- specific responses (Fig. 5A) . When the immunized animals were challenged with the homologous streptococcal strain Compton R, approximately 88% of the vaccinated animals survived (Fig. SB), whereas control mice were not protected (78% lethality). To evaluate whether the administration of the vaccine antigen by a different route can also lead to the elicitation of a protective response the animals were immunized by intranasal route using cholera toxin B subunit (CTB) as mucosal adjuvant. R5-specific antibody titres similar to those obtained by subcutaneous route were observed following intranasal vaccination (Fig. 5A) . Immunized and control mice were then challenged with the strain B176, a type la clinical isolate, to evaluate if vaccination with the R5 protein can also be effective against a heterologous strain. The obtained results confirmed that R5 is an efficient protective antigen, since 67% and 22% of the animals survived in the vaccinated and control group, respectively.

Discussion

In spite of advances in diagnosis and treatment, GBS infections remain a major cause of neonatal mortality and morbidity. Development of an effective vaccine to prevent GBS disease through maternal immunization seems to be a promising strategy for the control of GBS infections. A prerequisite for the development of an effective vaccine is the identification and characterization of potential cell-surface targets for therapeutic intervention. Because of the sub-optimal potential of capsular polysaccharides, the interest has shifted towards protein antigens as components of vaccine candidates. A number of surface proteins such as β-antigen (Jerlstrδm et al., 1991; Jerlstrom et al., 1996), α-protein (Michel et al., 1991), protein Rib (Stalhammar-Carlemalm et al., 1993) and R-proteins (Flores and Ferrieri, 1989,1996) have been described. Most of these protein antigens have been shown to be protective by either contributing towards resistance to opso- nization (Payne and Ferrieri, 1985) or by eliciting protecti- ve immunity (Lachenauer and Madoff, 1996; Michel et al., 1991). Elicitation of protection against encapsulated GBS strains (Larsson et al., 1996) underlines the importance of GBS surface proteins as vaccine candidates. The present invention describes yet another GBS R-like surface protein that was cloned from Compton R strain and sequence analysis showed that it possesses all the typical features of Gram-positive surface proteins such as signal sequence (Von Heijne, 1985) and the membrane anchor (Fischetti et al., 1990). The surface location was confirmed by immunoelectron microscopy. Sequence analysis showed no similarity on a protein level with any known GBS proteins indicating that this is a novel surface protein and was, therefore, designated R5. The protein consists of 979 amino acids and contains two identical repeats of 76 aminoacids separated by a 101 amino acid spacer in the C-terminal region. R5, therefore, belongs to a family of GBS surface proteins with repetitive structures (Wastfelt et al., 1996). Two other members of this family, namely protein Rib (Stalhammar-Carlemalm et al., 1993) and α-protein (Michel et al., 1992), are also trypsin-resistant and give a ladder-like pattern similar to R5 pattern in SDS-PAGE and Western blot analysis. R5, however, showed no sequence similarity to pro- tein Rib or α-protein. On the nucleotide level the repeats of R5 show some similarity to mrp-gene from Streptococcus suis (Smith et al., 1992). The function of the mrp gene product is not yet known.

To determine the prevalence of R5, the protein was expressed as a fusion, purified and used to raise polyclonal antiserum. Immunoprecipitation in agarose was useful to determine that R5 was indeed an integral, although up to now undetected, surface antigen of S. agalactiae Compton R and to identify it as a unique protein, separate from R3 and R4 proteins. In early (Wilkinson, 1972) and subsequent work on the R proteins of GBS (Flores and Ferrieri, 1989), the Compton R strain was identified as having only R3 and R4 proteins. When the HCl extracts of the invention were tested with Prague anti- Compton R serum, as was used in those studies, the R5 precipitin reaction was weak and practically fused with that of R3, making it difficult to recognize them as two distinct lines. However, separation and identification of three separate proteins in the HCl and trypsin extracts of Compton R was possible with the new polyclonal rabbit antiserum of the invention. Comparison of the reactions of the new antiserum of the invention with those of the one from Prague, showed that, although somewhat obscured, the Prague antiserum also contained antibodies to R5 (data not shown). This further con- firmed that R5 is a new protein that is separate from R3 and R4, the previously recognized proteins of Compton R. The results from the enzyme digestion studies were additional support for the fact that R5 was a separate protein of Compton R. DD results indicated that whereas R5 could be extracted by very mild trypsin treatment of bacterial cells, further digestion with even 0.2% trypsin eliminated its precipitin reaction. In contrast, the results from mild pepsin treatment of R5 at sub-optimal conditions (pH 4) indicated that R3 was more pepsin susceptible than R5. This reduced susceptibility of R5 to pepsin digestion is similar to that of R4 protein (Flores and Ferrieri, 1996) and protein Rib, since treatment with pepsin at sub-optimal pH conditions was used to characterize the latter and to compare it to the α component of the c protein (Stalhammar-Carlemalm et al., 1993), another trypsin-resistant protein of GBS (Ferrieri, 1988).

Finding R5 in GBS strains such as H4A-0 126 and H4A-0148, was important in showing that this protein of Compton R is found in wild GBS strains of other serotypes and protein profiles. Our results from examination of 1400 human GBS isolates, indicated that R5 was commonly found in serotype Ia/Rl isolates (Flores et al. 1999). The presence of both Rl and R5 in the serotype la isolates was analogous to the presence of both Rl and R4 in the majority of serotype V isolates (Ferrieri and Flores, 1997). Furthermore, these results indicated that R5 was a marker found in recently isolated colonizing human isolates and that, as such, it may be useful in their characterization (Ferrieri, 1988).

The results of challenge experiments demonstrated that the R5 protein is an antigen able to confer protective immunity against both homologous or heterologous strains of group B streptococci. Interestingly, vaccination by either subcutaneous or mucosal route triggered the elicitation of protective immunity. The natural portal of entry for group B streptococci is the mucosa from the respiratory and urogenital tracts. Thus, it seems particularly attractive to stimulate not only an efficient systemic but also a local mucosal response following vaccination. This may lead to a more efficient protection of newborns by two different mechanisms, namely (i) passive transfer of maternal antibodies leading to protection against disease, and (ii) reduction of the risk of maternal colonization (i.e. infection) by the presence of vaginal antibodies. In fact, it has been described that due to the mucosal network, antigen administration by intranasal route may also lead to the elicitation of local responses in the uroge- nital tract (Holmgren et al., 1992)

The following experiments and working examples are for illustration only and not to be construed as any limitation of the scope of the invention.

Experimental Procedures

Bacterial strains, phages, plasmids, and media

GBS strains were from the culture collection of the University of Minnesota (UM) Minneapolis, MN, USA. R protein prototype strains were Compton R (nontypeable/R3, R4, R5) (Compton 25/60, NCTC 09828, J. Jelinkova, Prague); 71-735 (III/Rl) (Lancefield D136C, R. Lancefield); and 76-043 (III/R4) (UM). Wild GBS strains were H4A-0126 (Ia/Rl, R5 ) , and H4A-0148 (Ia/Rl, R5), B176 (la, R5 ) colonizing human isolates. The E. coli strains XLl-Blue MRF and XLOLR were obtained from a com- mercial source (Stratagene) . GBS were grown in Todd Hewitt broth (Oxoid) whilst E. coli were grown in NZY medium, Luria Bertani medium (Sambrook et al., 1989) or Luria Bertani medium supplemented with 1 g/1 MgCl₂ and 4 g/1 maltose. Bacteria were grown at 37 °C unless otherwise stated. here appropriate, E. coli were grown in the presence of 100 μg/ml ampicil- lin, 15 μg/ml tetracycline, 50 μg/ml kanamycin and 1 mM iso- propyl-β-D-galactopyranoside (IPTG) .

Antisera

Rabbit antisera included polyvalent serum recognizing R3, R4 , and R5 (anti-Compton R from Jelinkova and UM), divalent serum for R3 and R5 (UM anti-Compton R absorbed with strain 76-043 to remove anti-R4 ) , monospecific anti-R3 (UM anti-Compton R absorbed with strains 76-043 and H4A-0148 to remove anti-R4 and anti-R5), monospecific anti-R4 (produced with strain 76- 043, UM), monospecific anti-Rl (produced with strain 71-735, UM), and anti-R5 (produced against the purified recombinant R5 protein). For screening of the gene library, polyvalent serum raised against the purified surface R proteins of Compton R was used.

Protein purification

For the purification of cell-surface R proteins from S. agalactiae Compton R, a 1 1 shaken overnight culture was centri- fuged (10,000 g, 10 min) and the pellet washed twice in 1.8% saline then resuspended to 0.33 g/ml wet weight in 50 mM glycine-NaOH pH 11. The pH was adjusted to pH 12 with 1 M NaOH. Alkali extraction of cell-surface R proteins was allowed to proceed for 2 h at 37 °C with shaking. The suspension was centrifuged (15,000 g, 20 min) and the supernatant adjusted to pH 7 using 1 M HCl. The supernatant (15 ml) was concentrated to 2 ml final volume and the buffer changed against 20 mM Tris-HCl pH 7.4 using a Centriprep-30 concentrator (Amicon) at 4°C. The preparation was applied to a MonoQ HR5/5 column (Pharmacia) with a flow rate of 1 ml/min using the same buffer. R proteins were eluted from the column using a 20 ml linear NaCl gradient (0-0.4 M NaCl in 20 mM Tris-HCI). Fractions containing R protein were detected by immunoblot- ting with antiserum raised against S. agalactiae Compton R whole cells. R protein-containing fractions were dialysed against 1/10 diluted PBS, lyophilised, then resuspended in an appropriate amount of 1/10 diluted PBS. Recombinant GST- tagged R5 protein was purified using glutathione-agarose af- finity chromatography in accordance with manufacturer's instructions (Pharmacia) . His-tagged R5 fusion protein was purified under native conditions according to Qiagen protocols. Protein concentration was determined by the method of Bradford (Bradford, 1979).

DNA manipulations

Chromosomal DNA from S. agalactiae Compton R was isolated according to the method of Talay et al., (1992). Purified chro- mosomal DNA was partially digested with the restriction enzyme Sau3AI and then subjected to NaCl gradient centrifugation. Isolated 4-8 kb DNA Fragments were cloned into the BamHI site of Lambda Zap-Express-arms (Stratagene) according to the manufacturer's instructions. The ligation mixture was packaged in vitro into lambda heads and tails (Gigapack Gold 11, Stratagene) and transfected into E. coli XLl-Blue MRF' according to the manufacturer's instructions. Positive clones were in vivo-excised to form pBKCMV derivatives using the helper strain E. coli XLOLR in accordance with the Stratagene manual. Plasmid DNA was prepared using the QIAwell plasmid extraction kit (Qiagen) and sequenced using the method of San- ger et al., (1977). Reactions were carried out using dye terminator ready reaction mix (Perkin Elmer) and electrophoresed on an ABI 373A DNA sequencer (Applied Biosystems). For complete sequencing of both strands of analysed DNA, universal and internal primers were generated and used to initiate se- quencing of DNA. Sequence analysis was undertaken using GEN- MON 4.4 software (GBF). The polymerase chain reaction using the 5'-BamHI primer 5 '-TTACATCTGGATCCACTCCAACAGGTG-3 ' and the 3'-SmaI primer 5 '-TAGTTGGAACCCGGGATTTATTGGTTGG-3 ' was performed in a thermocycier (MWGBiotech) ; the resulting PCR frag- ment was cloned into the BamHI and Smal sites of the expression vector pGEX2T (Pharmacia) then induced with IPTG using standard procedures (Sambrook et al., 1989). For His-tagged R5 fusion protein, the gene was cloned into pQE30 (Qiagen), overexpressed and purified as described previously (Molinari et al. , 1997) .

SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis

SDS-PAGE was performed as described by Laemmli (1970) then stained with Coomassie brilliant blue R250 (Sigma). Pre- stained high molecular weight markers were used to determine the apparent molecular weight of proteins (Sigma). Alternatively, Western blots of proteins electroeluted onto Immobilon P membranes (Millipore) were performed essentially as described by Burnette (1981). Immunoelectron microscopy

IgG fractions of the anti R5 serum were affinity purified using protein A sepharose (Sigma) and used in immunoelectron microscopic studies with whole cells of S. agalactiae and gold-labelled protein A. Cells were incubated with anti-R5 antibodies for 2 h at 30°C, washed, and incubated with protein A/gold complexes. Antibodies purified from preimmune serum as well as anti-GST antibodies served as control. Samples we- re fixed with glutaraldehyde, osmium tetroxide, and dehydrated with acetone, and finally embedded according to the method of Spurr (1969) .

Immunoprecipitin reactions in agarose

R proteins from GBS strains were detected in agarose slides by Ouchterlony double diffusion (DD) immunoprecipitation using Lancefield hot HCl or 0.1% trypsin cell extracts (Flores and Ferrieri). To examine the susceptibility of the va- rious R proteins to trypsin or pepsin digestion, HCl or 0.1% trypsin extracts were treated (1 hr/37°C, pH 8.2) with 5% or 0.2% trypsin or with 0.5% or 0.2% pepsin (pH 2.0 and pH 4.0, respectively) (Flores and Ferrieri, 1996; Stalhammar- Carlemalm et al., 1993; Wilkinson, 1972; Wilkinson and Eagon, 1971). Enzyme-treated or control (buffer only) samples were then tested to determine the effect of such treatment on the immunoprecipitin reactions.

Immunization and protection studies

Four weeks-old female BALB/c (H-2^d) mice (Harlan Winhelmann) were immunized with recombinant R5 protein on days 1, 3, 6 and 27 (30 μg/dose) by subcutaneous or intranasal route using aluminium phosphate (Adju-Phos, Axell Accurate Chemical & Scientific Corp.) or cholera toxin B subunit (CTB 10 μg/dose, Sigma) as adjuvant, respectively. Groups of immunized and control animals (n = 9) were challenged on day 37 with an inoculum of the streptococcal strain Compton R or B176, which corresponds to the 90% lethal dose (LD₉₀) for these strains in the non immunized mice, and mortality was recorded daily.

ELISA

Serum samples were collected on day 36 and monitored for R5- specific antibodies by an enzyme-linked immunosorbent assay (ELISA) . Briefly, 96 wells Nunc-ImmunoMaxiSorp™ assay plates (Nunc, Roskilde, Denmark) were coated with 50 μl/well of R5 in coating buffer (bicarbonate, pH 8.2). After overnight incubation at 4°C, plates were blocked with 10% fetal calf serum (FCS) in PBS for 1 h at 37 °C. Serial two-fold dilutions of serum in 10% FCS-PBS were added (100 μl/well) and plates were incubated for 2 h at 37 °C. After four washes with PBS- 0.05% Tween 20, secondary antibodies were added: biotinylated μ-chain specific goat anti-mouse IgM and μ-chain specific goat anti-mouse IgG (Sigma), and incubated for a further 2 h at 37 °C. After four washes, 100 μl of peroxidase-conjugated streptavidin (Pharmingen) were added to each well and plates were incubated at room temperature for 1 h. After four washes, reactions were developed using ABTS [ (2 , 2 '-azino- bis (3-ethylbenzthiazoline-6-sulfonic acid)] in 0.1 M citrate- phosphate buffer (pH 4.35) containing 0.01% H₂0₂. Results we- re expressed as endpoint titers and correspond to the last dilution which gave an optical density at 405 nm (OD₄₀s) of 0.1 unit above the OD₄₀s of negative controls after a 10 min incubation.

Summing up, Group B streptococci (GBS) express various sur- face antigens designated c-, R-, and X antigens. A new R-like protein has been identified from Streptococcus agalactiae strain Compton R using a polyclonal antiserum raised against the R protein fraction of this strain to screen a lambda Zap library. DNA sequence analysis of positive clones allowed the prediction of the primary structure of a 105 kDa protein designated R5 that exhibited typical features of streptococcal surface proteins such as a signal sequence and a membrane anchor region but did not show significant similarity with other known sequences. Immunogold electron microscopy using the R5 specific antiserum confirmed the surface location of R5 on S. agalactiae strain Compton R. Anti R5 antibodies did not cross-react with Rl and R4 proteins expressed by two variant type III GBS strains, but reacted with the parental streptococcal strain in Western blot and immunoprecipitin analysis. Separate R3 and R5 immunoprecipitin bands were observed when the cell extract of Compton R strain was tested with antiserum against Compton R previously cross-absorbed to remove R4 antibodies. In addition, R5 was considerably less resistant to trypsin but more resistant to pepsin than R3 protein. Immunization of mice with recombinant R5 protein either by subcutaneous or intranasal route gave an efficient antigen specific response and immunised animals survived challenge with lethal doses of homologous as well as hetero- logous GBS strains. Therefore, R5 protein represents a novel pathogenicity factor and a promising vaccine candidate against GBS. Literature References

Baker, C.J., and Edwards, M.S. (1995) Group B streptococcal infections. In: Infectious diseases of the fetus and newborn infant. Remington, J.S., and Klein, J.O. (eds.). Philadelphia: W.B. Saunders, pp. 980-1054.

Baker, C.J., and Kasper, D.L. (1976) Correlation of maternal antibody deficiency with susceptibility to neonatal group B streptococcal infections. N Engl J Med 294: 753-756.

Baker, C.J., Rench, M.A. , Edwards, M.S., Carpenter, R.J., Hays, B.M., and Kasper; D.L. (1988) Immunization of pregnant women with a polysaccharide vaccine of group B Streptococcus. N Eng. J Med 319: 1180-1220.

Bradford, M.M. (1979) A rapid and sensitive method for the quantitation of microgramm quantities of proteins utilizing the principle of protein-dye binding. AnalBiochem 72: 248- 254.

Burnette, W.N. (1981) "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrl- amide gels to unmodified nitrocellulose and radiographic de- tection with antibody and radiolabelled protein A. Anal Bio- chem 112: 195-203.

Farley, C, Harvey, C, and Stull, T. (1993) A population- based assessment of invasive disease due to group B strepto- coccus in non-pregnant adults. N Engl J Med 328: 1807-1811. Ferrieri, P. (1988) Surface-localized protein antigens of group B streptococci. Rev Infect Dis 10 (Suppl. 2): 363-366.

Ferrieri, P., and Flores, A.E. (1997) Surface protein expression in group B streptococcal invasive isolates. In: Streptococci and the Host. Proceedings of the Xlllth Lancefield International Symposium on Streptococci and Streptococcal Diseases. Horaud T., Bouvet, A., Leclercq, R. , de Montclos, H., and Sicard, M. (eds.). New York: Plenum Press, pp. 635-637.

Fischetti, V.A. , Pancholi, V., and Schneewind, 0. (1990) Conservation of a hexapeptide sequence in the anchor region of surface proteins from Gram-positive cocci. Mol Microbiol 4: 1603-1605.

Flores, A.E., and Ferrieri, P. (1989) Molecular species of R- protein antigens produced by clinical isolates of group B streptococci. J Clin Microbiol 27: 1050-1054.

Flores, A.E., and Ferrieri, P. (1996) Molecular diversity among the trypsin resistant surface proteins of group B streptococci. Zb Bakt 285: 44-51.

Flores, A.E., Erdogan, S., Chhatwal, G.S., Baker, C.J., Hil- lier, S., and Ferrieri, P. (1999) Expression of the R5 surface protein among polysacchanide serotypes of group B streptococci. XIV Lancefield International Symposium on Streptococci and Streptococcal Diseases. Abstract 02.8.

Gardam, M.A. , Low, D.E., Saginur, R. , Miller, M.A. (1998) Group B streptococcal necrotizing fasciitis and streptococcal toxic shock-like syndrom in adults. Arch Intern Med 158: 1704-1708.

Hall, R.T., Barnes, W. , Krishan, L., Harris, D.J., Rohdes, P.G., Fayez, J. , and Miller, G.L. (1976) Antibiotic treatment of parturient women colonized with group B streptococci. Am J Obstet Gynec 124: 630-634.

Hollingshead, S.K., Fischetti, V.A. , and Scott, J.R.(1986) Complete nucleotide sequence of type 6 M protein of group A Streptococcus. Repetitive structure and membrane anchor, J Biol Chem 261:1677-1686.

Holmgren J, Czerkinsky C, Lycke N, Svennerholm, A.M. (1992) Mucosal immunity: implication for vaccine development. Immu- nobiology 184: 157-179.

Jerlstrόm, P.J., Chhatwal, G.S., and Timmis, K.N. (1991) IgA- binding β antigen of the c protein complex of group B strep- tococci: sequence determination of its gene and detection of two binding regions. Mol Microbiol 5: 843-849.

Jeristrδm, P.J., Talay, S.R., Valentin-Weigand, P., Timmis, K.N., and Chhatwal, G.S. (1996) Identification of an immuno- globulin A binding motif located in the β-antigen of the c protein complex of group B streptococci. Infect Immun 64: 2787-2793.

Kvam, A.I., Bevanger, L.S., and Eversen, O.J. (1993) Charac- terization of a surface protein of group B streptococci resembling the α-antigen of C protein. In: Pathogenic streptococci: present and future. Totolian, A. (ed.). St. Petersburg: Lancer Publication.

Lachenauer, C.S., and Madoff, L.C. (1996) A protective sur- face protein from type V group B streptococci shares N- terminal sequence homology with the alpha c protein. Infect Immun 64: 4255-4260.

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.

Larsson, C, Stalhammar-Carlemalm, M. , and Lindahl, G. (1996) Experimental vaccination against group B streptococcus, an encapsulated bacterium, with highly purified preparations of cell surface proteins Rib and α. Infect Immun 64: 3518-3523.

Lin, F.-.Y.C., Clemens, J.D., Azimi, P.H, Regan, J.A., Weis- man, L.E., Philips III, J.B., Rhoads, G.G., Clark, P., Bren- ner, R.A., and Ferrieri, P. (1998) Capsular polysaccharide types of group B streptococcal isolates from neonates with early-onset systemic infection. J Infect Dis 177: 790-792.

Linden, V., Christensen, K.K., and Christensen, P. (1983) Correlation between low levels of maternal IgG antibodies to R protein and neonatal septicemia with group B streptococci carrying R protein. Int Arch Allergy Appl Immunol 71: 168- 172.

Madoff, L.C, Hori, S., Michel, J.L., Baker, C.J., and Kasper, D.L. (1991) Phenotypic diversity in the alpha C protein of group B streptococci. Infect Immun 59: 2638-2644. Michel, J.L., Madoff, L.W., Kling, D.E., Kasper, D.L. and Au- subel, F.M. (1991) Cloned alpha and beta C-protein antigens of group B streptococci elicit protective immunity. Infect Immun 59: 2023-2028.

Michel, J.L., Madoff, L., Olson, K. , Kling, D.E., Kasper, D.L., F.M., Ausubel, F.M. (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.

Molinari, G., Talay, S.R., Valentin-Weigand, P., Rohde, M., and Chhatwal, G.S. (1997) The fibronectin-binding protein of Streptococcus pyogenes, Sfbl, is involved in the internaliza- figroup A streptococci by epithelial cells. Infect Immun 65: 1357-1 363.

Pass, M.A., Gray, B.M., Khare, S., and Dillon, H.C. (1979) Prospective studies of group B streptococcal infections in infants. J Pediatr 95: 437-443.

Payne, N.R., and Ferrieri, P. (1985) The relation of the Ibc protein antigen to the opsonization differences between strains of type II group B streptococci. J Infect Dis 151: 672- 681.

Pearlman, M.D., Pierson, C.L., and Faix, R.G. (1998) Frequent resistance of clinical group B streptococci to clindamycin and erythromycin. Obstet Gynecol 92: 258-261. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning. A laboratory manual. Cold Spring Harbour, New York: Cold Spring Harbour Laboratory Press.

Sanger, F., Nicklen, S., and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467.

Sherman, M.P., and Lehrer, R. (1985) Oxidative metabolism of neonatal and adult rabbit lung macrophages stimulated with opsonized group B streptococci. Infect Immun 47: 26-30.

Smith, H.E., Vecht, U., Gielkens, L.J., and Smits, M.A.

(1992) Cloning and nucleotide sequence of the gene encoding the 136-kilodalton surface proteins (muramidase-released protein) of Streptococcus suis Type 2. Infect Immun 60: 2361- 2367.

Spurr, A.R. (1969) A low viscosity epoxy resin embedding me- dium for electron microscopy. J Ultrastruct Res 26: 31-43.

Stalhammar-Carlemalm, M., Stenberg, L., and Lindhal, G.

(1993) Protein rib, a novel group B streptococcal cell surface protein that confers protective immunity and is ex- pressed by most strains causing invasive infections. J Exp Med 177: 1593-1603.

Talay, S.R., Valentin-Weigand, P., Jerlstrόm, P.G., Timmis, K.N., and Chhatwal, G.S. (1992) Fibronectin binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells. Infect Immun 60: 3837-3844. Valtonen, M.V. , Kasper, D.L., and Levi, N.J. (1986) Isolation of a (Ibc) protein from a group B Streptococcus which elicits mouse protective antibody. Microb Pathogen 1: 191-204.

Von Heijne, G. (1985) Signal sequences. The limits of variation. J Mol Biol 184: 99-105.

Wastfelt, M., Stalhammar-Carlemalm, M., Delisse, A.M., Carbe- zon, T., and Lindahl, G. (1996) Identification of a family of streptococcal surface proteins with extremely repetitive structure. J Biol Chem 271: 18892-1 8897.

Wessels, M.R., Paoletti, L.C, Guttormsen, H.K., Michon, F., D'Ambra, A.J., Kasper, D.L. (1998) Structural properties of group B streptococcal type III polysaccharide cojugate vaccines that influence immunogenicity and efficacy. Infect Immun 66: 2186-2192.

Wilkinson, H.W. (1972) Comparison of streptococcal R antigens. Appl Microbiol 24: 669-670.

Wilkinson, H., and Eagon, R.G. (1971) Type-specific antigens of group B type Ic streptococcci. Infect Immun 4: 596-604.

Zangwill, K.M., Schuchchat, A. and Wenger, J.D. (1992) Group B streptococcal disease in the United States. 1990: report from a multistate active surveillance system. Morbid Mortal Weekly Rep 41: 25-32.

Claims

1. DNA sequence comprising a DNA sequence selected from the group consisting of:

(a) the following DNA sequence:

10 20 30 40 50 60 70 BO 90

1234557390 1234567890 1234SS7390 1234557390 1234567330 1234567890 12)45S7a90 12J4557390 1234567830

AAACrrcTCTT TAATTAGCTT TATATΓCGAT AGAAGTATCG AATCTGTTTT ATGTAAACTA CCAGCTCTTA GTATCTATTT TΓCAATTTAT 90

CTTGΛATGAC &-ACCTCTTT ACTTTTATGT AAACTCAAAA TATTCTTAAA TtTTrrAl-AT TTTCAAATTT CΓTTACCTTT -T-H-tTGACA 130

ACGCΓTTTAT TTΓΓTGTTCT TTTΓΓAAATG TAAAAACTCT TACAGGAAAG AOGAAGATAT TATCTΓTCGT CAATATAATT TTCAAAAAOG 270 K P R Q Y H F E K C

TTTAAATTTT TCCATTCCTA AATTTTCGGT TOGAATAGCT TCAGTTCCTA TAGCATCATT ATTATTCATT TTACCTCACG TACTTCCACA 360

L N F S I R K P S V C I A £ V A I G S C L P I L P O V L A D TGAGACAACT AGTGTTACAT CTGCAACAAC TCCAACAC T CTAACGACAA CACATGCAAA CTTGCTCΛAT C TAACAATT CAΛCTCCTAC 450

E T T S V T S A T T P T G V T T T D A N L V N P N N S T P T TTCCACTAAT ACCACTGCAA CTACCACTCA ACGAAGTAAT TTGAGTAATA CTTCAGAAAT CATTAAGCCT GCAACTTTAG CAGCAACATC 540

S T N R S A T S T Q C S N L S N T S E I I K P A T L A A T S ACCAACAACT GATAATGTAG CCCCATCAGT AGACAACACG ACGTATGCTA CTAGTGCCGA TTCGACCTTA CAAAA7CCAT ATGCTGACAG 630

P T T D N V A P S V D K R T Y A T S G D W T L Q N P Y A D S TGTTCGAAAT AAAAATATTT CTCCAAGTGT TCGTCATGAA TCATTTAAAA GTCCTGAAAC AACCG-RGGTT CG7CA7CATA ACTCAACCCT 720

V R N K N I S P S V R H E S P K S A E T T V V R H D N S T V TAAAGTGACT CCGACAATTA CTCCTGTAGA AGCGAATGAT CΛAGGTTCTC GTATTTTGAC AAATGCTCGT AATCAGTCAG AATATAAAGC ΘIO

K V T A T I T P V E G N D E G S G I L T N C G N Q S E Y K A AACATCAGAA ATGTTTGTTG GAAATGTAGA TCCTCCAAAA ATACCTGCTT TACGTGTCTA TACACAACCA GGACG7ACTG AACGAGGTAG 9CO

T S E H F V G N V D P A X I P A L G V Y T Q p G R τ e G G S

TAAACTATCT GATAAGCTCA ATTTTAATGG CAAGCCGCCA TCAACTATTT TAACATTGAA ATTTGACAAC CCACTTACAG ACCCTATTAT 990

K L S D K L N P N G K A P S T I L T L K F D K A V T D P I I TGAΓTΓATCT CGTGTTGGAG GTAATGCACG TCTATCATTT ACACAAACTG TCAΓGGAAAA TGGTAAGATT GTTGAAAAAT TTGACTATGC 1030

D L S G V G G N A R L S F T E T V M E N G K I V Ξ K P D Y A ACTTΓOC-TTCA TATAATTCAA CCTΓTCTΓTGA TGGAATAACT CCAC TATAA GCCTΓGAAAA AGCAAOTTCA CGAGTTAATT TAACAGTTAC 1170

R G S Y N S T P P D G I T P G I S L E K A S S G V N L T V T AGCGAATACG GTTGATGTAA CAGACAAGAA CACCTTTAAT GAGTCAGTTG TTAATCCATC TCACGAAGAC TTTCTCAATG GTCCAGATAG 12SO

A N T V D V T D K N T F N E S V V N P S D E D P V N G P D R AACGCCTGAT GCAGTCCCAG OGGGAACAGG CTCAATCCGT TTAAAAGCAA CCTTTACAGA CGCTRCATTT AAATTATATC ATCAAGCTGT 1350

T P D A V P A C T G S I R L K G T F T D A S F K L Y M O. A V TCCATCTACA GCATTTTCTA AAGAAAAATA TCATACAGGA CATGGATATT ATAATACTAT TGCGAATGTT CCACCAACTG TAGTTAAACC 1440

P S T A F S K E K Y H T C D G Y Y N S I A N V R P T V V K P AGATAGTATT AATGGTTTAA ATAAGCACGC AAGTGATGTT ATTGATTATA AAATTTCCGA TAATAATGAT ATTTCGAACC ATGACTTACT 1530

D S I N G L N K H A S D V I D Y K I S D N N D I S N D D L L TCGTTTATCT GTTCGCT AC AAAATCCCCG TGGATCAGTT CTTCTAAATT ATATTGATAC AGAAGGCAAT ATTATTGGTA CTGAATACAA 1620

R L S V R L Q N P R G S V V V N Y I D T E G N I I G T E Y K AGATACCACC GATGCAATTC CAGCAACACA TTACAATACG GCACAATCAT CAGGAGACCT TAATT AGAC CCCACAGTTG AGCCTCCTTC 1710

D T T D A I P G T H Y N T A E S S G D L N S D A T V E R P S CACTATTACT AAAGACOGTA-AGGTGTATGA TTTAGTAGCA GAAAATATTA CAGTGCCACT TGGTAACGTT AATTCGGATG GTACCTTGGC 1B0O

T I T K D G K V Y D L V A E M I T V P V G K V N _S D G T L A

TλCAAATGGG AGCTCATTTA ΛTTATCGTAC TGAΓCCAGCA TCTC AGAAG TTCCAGAAGG TACTAAGTCA GTAATTTATG TTATTAGCAT 1890 T N G S S P N Y C T D A A S A E V A E G T IC S V I Y V I S I

TAAACAAGAA TCA G22 ATGTTCATGC CCGCTATGTT ATTTTACGGA CAGAGACTGA CT-TGCAAGT GCTAAAACAG TTAAATCACA 1930 K Q E S G N V H A R Y V I L G T E T B L A S A K T V K S B

ACCTCCAATT GATGAGGCAT ATTCAGATAA AGCACCTGCA ACTCTTGAAA AAGATCCTAA CTTGTATGAA TTTGTACATO TC-CGTGATAA 2070

A P I D E A Y S D K A P A T L B K D G K L Y E F V H V R D N

TAAGGGCGAT CCTCCAGCAG ATGGTAAGG-Γ CACTCAACAA GATCAGACCA TTACATATGA ATATGTTGAA GTTCCTAAGG CTCGTGTGGT 2160

K G D A P A D G K V T E Q D Q T I T Y E Y V E V P K G R V V

TCTAGATTAT GTTATCGAGG GTACTGCTAC AAAACCTAAA GATACTTATG TCCATACCCC AACAGCTTAT ATTCCTGATA AAGAAC TCA 2250

V D Y V I B G T A T K P K D T Y V D T P T A Y I R D K E G Q

AGCTATTCCT TATAATACAG CTGAGAATGA TTCTCAGAAA CCATTGTTAC TIGACAAAGA TCCΛΛTΛΛΛΛ TATAACTAG TTTCAATTCA 2340

A T P Y N T A E N D S Z K P L L L D K D G I K Y E L V S I Q ACACGGGTCΛ CCACCGGAGA AAGCAACACT TCGAGAAGGG GAACAACATG TTGTCTATCA ATATCGTAAA GTCCTACACG TTCCAAGTGT 2130

E C S A A E K G T L R E G E Q H V V Y Q * R K V V E V P S V TAAΛGTTOΞ- AATGTTCATG CCCGCTATGT TATTTTAGGG ACAGAGACTC ACCTTCCAAG TC TAAAACA GTTAAATCAG AGCTCCAAT 2S20

K V G N V H A R Y V I L G T E T B L A S A R V JC S E A P I TGATGAGGCA TATTCAGATA AACCACCTCC AACTCTTCAA AAAGATCCTA ACTTCTATGA ATTTGTACAT GTGCCTOATA ATAAGGGCGA 2510

D E A Y S O K A P A T L E R D C K L Y E P V H V R D N I G D TCCTCCAGCA CATGGTAAGG TGACTGAACA AGATCAGACC ATTACATATG AATATAACCT TAACAAAGAT GACCCAGACG CTGTACCGAA 2700

A P A D G K V T E Q D Q T I T Y E Y K L K K D D A D A V G N TGTAGTCATT AACTATCTGG ACGAAACACG TAATCTTATC AAAAAACCAA TACTAGATAC ACACGAATCA AAGC-7TO33A CACCATATGA 2790

V V I N Y V D E T G N V I K K P I L D T H E S K V G T P Y D TACCACAGAT TATAAATTCG CTGAAATTAA ATTTAATGGT AAGATTTA7A AACTCGTTTC TC<-AAAAACT ATCCGAAATG AA-TCGGAAΛ 2330

T T D Y K P A E I K F N G K I K L V S A X T M C . E F G K CCTCACTGAA CCCACAACAG AACTCACTTA TGTGTATCCA CAATCAGTAA AATCTACCCT ACCAΛAGCAΛ ACCOCCATTT CTATTCCAAG 2970

V T E G T T E V T Y V Y R E S V S T L P R E T G I S I P S CCAATCAGAG TCT CTAAGT TTATATCTAC CCAAACTACA GΛλλλTAGAC CTAATAAAGG AGTATTAACT TCA7C7AAAA ATCCAACCλλ 3050

E S E S P K F 1 S T Q T T E N R P N G V L T S S K N P T N TAAATCTGTT CTTCCAACTA CVCCCCAGGA AACTAATAGA ATTCTCCGAG TTCTTCGGAT TACTCTAGTA GCAλ AA--G CrACTTTGCC 3150 S V L P T T G E E S N R I L G V V G I T L V A T T A T L A ACCTAGCAGT TTAAAACGAC GTAAAAATTA AAATTCTTCT GACTCGATCT GACCCAATλC CGTTA-TTATA AGATTCTTTT CTTGATAATC 3240

A S S L K R R K N . ACATCATACA TrTTCATCAA CTATTTGTTA TGGTATGTTT GCGTTGTAAT GACTGAATTA TACCTATAT Tλλλ^AOCAC CCTCTTACCA 3330

CT TACAAλλ AAGTCATCAT TT ΛTCCCTT TTTTTCCAAT TTATCATTTT Aλλλλ CGAA TAAAATATTA T7AOA: GC7 341 or its complementary strand,

(b) DNA sequences which hybridise under stringent conditions to regions of a DNA sequence according to (a) encoding pro- teins or to fragments of said DNA sequence,

(c) DNA sequences which hybridise to DNA sequences according to (a) and/or (b) because of a degeneration of the genetic code,

(d) allelic variants and mutants of DNA sequences according to (a) to (c) in which one or more nucleotide residues are deleted and/or substituted and/or inserted and/or one or more nucleotide segments are inverted, wherein said allelic vari- ants and mutants encode isofunctional expression products.

2. DNA sequence according to claim 1, wherein said fragments according to (b) have a degree of homology of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%.

3. DNA sequence according to claim 1 or 2 , wherein in said allelic variants and mutants according to (d) 1 to 300, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 10 or 1 to 5 residues are deleted and/or sub- stituted and/or inserted.

4. Recombinant expression vector comprising a DNA sequence according to claims 1 to 3.

5. Recombinant expression vector according to claim 4 deposited under EMBL accession number AJ133114.

6. Procaryotic or eucaryotic cell transformed or transfec- ted with a DNA sequence according to claims 1 to 3 or a recombinant expression vector according to claim 4 or 5 or having a DNA sequence according to claim 1 to 3 stably integra- ted into the chromosomal DNA.

7. Cell according to claim 6 which is Escherichia coli.

8. Expression product or partial expression product of a DNA sequence according to claims 1 to 3 or of a recombinant expression vector according to claim 4 or 5.

9. Synthetic protein or polypeptide having the amino acid sequence of an expression product or partial expression pro- duct according to claim 8.

10. Process for the preparation of an expression product or partial expression product according to claim 8 comprising the steps of cultivating cells according to claim 6 or 7 in a suitable culture medium and isolating the expression product or partial expression product from the cells and/or the culture medium.

11. Protein or polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) the following amino acid sequence: 10 20 30 40 50 60 70 80 90

1234567890 1234567890 1234567390 1234567830 1234567390 12345S7890 1234567890 1234557990 1231567890 AAAGTCTCTT TAATTACCTT TATATTGGAT AGAACTATCG AATCTGTTTT ATGTAAACTA CCAGGTCTTλ GTAT TATTT TTCAATTTAT 90

CTTGAATGAC C ACCTCTTT AGTTTTATGT AAAGTCAAAA TATTCTTA\A TtT AlAAT TTTCAAATTT CTTT - TTF TA TrC CA 130 TATCTTTCGT CAATATAATT 270

M P R Q Y N P E K G TTTAAATTTT TCCATTCGTA AATTTTCGGT TTACCrCACC TACTTCCACA 360

L N F S I R K P S V G I A S V A I G S L L P I L P Q V L A D TGAGACAACT ACTCTTACAT CTGCAACAAC TCCAACACCT GTAACGACAA CAGATGCAAA CTTGCTCAAT CC7AACAATT CAACTCCTAC 450

E T T S V T S A T T P T G V T T T D A N L V N P N S T P T TTCCACTAAT AGGAGTGCAA CTAGCACTCA AGGAΛGTAAT TTGAGTAAT CTTCAGAAAT CATTAAGCCT GCAACTTTAC CACCAACATC 5 0

S T N R S A T S T Q G S N L S N T S E I I K P A T L A A T S ACCAACAACT GATAATGTAG CCCCATCAGT AGACAACAGG ACGTATGCTA CTAGTGGCGA TTCGACGTTA CAAAATCCAT ATGCTGACAG 630

P T T D N V A P S V D K R T Y A T S G D T L Q N P Y A D S TCTTCGAAAT AAAAATATTT CTCCAAGTGT TCGTCATGAA TCATTTAAAA GTGCTGAAAC AACGGTGGTT CGTCACGATA ACTCAACCGT 720

V R N K N I S P S V R H E S P K S A E T T V V R H D N S T V TAAAGTGACT CCGACAATTA CTCCTGTAGA ACGCAATCAT CAAGGTTCTG GTATTTTCAC AAATGGTGGT AATCAGTCAG AATATAAAGC 810

K V T A T I T P V E G N D E G S G I L T N G G N Q S B Y K A AACATCAGAA ATGTTTGTTG GAAATGTAGA TCCTCCAAAA ATACCTGCTT TAGGTGTCTA TλCACAACCA GGACGTACTG AAOGAOGTAG 90O

T S E M P V G N V D P A K I P A L G V Y T y P G R T E G G S TAAACTATCT GATAAGCTCA ATTTTAATGG GAACCCGCCA TCAACTATTT TAACATTGAA ATTTGACAAG GCAGTTACAG ACCCTATTAT 990

K L S D K L N F N G K A P S T I L T L K F D K A V T D P I 1 TGATTTATCT GGTGTTGGAG GTAATGCACG TCTATCATTT ACAGAAACTG TCATGGAAAA TGGTAAGATT GTTGAAAAAT TTGACTATGC 1030

D L S G V C C N A R L S F T E T V M E N G K I V E K F D Y A ACGTGCTTCA TATAATTCAA CGTTCTTTGλ TGGAATAACT CCAGGTATAA GCCTTGAAAA AGCAAGTTCA CCAGTTAATT TAACAGTTAC 1170

R G S _ N S T F F D G I T P G I S L E K A S S G V N L T V T AGCGAATACG CTTGATGTAA CACACAAGAA CACCTTTAAT GAGTCAGTTG TTAATCCATC TGACGAAGAC TTTGTCAATG GTCCAGATAG 1250

A N T V D V T D K N T F N E S V V N P S D S D P V N G P D R AACGCCTGAT GCAGTCCCAG CGGGAACAGG CTCAATCCGT TTAAAAGGAA CCTTTACAGA CGCTTCATTT AAATTATATC ATCAAGCTGT 1350

T P D A V P A G T G S I R L K C T F T D A S K K L Y H Q A V TCCATCTACA GCATTTTCTA AAGAAAAATA TCATACAGGA GATCGATATT ATAATACTAT TGCGAATGTT CGACCλλCTC TAGTTAAACC 14 0

P S T A F S K E K Y H T G D G Y Y N S I A N V R P T V V K P AGATAGTATT AATOGTTTAA ATAAGCACCC AAGTGATGTT ATTGATTATA AAATTTCCGA TAATAATGAT ATTTCGAACG ATGACTTACT 1530

D S I N G L N K H A S D V I D Y K I S D N N D I S N D D L L TCGTTTATCr C^TTCGCTTAC AAAATCCGCG TGGATCAGTT CTTGTAAATT ATATTGATAC AGAAGGCAAT ATTATTGGT CTGAATACAA 1620

R L S V R L Q N P R C S V V V N Y I D T E β N I I G T E Y K AGATACCACC GATGCAATTC CAGGAACACA TTACAATACG GCAGAATCAT CAGGAGACCT TAATTCAGAC CCCACAGTTG AGCGTCCTTC 1 10

D T T D A I P G T H Y N T A E S S G D L N S D A T V E R P S CACTATTACT AAAGACOGTA AGGTGTATGA TTT GTAGCA GAAAATATTA CAGTGCCAGT TGGTAAGGTT AATTCGGATG GTACCTTGGC 1B0O

T I T K D G K V Y D L V A E M I T V P V C K V N S D C T L A TACAAATGGG AGCTCATTTA ATTATGGTAC TGATGCAGCA TCTGCAGAAG TTGCAGAACG TACTAACTCA GTAATTTATG TTATTAGCAT 1830

T N G S S P H Y C T D A λ S A E V A E G T K S V I Y V I S I TAAACAAGAA CCGCTATGTT GCTTGCAAGT GCTAAAACAC 1980

K Q E S K G N V H A R Y V I L G T E T B L A S A K T V K S E AGCTCCAATT GATGAGGCAT ATTCACATAA AGCACCTCCA ACTCTTGAAA AAGATGGTAA GTTGTATGAA TTTGTACATG TGCGTGATAA 2070

A P I D E A Y S D K A P A T L E K D C K L Y E F V H V R D N TAAGCCCGAT GCTCCAGCAG ATGGTAAGGT GACTGAACAΛ GATCAGACCA T7ACATATGA ATATGTTGAA GTTCCTAAGG CTCGTGTGGT - 2160

K G D A P A D G K V T E Q D Q T I T Y E Y V E V P K G R V V TCTAGλTTAT GTACTGCTAC GATACTTATG AACAGCTTAT ATTCCTGATA 2250

V D Y V I B G T A T K P K D T Y V D T P T A Y I R D K E G O. ACCTATTCCT TATAATACAC CTGAGAATGA TTCTGAGAAA CCATTGTTAC TTGACAAAGA TGGAATAAAA TATGAACTAG TTTCAATTCA 23 0

A I P Y N T A E N D S Z K P L L L D K D G I K Y 8 L V S I Q AGAGGCGTCA GCAGCGGAGA AAGGAACACT TCGAGAAGGG GAACAACATG TTGTCTATCA ATATCGTAAA CTCGTAGA . TTCCAAGTGT 21 0

E G S A A E K G T L R E G E Q H V V Y Q Y R K V V S V P S V TAAACπffiH AATGTTCATG CCCGCTATGT TATTTTAGGG ACAGAGACTC ACCTTCCAAG TGCTAAAACA GTTAAATCAG AAGCTCCAAT 2520

K V G N V H A R Y V I L G T E T E L A S A K T V K S B A P I TGATGAGGCA TATTCAGATA AAGCACCTGC AACTCTTGAA AAAGATGGTA ACTTGTATGA ATTTGTACAT GTG GTGATA ATAAGGOCGA 2610

D E A Y S D K A P A T L E K D G K L Y E P V H V R D N K G D TCCTCCAGCA CATGGTAAGG TGACTGAACA AGATCAGACC ATTACATATG AATATAACCT TAACAAAGAT GACGCAGACG CTGTAGGGAA 2700

A P A D G K V T E Q D Q T I T Y E Y K L K K D D A D A V G N TGTAGTCATT AACTATGTGG ACGAAACACG TAATGTTATC AAAAAACCAA TACTAGATλC ACACGAATCA AAGGTTGGGA CACCATATGA 2790

V V I N Y V D E T C N V I K K P I L O T H E S K V G T P Y D TACCACAGAT TA AAATTCC CTCAAATTAλ ATTTAATGCT AACATTTATA AACTCCTTTC TGCAAAAACT ATCC AAATG AATTCGGAAA 2330

T T D Y K F A E I K F N G K I Y K L V S A K T M G N E F G K C<7rCACTGAA OGCACAACAG AAGTGACTTA TGTGTATCGA GAATCAGTAA AATCTACCCT ACCAAAGGAA ACCCGCATTT CTATTCCAAG 29 0

V T E C T T E V T Y V Y R E S V K S T L P R E T G I S I P S CCAATCAGAG TCTCCTAAGT TTATATCTAC CCAAACTACA CAAAATAGAC CTAATAAACG AC7ATTAACT TCA7 TAAAA ATCCAACCAA 3060

E S E S P K F 1 S T Q T T E N R P N K G V L T S S K N P T N TAAATCTGTT CTTCCAACTA CAC CGAGGA AAGTAATAGA ATTCTCCGAG TTGTTGCGAT TACTCTACTA CCAACAACAG CTACTTTGGC 1 0

K S V L P T T G E E S N R I L G V V G I T L V A T T A T L A AGCTACCλCT TTAAAACGAC CTAAAAATTA AAATTCTTCT GACTCGATCT CACCCAATAC CGTTATTATA AGA7TCTTTT GTTGATAATC 3240

A S S L K R R K N . ACATCATAGA TTTTGATGAA CTATTTC-TTA TGCTATC7TT GGGTTGTAAT CAGTCAATTA TACCTATATC TAΛACACCAC CCTCTTACCA 3330

GTCTACAAAA AACTCATCAT TTGATCGCTT TTTTTGCAAT T7ATCATTTT AAAAAGGGAA TAAAATATTA T AGADGGCT 3410 (b) allelic variants and mutants of the amino acid sequence according to (a) in which one or more amino acid residues are deleted and/or substituted and/or inserted and/or one or more amino acid segments are inverted, wherein said allelic vari- ants and mutants are isofunctional,

(c) an amino acid sequence having fused to its C-terminus and/or N-terminus an amino acid sequence, wherein the resulting fusion protein or fusion polypeptide is isofunctional and/or the additional C-terminal or N-terminal amino acid sequences may be easily eliminated under physiological conditions.

12. Protein or polypeptide according to claim 11, wherein in said allelic variants and mutants according to (b) 1 to 100,

1 to 80, 1 to 50, 1 to 30, 1 to 15, 1 to 10 or 1 to 5 residues are deleted and/or substituted and/or inserted.

13. Polyclonal or monoclonal antibody specifically directed against an expression product or partial expression product according to claim 8 or against a synthetic protein or polypeptide according to claim 9 or against a protein or polypeptide according to claim 11 or 12.

14. Hybridoma cell producing a monoclonal antibody according to claim 13.

15. Polyclonal or monoclonal antibody according to claim 13 which is detectably labeled.

16. Polyclonal or monoclonal antibody according to claim 15 wherein the label is selected from one or more members of the group consisting of radioactive, coloured or fluorescent groups, groups for immobilisation to a solid phase and groups for an indirect or direct reaction, particularly by means of enzyme-conjugated secondary antibodies, the biotin/avi- din(streptavidin) system or the collodial gold system

17. Use of a DNA sequence according to claims 1 to 3 or an expression product or partial expression product according to claim 8 or a synthetic protein or polypeptide according to claim 9 or a protein or polypeptide according to claim 11 or 12 or a polyclonal or monoclonal antibody according to claim 13, 15 or 16 in an assay for diagnosting an infection with group B streptococci.

18. Kit for use in an assay for diagnosting an infection with group B streptococci comprising a DNA sequence according to claim 1 to 3 or an expression product or partial expression product according to claim 8 or a synthetic protein or polypeptide according to claim 9 or a protein or polypeptide according to claim 11 or 12 or a polyclonal or monoclonal antibody according to claim 13, 15 or 16.

19. Composition comprising a DNA sequence according to claims 1 to 3 or an expression product or partial expression product according to claim 8 or a synthetic protein or polypeptide according to claim 9 or a protein or polypeptide according to claim 11 or 12 as an antigen for prophylactic or therapeutic vaccination against an infection with group B streptococci.

20. Composition according to claim 19 wherein said DNA sequence according to claims 1 to 3, said expression product or partial expression product according to claim 8, said synthetic protein or polypeptide according to claim 9 or said protein or polypeptide according to claim 11 or 12 as an antigen is incorporated into liposomes, ISCOMs or microparticles .

21. Composition according to claim 19 or 20 wherein said expression product or partial expression product according to claim 8, said synthetic protein or polypeptide according to claim 9 or said protein or polypeptide according to claim 11 or 12 as an antigen is conjugated with a carbohydrate moiety to increase the immunogenicity and/or efficacy of the composition.

22. Composition according to any one of claims 19 to 21 for subcutaneous or mucosal administration.

23. Composition according to claim 22 wherein said mucosal administration is intranasal.

24. Composition comprising a polyclonal or monoclonal antibody according to claim 13 for immunotherapy of an infection with group B streptococci.