A list of the referenced literature with detailed biblio- graphic 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 inade- quately with antibiotics (Hall et al., 1976). Since many wo- men 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 ma- ternal 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 Ia, lb, II and III have been more prevalent. Approximately 40% of the isolates from cases with invasive GBS disease express Ia 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 vaccinati- on 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. Howe- ver, 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 vacci- nes, interest has shifted towards the surface proteins anti- gens of GBS as vaccine candidates or carrier proteins for specific GBS polysaccharides. These antigens include a and B antigen of the c protein complex (Jerlström et al., 1991 ; Ma- doff et al., 1991), an a-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 Ia and lb isolates and by 50% of type II invasive isolates, however, they are rarely expressed by type III GBS (Ferrieri and Flo- res, 1997).

Rib protein also confers protective immunity in mice (Stål- hammar-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 80 90 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 AAAGTCTCTT TAATTAGCTT TATATTGGAT AGAAGTATCG AATCTCTTTT ATGTAAACTA CCACGTCTTA GTATCTATTT TTCAATTTAT 90 CTTGAATGAG CCACCTCTTT AGTTTTATGT AAAGTCAAAA TATTCTTAAA TTTCGATAAT TTTCAAATTT CTTTATCTTT ATAATTGACA 180 ACCCTTTTAT TTTTTGTTGT TTTTTAAATG TAAAAACTGT TACACGAAAG AOGAAGATAT TATGTTTCGT CAATATAATT TTGAAAAAGG 270 M P R Q Y N F E K G TTTAAATTTT TCCATTCGTA AATTTTCGGT TGCAATAGCT TCAGTTOCTA TAOGATCATT ATTATTCATT TTACCTCAOG TACTTOCAGA 360 L N P S I R K F S V C I A S V A I G S L L F I L P Q V L A D TGAGACAACT AGTGTTACAT CTGCAACAAC TCCAACAGGT GTAACGACAA CAGATOCAAA CTTGGTCAAT CCTAACAATT 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 N S T P T TTCCACTAAT AGGAGTGCAA CTAGCACTCA AGGAAGTAAT TTGAGTAATA CTTCAGAAAT CATTAAOCCT GCAACTITAG CACCAACATC 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 AGACAAGAGG ACGTATGCTA CTAGTCCCCA TTOGACGTTA CAAAATCCAT ATOCTGACAG 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 GTGCTGAAAC AACOGTGGTT CGTCATCATA AZICAACCGT 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 GCGACAATTA CTCCTGTAGA AGGGAATGAT GAAGGTTCTG GTATTTTGAC AAATGGTGGT AATCAITCAG AATATAAAGC 810 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 TCCTGCAAAA ATACCTGCTT TAGGTGTCTA TACACAACCA CGACGTACTG AAOCAGGTAG 900 T S E M F 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 TAAACTATCT GATAAGCTCA ATTTTAATGG GAAGGCGCCA TCAACTATTT TAACATTGAA ATTTGACAAG GCAITTACAG 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 TGATTTATCT CGTGTTGGAG GTAATOCACG TCTATCATTT ACAGAAACTG TCATGGAAAA TGGTAAGATT GTTGAAAAAT TTGACTATGC 1080 D L S G V G G N A R L S F T E T V M E N G K I V E K P D Y A ACGTGGTTCA TATAATTCAA CGTTCTTTGA TGGAATAACT CCAGGTATAA GCCTTGAAAA AGCAAGTTCA GGASTTAATT TAACAGTTAC 1170 a c s y N s T P P D 0 1 T P G I S L £ R 1 S S S C ! i L T V T AGCGAATACG GTTGATGTAA CAGACAAGAA CACCTTTAAT GAGTCAGTTG TTAATCCATC TGACGAAGAC TTTOTCAATC GTCCAGATAG 1250 ANT VDVT DKM TPN ESVV MRS DED FVUG PDR AACGCCTGAT OCAGTCCCAG COOCAACACG CTCAATCCGT TTAAAAGGAA CCTTTACAGA CCCTTCATTT AAATTATATC ATCAAGCTGT 1350 TPD AVPA CTC SIR LKCT FTD ASF KLYH QAV TCCATCTACA GCATTTTCTA AAGAAAAATA TCATACAGGA GATGGATATT ATAATAGTAT TGCGAATGGT CGACCAACTG 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 P T V V K P AGATAGTATT AATGGTTTAA ATAAGCACGC 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 TCGTTTATCT GTTCGCTTAC AAAATCCGCG TGGATCAAGTT GTTGTAAATT ATATTGATC AGAACGCAAT ATTATTGGTA CTGAATACAA 1620 R L s V R L 0 N P R C S V V V N Y I D T c E Y K AGATACCACC GATGCAATTC CAGGAACACA TTACAATACG GCAGAATCAT CAGGAGACCT TAATTCAGAC GCGACAITTG AGCGTCCTTC 1710 DTT DAIP CTH YMT AESS GDL t7SD A\'VE RPS CACTATTACT AAAGACOGTA AGGTGTATGA TTTAGTAGCA GAAAATATTA CAGTGCCAGT TGGTAAGGTT AATTCGGATG GTACGTTGGC 1800 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 TACAAATGGG AGCTCATTTA ATTATGGTAC TGATGCAGCA TCTGCAGAAG TTOCAGAAOG TACTAAGTCA CTAATTTATG TTATTAGCAT 1990 T N C S S P H Y C T D A A S A E V A E C T K S V I Y V I S I TAAACAAGAA TCAAAGGGEA ATGTTCATGC CCGCTATGTT ATTTTAGGGA CAGAGACTGA CCTTGCAAGT GCTAAAACAS TTAAATCAGA 1980 X Q SKGH VHA RYV ILCT ETE LAS AKTV KSZ ACCTCCAATT GATGAGGCAT ATTCAGATAA AOCACCTGCA ACTCTTGAAA AAGATGGTAA GTTGTATGAA TTTGTAZATG TGCGTGATAA 2070 A P I D E A Y D S D K A P A T L E K D C K L Y E F V E V R D N TAAGOOCGAT GCTCCAGCAG ATGGTAAGGT GACTGAACAA GATCAGACCA TTACATATGA ATATGTTGAA GTTCCTAAGG GTCGTGTGGT 2160 x GD APAD CKV TEQ DQTI TYE YVE VFKC RVV TGTAGATTAT GTTATCGAGG GTACTGCTAC AAAACCTAAA GATACTTATG TCGATACCCC AACAGCTTAT ATTCGTGATA AAGAAOGTCA 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 R D K E G Q AGCTATTCCT TATAATACAG CTGAGAATGA TTCTGAGAAA CCATTGTTAC TTGACAAAGA TGGAATAAAA TATGAACTAG TTTCAATTCA 2340 A I P Y N T A E N D S E K P L L L D K D G I K Y E L V S I Q ACACGCGTCA GCACCGGAGA AAGGAACACT TCGAGAAGCC GAACAACATG TTGTCTATCA ATAGCGTAAA GTCGTATACG TTCCAAGTGT 2410 EGS AAEK GTL REC EQHV VYO YRK V\'.\'2V PSV TAAAGTTCGA AATGTTCATG CCCGCTATGT TATTTTAOCG ACAGAGACTC AGCTTGCAAG TGCTAAAACA GTTAAATCAS AAOCTCCAAT 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 E A P I TGATGAGGCA TATTCAGATA AAGCACCTOC AACTCTTGAA AAAGATGGTA AGTTGTATGA ATTTGTACAT GTGCGTGATA ATAACCOCGA 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 H K G D TOCTCCAGCA GATGGTAAGG TGACTGAACA AGATCAGACC ATTACATATG AATATAAOCT TAAGAAAGAT GACTTASACS CTGTAGGGAA 2700 A P A D G K V T E Q D Q T I T Y E Y E L E K D D A D A V G N V GACTTACATG 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 CTAGTGATT AACTATGTGG ACGAAACAGG TAATGTTAGTC AAAAAACCAA TACTAGATAC ACACGAATCA AACGTTGOGA CACCATATG V V I H Y V D E T G N V I K K P I L D T H E S K V S T P Y D : :-. "C ;. \' : To 1. t i\'" : C :". :.. i. Z\' : :. a Al\'si\'LTx : : .. :, T\' : a.\' : _^ ; C\' :\' :\'i \' : , ^. a : ^_i\' : : ?. a. :\'I GCrla.. 23 3 J 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 CCTCACTGA GGCACAACAG AAGTGACTTA TGTGTATCGA GAATCAGTAA AATCTACCCT ACCAAAGGAA ACGGOCATTT CTATTCCAAG 2970 V T E C T T E V T Y V Y R E S V K S T L P K E T C I S I P S C\'..),. 1 :. >. : : TT_ : : a 1W TT, aT ?. TCi . ;. CC ?. L. ^. :. :. GL1,. > :. : A : :, CT. : : :. ?. > :.\'v : .-.. 1M :. 1. : T ? _ :.. :. J L : a :\'C1. ICCLI 3 : G E S E S P K F I S T Q T T E N R P N K G V L T S S K N P T N T AAATCTGTT CTTCCAACTA CAGGCCAGGA AAGTAATAGA ATTCTCCGAG TTGTTOCGAT TACTCTAGTA OCAATAACAG CTACTTTGGC 3150 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 a : c ; . . : : ^. ;, aa. c ;, . au : a r : c ;. : r carr :- :,- : ;. vca. a : cr-rr ; x ; x : :.. _...,. c--crrc \'= :- a S S L K R R K N A ; \' :. :. 1U u :, T"\',AJ. :"". J,. 1TC : ?,. I\'"\'\'C 1AT^., G- :.\'. r.\'T i G : :,,\'^ : :A;. La.\'G ; :.\' : \'.,. . a : ^, a T. A :, 5 :. A\'. a i _ T. LL. ; _,\' :. :, aCCa 71 3 J GTCTACAAAA AAGTCATCAT TTGATGGCTT TTTTTGCAAT TTATCATTT s AAAAACGGAA TAAAATATTA TTATAICCGT 3410

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 ex- pression 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 sequen- ces 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 prepara- tion 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 2O 90 1134557390 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 AAACTCTCTT TAATTAGCTT TATATTGGAT AGAAGTATCG AATCTGTTTT ATGTAAACTA CCAGGTCTTA GTATCTATTT TTCAATTTAT 90 CTTGAATGAC CCACCTCTTT ACTTTTATGT AAAGTCAAAA TATTCTTAAA TTTGCATAAT TTTCAAATTT CTTTACCTTT ATAATTCACA 180 ACCCTTTTAT TTTTTGTTGT TTTTTAAATG TAAAAACTGT TACACGAAAG AOGAAGATAT TATGTTTCGT CAATATAATT TTGAAAAAGG 270 M F R Q Y ru P E K G TTTAAATTTT TCCATTCGTA AATTTTCGGT TGGAATAGCT TCAGTTGCTA TAGGATCATT ATTATTCATT TTACCTCAOG TACTTGCACA 350 L N F S I R K F S V C I A S V A I G S L L F I L P Q V L A D TGAGACAACT AGTGTTACAT CTGCAACAAC TCCAACACGT GTAACGACAA CAGATGCAAA CTTGGTCAAT CCTAACAATT 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 N S T P T TTCCACTAAT AGGAGTGCAA CTAGCACTCA AGGAAGTAAT TTGAGTAATA CTTCAGAAAT CATTAAGCCT GCAACTTTAC CACCAACATC 540 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 AGACAAGAGG ACGTATGCTA CTAGTOGCGA TTOGACGTTA CAAAATCCAT ATOCTGACAG 630 PTT DMVA PSV DKR TYAT SGD WTL Qt-PY ADS TGTTCGAAAT AAAAATATTT CTCCAAGTGT TCGTCATGAA TCATTTAAAA GTGCTGAAAC AACGGTGGTT COTCATGATA ACTCAACCGT 720 VRM KMIS PSV RHZ SPKS AET TVV RHDK STV TAAAGTGACT GCGACAATTA CTCCTGTAGA AGGGAATGAT GAAGGTTCTG GTATTTTGAC AAATGGTGGT AATCAGTCAG AATATAAAGC 810 KVT AT IT PVE GND EGSG ILT NCC MQSE YKA AACATCAGAA ATGTTTGTTG GAAATGTAGA TCCTGCAAAA ATACCTGCTT TAOGTGTCTA TACACAACCA GGACGTACTG AAOGAGGTAG 900 T S E M F 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 TAAACTATCT GATAAGCTCA ATTTTAATGG GAACGCGCCA 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 I TGATTTATCT GGTGTTGGAG GTAATGCACG TCTATCATTT ACAGAAACTG TCATGGAAAA TGGTAAGATT GTTGAAAAAT TTGACTATGC 1080 DLS GVGG NA. R LSF TETV MEN CKI VEKF DYA ACGTGGTTCA TATAATTCAA CGTTCTTTGA TGGAATAACT CCAGGTATAA GCCTTGAAAA AGCAAGTTCA CCATTTAATT TAACAGTTAC 1170 PCS YMST PFD GIT PGIS LEK ASS GVML TVT AGCGAATACC GTTGATGTAA CAGACAAGAA CACCTTTAAT GAGTCAGTTG TTAATCCATC TGACGAAGAC TTTGTCAATG GTCCAGATAG 1260 ANT VDVT DKN T ? N ESVV UPS DED FVNC PDR AACCCCTCAT GCAGTCCCAG CGGGAACAGG CTCAATCCGT TTAAAAGGAA CCTTTACAGA CGCTTCATTT AAATTATATC ATCAAGCTGT 1350 TPD AVPA CTC SIR LKGT FTD ASF KLYH QAV TCCATCTACA GCATTTTCTA AAGAAAAATA TCATACAGGA GATGGATATT ATAATAGTAT TGCGAATGTT CGACCAACTG TAGTTAAACC 1440 PST AFSK EKY HTG DGYY MSI AMV R ? T V V K P AGATAGTATT AATGGTTTAA ATAAGCACGC AAGTGATGTT ATTGATTATA AAATTTCCGA TAATAATGAT ATTTCGAACG ATGACTTACT 1530 DSI NGLM KHA SDV IDYK ISD NMD ISKD DI. L TCGTTTATCT CTTCGCTTAC AAAATCCCCG TGGATCAGTT GTTGTAAATT ATATTGATAC AGAAGGCAAT ATTATTGGTA CTGAATACAA 1620 RLS VRLQ NPR GSV VVMY IDT EON IIGT EYK AGATACCACC GATGCAATTC CAGGAACACA TTACAATACG GCAGAATCAT CAGGAGACCT TAATTCAGAC GCCACAGTTG AGCGTCCTTC 1710 DTT DAIP GTH YHT AESS GDL NSD ATVE RPS CACTATTACT AAAGACGGTA AGGTGTATGA TTTAGTAGCA GAAAATATTA CAGTGCCAGT TGGTAAGGTT AATTCGGATG GTACGTTGGC 1800 TIT KDGK VYD LVA EMIT VPV CKV NSDG TLA TACAAATOOC AGCTCATTTA ATTATGGTAC TGATGCAGCA TCTGCAGAAG TTGCAGAAGC TACTAAGTCA GTAATTTATG TTATTAGCAT 1990 T N G S S F N Y G T D A A S A E V A E G T K S V I Y V I S I TAAACAAGAA TCAAAGGGEA ATGTTCATGC CCGCTATGTT ATTTTAGGGA CAGAGACTGA GCTTGCAAGT GCTAAAACAG TTAAATCAGA 1980 KQE SKGM VHA RYV ILGT ETE LAS AKTV KSB ACCTCCAATT GATGAGGCAT ATTCAGATAA ACCACCTGCA ACTCTTGAAA AAGATCGTAA GTTGTATGAA TTTGTACATG TCCGTGATAA 2070 API DEAY SDK APA TLEK DCK LYE FVHV RDM TAAGGCCGAT GCTCCAGCAG ATGGTAAGGT GACTGAACAA GATCAGACCA TTACATATGA ATATGTTGAA GTTCCTAAGG GTCGTGTGGT 2150 K G D A P A D C K V T E Q D Q T I T Y E Y V e KG RVV TCTACATTAT GTTATCGAGG GTACTGCTAC AAAACCTAAA GATACTTATG TCGATACCCC AACAGCTTAT ATTCGTGATA AAGAAGGTCA 2250 VDY VIBG TAT KPK DTYV DTP TAY IXDK ECQ AGCTATTCCT TATAATACAC CTGACAATGA TTCTGAGAAA CCATTGTTAC TTGACAAAGA TGGAATAAAA TATGAACTAG TTTCAATTCA 2340 AIP YNTA END SZK PLLL DKD CtK YSLV 5IQ AG AGGCGTCA GCACCGGACA AAGGAACACT TCGAGAAGCC GAACAACATG TTGTCTATCA ATATCGTAAA GTCCTACACC TTCCAAGTGT 2430 E G s A A e K G T L R E G E Q H V V Y Q Y R K V f E V P S V TAAAGTTTGA AATGTTCATG CCCGCTATGT TATTTTAGCC ACAGAGACTC ACCTTCCAAG TGCTAAAACA GTTAAATCAC AACCTCCAAT 2520 K MVHA RYV ILG TETE LAS AKT VXSE API TGATGAGGCA TATTCAGATA AAGCACCTGC AACTCTTGAA AAAGATGGTA AGTTGTATGA ATTTGTACAT GTGCGTGATA ATAAGGGCGA 2510 DEA YSDK APA TLE KDGK LYE FVH VXDN KGD TOCTCCAGCA CATGGTAAGG TGACTGAACA AGATCAGACC ATTACATATC AATATAAGCT TAAGAAAGAT GACGCAGACG CTGTAGCGAA 2700 APA DCKV TEQ DQT ITYE YKLXKD DAOAVCH TGTAGTGATT AACTATGTGG ACGAAACACG TAATGTTATC AAAAAACCAA TACTAGATAC ACACGAATCA AACGTTGOCA CACCATATGA 2790 VVI MYVD ETC NVI KKPI LOT HES K.\'GT PYD T \'Ci.. AT. \' : T7vT. A. l i\'L ; : Ci\'"YiTT1 AT\'i I\'LT\'CCA T\'i C. 1\' : a CCL\' :\'irC i . A : : ? _L : L : TCCG ?. 23 i J T T D Y K F A E I K F N G K I Y K L V S A K T H C N E F G K GGTCACTGAA OGCACAACAG AAGTGACTTA TGTGTATCGA GAATCAGTAA AATCTACCCT ACCAAAOGAA ACCGOCATTT CTATTCCAAG 2970 V T E C T T T E V T Y V Y R E S V K S T L P K E T C I S I P S CGAATCAGAG TCTCCTAAGT TTATATCTAC CCAAACTACA CAAAATAGAC CTAATAAAGG AGTATTAACT TCATTTAAAA ATCCAACCAA 3050 E S E S P K F I S T Q T T E N R P N K C V L T S S K N P T N TAAATCTGTT CTTCCAACTA CACGCGAGGA AAGTAATAGA ATTCTCCGAG TTGTTGCGAT TACTCTAGTA CCAACAACAG CTACTTTGCC 3150 K5V LPTT GEE SHR ILGVVGI TLV A--ATLA ACCTAGGACT TTAAAACGAC GTAAAAATTA AAATTCTTCT CACTCGATGT GACCCAATAC CGTTATTATA AGATTCTTTT GTTGATAATC 3240 CTCTACAAAA S S L K R R K N AG. ? GT. 1U T\'I\'iI"1 :\'\'.,. 1.. 1 CsJII\'I\'ICII\'n1 T\'". G : :. : . i\'L\' G :, :. T\' \'.\'riii C :, G\' : Cla : ^.\'A T. l : C\' : ?. : a : _\'.. L1 : :. t, t. C\' : \'TT1CW J337


GTCTACAAAA AAGTCATCAT TTGATGGCTT TTTTTGCAAT TTATCATTTT AAAAACCGAA TAAAATATTA TTACATOCGT 3410

(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 resul- ting fusion protein or fusion polypeptide is isofunctional and/or the additional C-terminal or N-terminal amino acid se- quences may be easily eliminated under physiological conditi- ons.

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 abo- ve.

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 par- tial expression products as defined above or synthetic pro- teins or polypeptides as defined above or proteins or poly- peptides as defined above or polyclonal or monoclonal antibo- dies as defined above in an assay for diagnosting an infec- tion 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 pro- ducts or partial expression products as defined above or syn- thetic 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 car- bohydrate 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 ex- traction 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 rab- bit serum (lanes 4-6).

B. A Western blot analysis of GBS strains using monospeci- fic 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 Comp- ton 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 : HC1 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 (l hr/37°C) on the immuno- precipitin reactions of the R3 and R5 proteins from the HC1 extract of S. agalactiae Compton R. Center well : antiserum to the R3 and R5 proteins of Compton R. Well 1 : untreated HC1 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 antibo- dies 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 LDgoof eith- er Compton R (CR) or B176 strain, respectively, at day 37 af- ter the primary immunization, and mortality was daily recor- ded.

Cloning and nucleotide sequence analysis of the gene encoding R5 protein A lambda Zap gene library of S. agalactiae Compton R chromo- somal 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, desi- gnated pSE3. The 5. 0 kb DNA insert of the plasmid was subjec- ted 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 re- peats 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 R1, R3 and R4 (see below) and thus desi- gnated 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\'-SmaI 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 XL1-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 reco- gnized 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 XL1-Blue MRF\' (pGEX-2T) whole cell extracts and the 158 kDa degraded recom- binant 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 an- tigen produced by S. agalactiae Compton R, partially purified Compton R surface proteins and purified recombinant GST- tagged R5 are degraded following sample preparation, resul- ting in multiple bands being detected (Figure 2B, lanes 1, 2, 4, 6). However, lack of reactivity of the R5 antiserum against E. coli XL1-Blue MRF\'confirms the lack of cross- reactive antibodies and the purity of the GST-tagged R5 pre- paration 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 R1 and R4. S. agalactiae H4A-0126 (serotype la/R1) 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 distinc- tion between R1, 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 re- veal 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. Fi- gure 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 an- tiserum 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 produ- ced 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 pre- cipitin reaction of identity was observed with the purified recombinant protein and the HC1 extract of the Compton R strain, while the extracts of strains 71-735 (III/R1) 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 HC1 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, ligh- ter) 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) elimina- ted R5 but did not affect the R3 reaction, while 5% trypsin greatly diminished the R3 reaction (well 4). By contrast, on- ly 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 opti- mum 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. Immuniza- tion 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 adju- vant. 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 Ia 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 infecti- ons 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 stra- tegy 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 potenti- al of capsular polysaccharides, the interest has shifted to- wards protein antigens as components of vaccine candidates. A number of surface proteins such as B-antigen (Jerlström et al., 1991 ; Jerlström et al., 1996), a-protein (Michel et al., 1991), protein Rib (Stalhammar-Carlemalm et al., 1993) and R-proteins (Flores and Ferrieri, 1989, 1996) have been de- scribed. 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 in- vention 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 con- sists 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 a-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 a-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 pro- duct 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 HC1 extracts of the invention were tested with Prague anti- Compton R serum, as was used in those studies, the R5 preci- pitin reaction was weak and practically fused with that of R3, making it difficult to recognize them as two distinct li- nes. 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 in- vention. 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 con- tained 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 ex- tracted by very mild trypsin treatment of bacterial cells, further digestion with even 0. 2% trypsin eliminated its pre- cipitin 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 a 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, in- dicated that R5 was commonly found in serotype Ia/Rl isolates (Flores et al. 1999). The presence of both R1 and R5 in the serotype Ia isolates was analogous to the presence of both R1 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 iso- lates and that, as such, it may be useful in their characte- rization (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 subcutane- ous or mucosal route triggered the elicitation of protective immunity. The natural portal of entry for group B streptococ- ci 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 fol- lowing vaccination. This may lead to a more efficient protec- tion of newborns by two different mechanisms, namely (i) pas- sive 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 an- tibodies. In fact, it has been described that due to the mu- cosal 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 illu- stration 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 Universi- ty of Minnesota (UM) Minneapolis, MN, USA. R protein prototy- pe strains were Compton R (nontypeable/R3, R4, R5) (Compton 25/60, NCTC 09828, J. Jelinkova, Prague) ; 71-735 (III/R1) (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 (Ia, 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 medi- um supplemented with 1 g/1 MgCl2 and 4 g/1 maltose. Bacteria were grown at 37°C unless otherwise stated. Where appropria- te, E. coli were grown in the presence of 100 pg/ml ampicil- lin, 15 Hg/ml tetracycline, 50 pg/ml kanamycin and 1 mM iso- propyl-B-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. aga- lactiae 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 allo- wed to proceed for 2 h at 37°C with shaking. The suspension

was centrifuged (15, 000 g, 20 min) and the supernatant adju- sted to pH 7 using 1 M HC1. The supernatant (15 ml) was con- centrated 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 sa- me 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 in- structions (Pharmacia). His-tagged R5 fusion protein was pu- rified under native conditions according to Qiagen protocols.

Protein concentration was determined by the method of Brad- ford (Bradford, 1979).

DNA manipulations Chromosomal DNA from S. agalactiae Compton R was isolated ac- cording to the method of Talay et al., (1992). Purified chro- mosomal DNA was partially digested with the restriction enzy- me 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 ma- nufacturer\'s instructions. The ligation mixture was packaged in vitro into lambda heads and tails (Gigapack Gold 11, Stra- tagene) and transfected into E. coli XL1-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 manu- al. Plasmid DNA was prepared using the QIAwell plasmid ex- traction kit (Qiagen) and sequenced using the method of San- ger et al., (1977). Reactions were carried out using dye ter- minator ready reaction mix (Perkin Elmer) and electrophoresed on an ABI 373A DNA sequencer (Applied Biosystems). For com- plete 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 perfor- med in a thermocycier (MWGBiotech) ; the resulting PCR frag- ment was cloned into the BamHI and SmaI sites of the expres- sion 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). Alternati- vely, western blots of proteins electroeluted onto Immobilon P membranes (Millipore) were performed essentially as descri- bed 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 prote- in 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 dehydra- ted with acetone, and finally embedded according to the me- thod 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 HC1 or 0. 1% trypsin cell extracts (Flo- res and Ferrieri). To examine the susceptibility of the va- rious R proteins to trypsin or pepsin digestion, HC1 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-2d) mice (Harlan Winhelmann) were immunized with recombinant R5 protein on days 1, 3, 6

and 27 (30 pg/dose) by subcutaneous or intranasal route using aluminium phosphate (Adju-Phos, Axell Accurate Chemical & Scientific Corp.) or cholera toxin B subunit (CTB 10 pg/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 (LDgo) 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 pl/well of R5 in coating buffer (bicarbonate, pH 8. 2). After overnight in- cubation at 4°C, plates were blocked with 10% fetal calf se- rum (FCS) in PBS for 1 h at 37°C. Serial two-fold dilutions of serum in 10% FCS-PBS were added (100 pl/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 p-chain specific goat anti-mouse IgM and p-chain specific goat anti-mouse IgG (Sigma), and incubated for a further 2 h at 37°C. After four washes, 100 pl 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 (OD405) of

0. 1 unit above the OD405 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 de- signated R5 that exhibited typical features of streptococcal surface proteins such as a signal sequence and a membrane an- chor 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 R1 and R4 proteins expressed by two va- riant type III GBS strains, but reacted with the parental streptococcal strain in Western blot and immunoprecipitin analysis. Separate R3 and R5 immunoprecipitin bands were ob- served 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.

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