INTRODUCTION

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Group B streptococci (GBS) are the major cause of life-threatening bacterial infections in neonates and very young infants (38). Approximately 70 to 80% of infant infections occur in the first few days of life, so-called early-onset disease, while late-onset infections occur in infants between 1 week and 3 months of age. Newborns with early-onset GBS disease usually acquire the organism during delivery from their GBS-colonized mothers. In order to substantially reduce the incidence of early-onset GBS disease, prenatal screening for GBS and intrapartum antimicrobial prophylaxis are now highly recommended in the United States (9, 39). However, since these strategies require the frequent use of antibiotics, antibiotic-resistant GBS or other bacterial agents might emerge during the perinatal period (14). In addition, these measures are unlikely to prevent late-onset infections, prematurity, and stillbirths related to GBS, while obviously not addressing GBS disease in nonpregnant adults. Indeed, GBS are also a frequent cause of infections in pregnant women and in clinically ill and older adults, such as those suffering from diabetes, cirrhosis, malignancies, and immunodeficiencies (38). For all these reasons, vaccination is a very important alternative for disease prevention. Already, Baker et al. (2) have demonstrated a correlation between maternal antibody deficiency at delivery and susceptibility to neonatal GBS infection. That finding suggests that vaccination of pregnant women could become a very efficient prophylactic strategy to prevent GBS infection in neonates since it could stimulate transplacental transfer of GBS-specific antibodies from the mother to the fetus, thus considerably increasing the level of protective antibodies present at the time of delivery (3).

All clinical isolates of GBS express a polysaccharide capsule, with nine capsular serotypes identified so far. The major invasive disease-causing serotypes are Ia, Ib, II, and III (38). Recent population-based surveillance studies have indicated an increasing importance of serotype V strains, which were reported to account for a substantial proportion of adult cases (7, 17, 30). The evolution in serotype distribution and target population will have a major impact on the formulation and efficiency of the multivalent polysaccharide-based vaccines currently under development (21, 45, 46). Indeed, it was observed that the protection conferred by capsular polysaccharides is type specific (21). Based on current information on serotype distribution, a tetanus toxoid conjugate vaccine would have to contain types Ia, Ib, II, III, and V to prevent the majority of disease in North America, but would also have to be modified to be efficient in other parts of the world, such as Japan, where other serotypes, such as VI and VIII, are more prevalent (25).

An alternative strategy for protecting neonates and infants would be to develop a GBS vaccine based on a ubiquitous protein. Bacterial surface proteins have numerous advantages for vaccine development. Indeed, such bacterial proteins were shown for other bacterial pathogens to be present in most pathogenic strains and to induce cross-protective immunity (11, 32). Furthermore, these proteins do not need to be conjugated to other molecules, since they elicit an effective T-cell-dependent antibody response resulting in long-term immunity. GBS surface proteins already being investigated as potential vaccine candidates are the R protein, the  and  subunits of the c protein, and the Rib protein (12, 15, 41). All these proteins are capable of eliciting antibodies in mice and to some extent prolong life and protect against lethal bacterial challenges.

Here, we report the discovery of a unique 53-kDa protein called Sip, for surface immunogenic protein. This protein is produced by all GBS isolates examined to date and is capable of conferring protection against experimental infection with GBS strains representing the five major disease-causing serotypes.

	MATERIALS AND METHODS

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Bacterial strains and media. A collection of 69 strains of GBS representing the nine capsular serotypes were used in this study. The panel of strains included 14 isolates of serotype Ia, 3 isolates of serotype Ib, 1 isolate of serotype Ic, 4 isolates of serotype II, 14 isolates of serotype III, 2 isolates of serotype IV, 12 isolates of serotype V, 2 isolates of serotype VI, 2 isolates of serotype VII, 1 isolate of serotype VIII, 11 isolates not serotyped, and 3 bovine strains. These strains were obtained from the American Type Culture Collection (Rockville, Md.), centre de recherche en infectiologie of the Centre Hospitalier de l'Université Laval (Ste-Foy, Canada), Children's Hospital and Medical Center (Seattle, Wash.), Laboratoire de Santé Publique du Québec (Montreal, Canada), and National Centre for Streptococcus, Provincial Laboratory of Public Health for Northern Alberta (Edmonton, Canada). The sip gene was initially identified from GBS strain C388/90 (Ia/c), which had been isolated from the cerebrospinal fluid of a child with meningitis and was obtained from the Children's Hospital of Eastern Ontario (Ottawa, Canada).

ZM15

SDS-PAGE and Western immunoblotting. To prepare the GBS whole-cell (WC) preparation used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots, approximately 15 mg (wet weight) of an overnight culture was added to 500 µl of sample buffer (0.2 M Tris-HCl [pH 6.8], 1% SDS, 2% mercaptoethanol, 10% glycerol, 0.001% bromophenol blue) and then boiled for 5 min, and after centrifugation, 15 µl of supernatant was applied to an SDS-10% PAGE gel. GBS overnight cultures were also digested with a final concentration of 350 U of mutanolysin (Sigma) per ml for 15 min at 37°C with gentle agitation before the addition of sample buffer (41). After electrophoresis, the proteins were stained with Coomassie brilliant blue (Bio-Rad), silver stained (Bio-Rad), or transferred to nitrocellulose membranes for Western immunoblotting as previously described (32).

ELISA. The antibody response of immunized mice was determined by enzyme-linked immunosorbent assay (ELISA) using either purified recombinant Sip at a concentration of 1 µg/ml in carbonate buffer (15 mM Na₂CO₃, 35 mM NaHCO₃ [pH 9.6]) or formaldehyde-killed GBS WC preparations as coating antigens. To obtain the WC preparations, a final concentration of 0.3% formaldehyde was added to the GBS culture and incubated overnight at 4°C. After several washes in phosphate-buffered saline (PBS), these killed GBS preparations were spectrophotometrically adjusted to an optical density at 600 nm (OD₆₀₀) of 1.0, and 100 µl was dispensed into the wells of the ELISA plates (Falcon 3915; Becton Dickinson, Franklin Lakes, N.J.). The ELISA procedure was described previously (37). The serum dilution for which an absorbance reading of 0.1 ( = 410/630 nm) was recorded after background subtraction was considered the titer of this serum.

Identification, cloning, and sequencing of the sip gene. Chromosomal DNA was isolated from different GBS strains as previously described (18). A ZAPII genomic library was constructed using chromosomal DNA purified from the GBS strain C388/90 (Ia/c) and screened according to the manufacturer's instruction (Stratagene, La Jolla, Calif.) with a pool of human normal sera collected from volunteers with no known history of GBS disease. These sera were selected by ELISA and Western immunoblots for their high reactivity with GBS WC preparations. Briefly, the purified chromosomal DNA was partially digested with tsp509I restriction enzyme, and the resulting fragments were electrophoresed on a 1% agarose gel (Bio-Rad). Fragments in the 5- to 10-kb size range were extracted from the gel and ligated to the EcoRI arms of ZAPII vector, and the vector was encapsidated using the Gigapack II packaging extract (Stratagene). The recombinant phages were used to infect E. coli XL1-Blue MRF', which was then plated onto LB agar. The resulting plaques were lifted onto Hybond-C nitrocellulose membranes (Amersham Pharmacia Biotech, Baie d'Urfée, Canada) impregnated with 10 mM isopropyl--D-thiogalactopyranoside (IPTG; ICN Biomedicals Inc., Costa Mesa, Calif.). The membranes were blocked using PBS with 3% skim milk and were sequentially incubated with pooled human sera or hyperimmune mouse sera collected after immunization with GBS WC preparations, corresponding peroxidase-labeled goat anti-human or anti-mouse immunoglobulin G (IgG) antisera (Jackson Immunoresearch Laboratories Inc., West Grove, Pa.), and substrate. Positive plaques were isolated and purified twice, and the recombinant pBluescript plasmids were excised with the ExAssist helper phage (Stratagene) according to the manufacturer's instructions. The sequence of the insert was determined using the Taq Dye Deoxy Terminator Cycle Sequencing kit with an Applied Biosystems Inc. (Foster City, Calif.) automated sequencer (model 373A) according to the manufacturer's recommendations.

Production and purification of recombinant Sip protein. The fusion protein was purified by affinity chromatography using a nickel-charged resin (Ni-nitrilotriacetic acid Superflow: Qiagen Inc., Mississauga, Canada) following the manufacturer's instructions. The purified recombinant plasmid pURV32 was used to transform E. coli B strain BLR by electroporation with the Genepulse II apparatus (Bio-Rad) following the manufacturer's recommendations. This recombinant strain was inoculated in LB broth (Gibco-BRL) containing kanamycin (40 µg/ml) and was first incubated at 34°C for approximately 3 h with agitation (OD₆₀₀ = 0.6), after which time the temperature was increased to 39°C for an additional 4 to 5 h in order to induce production of the recombinant protein. After the induction period, it was observed that the recombinant Sip protein could be found inside the bacterial cells as well as in the culture supernatant. It was decided to use the protein present in the supernatant for purification of the Sip protein. The bacterial cells were removed from the culture medium by centrifugation at 12,000 × g for 30 min at 4°C. The supernatant was then filtered onto a 0.22-µm membrane and concentrated using an ultrafiltration apparatus and a Diaflo ultrafiltration membrane YM10 (Amicon Inc., Beverly, Mass.). The concentrated supernatant was subjected to 50% (wt/vol) ammonium sulfate precipitation, and the precipitated proteins were collected by centrifugation and suspended in 50 mM Tris-HCl buffer (pH 8.5). The Sip protein was purified from the other proteins by two successive chromatographic steps, hydrophobic interaction chromatography using Sepharose HP (Amersham Pharmacia Biotech) and anion-exchange chromatography using Hi-load Q-Speharose high-performance resin (Amersham Pharmacia Biotech). The purity of the recombinant Sip protein was evaluated by SDS-PAGE, and the amount of protein was determined by the bicinchoninic acid assay according to the manufacturer's instructions (Pierce Chemical Company, Rockford, Ill.). The NH₂-terminal amino acid sequence of the recombinant Sip which was transferred onto polyvinylidene difluoride membrane (Bio-Rad) was determined using Edman degradation on an Applied Biosystems 473A protein sequencer (Applied Biosystems, Norwalk, Conn.) and was performed by the Service de Séquence des Protéines de l'Est du Québec (Quebec, Canada).

Generation of a Sip-specific MAb. BALB/c mice (Charles River Laboratories, Montreal, Canada) were injected subcutaneously (s.c.) three times at 3-week intervals with 20 µg of purified recombinant thioredoxin-Sip protein in 0.1 ml of PBS mixed with 20 µg of QuilA adjuvant (Cedarlane Laboratories, Hornby, Canada). Three days before the fusion procedure, the selected mouse received a final intravenous injection of purified recombinant Sip protein. The fusion protocol used to produce the hybridoma cell lines was described previously (37). Hybrid clone supernatants were tested for Sip-specific antibody production by ELISA and by immunoblots using purified recombinant Sip protein and GBS WC preparations. Specific hybrids were cloned by sequential limiting dilutions, expanded, and frozen in liquid nitrogen. The class, subclass, and light-chain type of the monoclonal antibodies (MAbs) were determined by ELISA with commercially available reagents (Southern Biotechnology Associates Inc., Birmingham, Ala.).

Immunization and protection model. Groups of 10 female CD-1 mice (Charles River), 5 to 7 weeks old, were injected s.c. three times at 3-week intervals with either 20 µg of purified recombinant Sip protein in 0.1 ml of PBS mixed with 20 µg of QuilA adjuvant (Cedarlane), 15 µg of formaldehyde-killed GBS WC with 20 µg of QuilA as positive controls, or 20 µg of QuilA in PBS as a negative control. Serum samples from each mouse were taken before each immunization and 2 weeks after the third injection. To enhance virulence, GBS strains were passaged by intraperitoneal (i.p.) injection several times in female CD-1 mice as described by Lancefield (27), and early-log-phase stock cultures were frozen at 80°C in THB containing 20% glycerol. Depending on the strain, the number of passages necessary to increase the virulence varied from 6 to 15. To evaluate the challenge dose required for each GBS strain, between 10⁴ and 10⁸ CFU/mouse was injected i.p. into groups of CD-1 female mice (14 to 16 weeks old). Mortality was recorded for the next 7 days. An animal infection model which was previously described by Baltimore et al. (4) was performed 3 weeks after the last injection. Briefly, before each challenge experiment, an aliquot from the appropriate mouse-passaged frozen stock culture was thawed and diluted with THB to obtain the predetermined 90% lethal dose (LD₉₀), which varied between 10³ and 10⁵ CFU. Mortality was recorded daily for the next 14 days. Statistical significance was estimated by Fisher's exact test. The protective efficacy of Sip immunization was estimated as previously described (29).

Nucleotide sequence accession numbers. The nucleotide sequences of the GBS sip genes described in this report were assigned the following accession numbers: strain C388/90, AF151357; strain AF151358, COH1; strain NCS246, AF151359; strain NCS535, AF151361; and strain NCS915, AF151362.

	RESULTS

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Identification and sequencing of the sip gene. A GBS chromosomal library was constructed from the GBS serotype Ia/c strain C388/90 in ZAPII. Chromosomal fragments in the 5- to 10-kb size range that were obtained after partial digestion with tsp509I restriction enzyme were ligated to the EcoRI bacteriophage arms. Of 25,000 plaques tested, a total of 38 positive plaques were identified after immunoscreening of the library with the pool of normal human sera. These sera were selected based on their reactivity with GBS WC preparations. After two purifications, these clones were screened a second time with several mouse polyvalent sera collected after immunization with GBS WC preparations from the GBS serotype Ia/c strain C388/90. One clone, identified as 32, which reacted strongly with all the mouse sera tested was selected for further characterization. Immunoblots using phage lysates revealed that the mouse sera as well as the pool of human sera reacted with a protein band with an approximate molecular mass of 53 kDa. The expression of the protein was not affected by IPTG induction, suggesting that the gene coding for the protein of interest was under the control of its own promoter. The recombinant pBluescript plasmid pSag32 was excised from the bacteriophage arms, the sequence of the GBS 3,480-bp insert was determined, and the schematic organization is presented in Fig. 1. Two complete open reading frames (ORFs), ORF2 and ORF3, and one incomplete gene encoding ORF1 were predicted after sequence analysis. The deduced amino acid sequence of ORF1 showed homology (54% identity) with a bacteriocin-like inhibitory substance from Streptococcus zooepidemicus which was described previously (40), while 45% identity was noted for ORF3 with a putative N-acetylmannosamine-6-phosphate epimerase of Clostridium perfringens (43).

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Schematic representation and partial restriction map of the DNA insert of recombinant plasmid pSag32. Numbers indicate the distance (in base pairs) from the 5' end.

View larger version (45K): [in this window] [in a new window]   FIG. 2.   Comparison of the predicted amino acid sequences of Sip proteins from one serotype Ia/c strain (C388/90), one serotype II strain (NCS 246), three serotype III strains (COH1, NCS 915, and NCS 945), and one serotype V strain (NCS 535). Differences are indicated in the one-letter code, and identities are marked by a period. A 25-amino-acid-residue leader peptide is underlined.

Comparison of sip genes. To evaluate the level of molecular conservation, sip genes were amplified by PCR from five additional GBS strains, three of serotype III, one of serotype II, and one of serotype V. The nucleotide and deduced amino acid sequences of these six sip genes were found to be highly conserved. Indeed, at the nucleotide level, those six sip genes showed differences in only 19 positions out of the 1,305 bp, which makes them >98% identical (data not shown). Similarly, at the amino acid level these predicted proteins differ at only 8 of 434 residues, making them 98% identical. These differences are not clustered in any particular region of the Sip protein (Fig. 2).

Production and purification of the GBS Sip protein. The sip gene from GBS strain C388/90 (Ia/c) was amplified by PCR, and the purified product was ligated first into the IPTG-inducible pET32 vector to generate sufficient recombinant protein to immunize the mice required for MAb production. The resulting recombinant plasmid was named pURV32.2. This thioredoxin-Sip fusion protein was purified by affinity chromatography with a nickel-charged resin. As expected, since the sip gene was inserted after a thioredoxin insert, the resulting product migrated after SDS-PAGE at an approximate molecular mass of 70 kDa (Fig. 3, lanes 1 and 2). This purified thioredoxin-Sip fusion recombinant protein was recognized by the pooled human sera (Fig. 3B) used for the initial screening of the genomic library and by the Sip-specific MAb 5A12 (Fig. 3C).

View larger version (51K): [in this window] [in a new window]   FIG. 3.   Coomassie blue-stained (A) SDS-10% PAGE gels and corresponding Western immunoblots probed with (B) pooled human sera diluted 1:500 and (C) Sip-specific MAb 5A12, showing the reactivity with WC of E. coli AD494(DE3) transformed with pURV32.2 (lane 1); 400 ng of purified thioredoxin-Sip fusion protein (lane 2); WC of E. coli BLR transformed with pURV32 (lane 4); culture supernatant obtained after production of recombinant Sip protein by E. coli BLR transformed with pURV32 (lane 5); and 400 ng of purified Sip recombinant protein (lane 6). The open triangles and arrows indicate the locations of the thioredoxin-Sip fusion and recombinant Sip proteins, respectively. Size standard proteins (200,000, 97,400, 68,000, 43,000, 29,000, 18,400, and 14,300 Da) were deposited in lane 3.

Generation of Sip-specific MAbs and identification of the Sip protein in GBS strains. To generate hybridoma clones secreting Sip-specific MAbs, mice were immunized with purified recombinant thioredoxin-Sip protein. Eight hybridoma cell lines were found to secrete Sip-specific MAbs. After the initial characterization, one Mab, named 5A12, an IgG1, which was found by immunoblots to react efficiently with purified recombinant Sip, purified thioredoxin-Sip fusion protein, as well as the native Sip present in GBS WC preparations, was selected to further evaluate the distribution of the Sip protein among GBS isolates (Fig. 3C). The GBS cells were boiled for 5 min in SDS-PAGE sample buffer without any mutanolysin treatment and then centrifuged to remove the larger fragments that were not solubilized. Since this treatment only released small quantities of protein from the bacteria, the gels were silver stained in order to vizualize most of the proteins present in the GBS WC preparations. The addition of mutanolysin during sample preparation increased the amount and diversity of protein released from the GBS cells (data not shown). However, visual examination of the migration profile on an SDS-PAGE gel of the proteins released from GBS WC preparations when they were not treated with mutanolysin indicated that the Sip protein was one of the major proteins. For that reason it was decided that boiling in SDS-PAGE buffer was sufficient to obtain suitable antigenic preparations for immunoblots. MAb 5A12 reacted with a protein band with an approximate molecular mass of 53 kDa which was present in WC prepared from 69 GBS strains. Representative results are presented in Fig. 4. This MAb recognized this protein band in all 69 GBS WC preparations tested, and results with representative strains are presented in Fig. 4. MAb 5A12 also revealed the presence of the Sip protein in the GBS culture supernatant after the bacteria were removed by centrifugation. This MAb did not react in immunoblots with S. pyogenes or S. pneumoniae for which a putative sip gene was identified (Fig. 4, lanes 11 and 12). The lack of reactivity of this MAb with WC preparations obtained from these two species may be explained either by the absence of the 5A12-specific epitope on those Sip homologues or by the silence of these putative sip genes in these two species, not being expressed under the growth conditions tested.

View larger version (46K): [in this window] [in a new window]   FIG. 4.   Immunoblots showing the reactivity of the Sip-specific MAb 5A12 with GBS WC preparations obtained from strains C388/90 (Ia/c), ATCC12401 (Ib), NCS 246 (IIR), COH1 (III), NCS 97R331 (IV), NCS 535 (V), NCS 9842 (VI), NCS 7271 (VII), NCS 970886 (VIII), ATCC27956 (bovine isolate), S. pneumoniae SP64, S. pyogenes LSPQ 2698, and E. coli and from 1 µg of purified recombinant Sip (lanes 1 to 14, respectively). Size standards are marked on the left (in kilodaltons).

Immunization with purified recombinant Sip confers protection against GBS infection. Groups of 10 CD-1 mice were immunized three times with either 20 µg of purified recombinant Sip, 15 µg of GBS WC prepared from the strain used for challenge, or adjuvant only. The development of Sip-specific antibodies was evaluated using ELISA with either recombinant Sip protein or GBS WC preparations as the coating antigen. The reciprocal serum titers determined for sera collected after three immunizations with recombinant Sip protein were higher than 640,000 when purified recombinant Sip or thioredoxin-Sip proteins were used as the coating antigen. Analysis of the corresponding sera obtained before immunization clearly indicated that there were no Sip-specific antibodies, since only background ELISA values were recorded at the lowest dilution tested (1:200). Only 3 weeks after the first injection (Fig. 5), antibodies directed against the native Sip protein in formaldehyde-killed GBS WC preparations were detected in the sera of mice immunized with the purified recombinant Sip protein. To verify that the reactivity of the Sip-specific antibodies induced by immunization was not restricted to the homologous strain C388/90 (Ia/c), GBS WC prepared from three serologically distinct strains, ATCC12401 (Ib), NCS 954 (Ib), and NCS 535 (V), were also used as coating antigens for ELISA, and representative results are presented in Fig. 5. The presence of Sip-specific antibodies in the sera of mice immunized with the recombinant protein was confirmed by immunoblots (data not shown). As shown by the increase in ELISA titers, the second immunization clearly boosted the specific humoral response by a factor of 10 or more. A similar increase in ELISA titers was not detected after the third immunization.

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Analysis of mouse antisera collected after immunization with purified recombinant Sip. BALB/c mice were immunized with 20 µg of purified recombinant Sip of GBS strain C388/90 (Ia/c) three time at 3-week intervals. Twenty percent QuilA was added to the purified recombinant Sip as an adjuvant. The sera were collected before each immunization and 2 weeks after the third immunization. The anti-GBS titers were measured by ELISA, using as the solid-phase antigens the formaldehyde-killed WC preparations obtained from strains (A) C388/90 (Ia/c), (B) ATCC12401 (Ib), (C) NCS954 (III), and (D) NCS535 (V). Each spot represents one mouse. These mice were subsequently challenged with the corresponding GBS strains, and the protection data are presented in Fig. 6.

View larger version (28K): [in this window] [in a new window]   FIG. 6.   Immunization of CD-1 mice with purified recombinant Sip protein confers protection against lethal infection with six different GBS strains. The mice were immunized s.c. three times with 20 µg of purified recombinant Sip (S, solid square), 15 µg of formaldehyde-killed GBS WC prepared from the strain used for challenge (WC, open triangle), or adjuvant only (C, open circle). After immunization, the mice were challenged i.p. with an LD90 dose of a GBS strain. Each panel represents the results obtained with one bacterial strain. The identification and serotype of the GBS strains are presented in the upper left corner of each panel. Deaths were recorded daily for up to 14 days. The number of mice surviving the challenge and the calculated P values in each group are also presented. Fisher's exact test was used for calculation of P values. No mice were immunized with formaldehyde-killed GBS WC from strain NCS 246.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In addition to the polysaccharide antigens, protective immunity is also induced by GBS surface proteins such as the  and  proteins (6, 26, 47), Rib (41), -like proteins purified from serotype III (24), and proteins from serotype V (1). Two of these, the  and Rib, are structurally related and are members of a family of streptococcal surface proteins with extremely repetitive structures (35, 44). It was reported that these proteins could be used for vaccine development (28, 29). The  protein was found to be present in approximately 50% of all clinical isolates (10, 20), while the Rib protein was found to be expressed by most serotype III GBS strains and was rarely found in strains of other serotypes (41). However, protection was shown to be restricted to strains that produce the specific protein (28). Both of these proteins were found to possess repetitive structures (35, 44) and to have extensive identity, but they were not found to cross-react immunologically (41, 44). Interestingly, the molecular size of these proteins was shown to vary from strain to strain according to the number of repeats (31, 33). The immunogenic and protective properties of the C protein were shown to be in direct relation to the number of repeats (1, 23).

We report the identification and partial characterization of a new GBS protein, which is distinct from the other known surface GBS proteins. Several studies indicated that the expression of  and  proteins (20, 34, 41), the Rib protein (28, 41), and the newly identified Fbs and Rib-like proteins found in certain serotype V strains (1) was linked to the capsular type expressed by the strains. A characteristic ladder-like pattern after SDS-PAGE was often reported for certain GBS surface proteins, which possess repetitive structures. In contrast to these proteins, a band with an approximate molecular mass of 53 kDa which corresponded to the Sip protein was identified with a Sip-specific MAb in every GBS strain tested, which included representatives of all nine serotypes (Fig. 4). In addition, sequence analysis clearly confirmed that the Sip protein does not have any homology with the  and Rib proteins and did not reveal the presence of anchoring and IgA-binding motifs or repetitive structures which are often present in other GBS surface proteins (19, 35, 44).

Many gram-positive surface-exported proteins are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved anchoring motif (36). In addition, these proteins are also usually synthesized with cleavable N-terminal extensions, termed signal peptides, and are exported outside the bacterial cells by specialized mechanisms (5). Analysis of the predicted amino acid sequence of the Sip polypeptide did not reveal the presence of an anchoring motif at the C-terminal region. However, after culture, the Sip protein was found in the GBS culture supernatant, which suggested that a portion of the Sip protein could be secreted. The C protein was also isolated from GBS culture supernatant (42). The identification of a signal peptide at the N terminus of the Sip protein is an additional indication that this protein is exported outside the cell, where it could be associated with the cell wall of the bacteria. The exact mechanism which mediates this association has yet to be identified. However, preliminary electron microscopy and flow cytometry experiments confirmed that the Sip protein is exposed at the surface of intact GBS cells, where it is accessible to specific antibodies (S. Rioux, D. Martin, H. W. Hackermann, J. Hamel, F. Couture, J. Dumont, P. Desjardins, and B. R. Brodeur, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., 2000, p. 298).

The sip gene was inserted into an expression vector in order to obtain sufficient amounts of purified recombinant Sip protein to conduct immunization and protection experiments. CD-1 mice immunized with the purified recombinant Sip protein developed a strong humoral immune response with antibodies reactive against the recombinant Sip protein as well as the native Sip protein present in GBS WC preparations. Importantly, these Sip-specific antibodies were found by ELISA and immunoblots to cross-react with native Sip proteins produced by representative strains of every GBS serotype. Since human sera were initially used to identify this GBS protein, this indicated that the native Sip protein is also recognized by the human immune system. In an effort to evaluate if there is a correlation between the presence of these Sip-specific antibodies in human sera and colonization, we are presently evaluating the levels of Sip-specific antibodies present in sera collected from GBS-colonized and noncolonized women. The immune response induced after immunization with the recombinant Sip protein efficiently protected CD-1 mice against representative strains of serotypes Ia/c, Ib, II/R, III, V, and VI. This result clearly indicates that the protection induced by immunization is not limited to GBS strains that express a particular capsular serotype. This is not surprising, since analysis of the deduced amino acid sequences obtained from six GBS strains clearly indicated that the Sip protein is highly conserved, with 98% identity. As was recently reported for Neisseria meningitidis (32), S. pneumoniae (8), and Borrelia burgdorferi (11), the protection data presented in this report confirmed that recombinant proteins which are produced in E. coli have enough characteristics in common with the native GBS protein to induce a protective response.

In this report, we have presented results that clearly demonstrate that immunization of mice with purified recombinant Sip protein can induce the development of a cross-reactive immune response that protects against lethal GBS infection. This protein was shown to be present in every GBS strain and is efficiently recognized by specific antibodies. We are presently constructing mutant strains in order to identify and study the function of this protein and hopefully to determine its role in the pathogenesis of streptococcal disease. In addition, we recently reported that passive administration of rabbit anti-Sip serum to pregnant mice or immunization of female mice before pregnancy with purified recombinant Sip conferred protective immunity to their offspring against GBS infection (D. Martin, M. Boyer, J. Hamel, S. Rioux, F. Couture, and B. R. Brodeur, Abstr. 100th Gen. Meet. Am. Soc. Microbiol. 2000, p. 300). These results, which involved the transfer of functional antibodies from pregnant mice to their pups, suggest that Sip-specific antibodies could play an important role in protection against GBS disease.