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Infect Immun. Oct 2000; 68(10): 5610–5618.
PMCID: PMC101513
Identification of Group B Streptococcal Sip Protein, Which Elicits Cross-Protective Immunity
Bernard R. Brodeur,* Martine Boyer, Isabelle Charlebois, Josée Hamel, France Couture, Clément R. Rioux, and Denis Martin
Unité de Recherche en Vaccinologie, Centre Hospitalier Universitaire de Québec, et Université Laval, Ste-Foy, Canada G1V 4G2
Editor: E. I. Tuomanen
*Corresponding author. Mailing address: Unité de Recherche en Vaccinologie, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, Édifice T-367, 2705 Boul. Laurier, Ste-Foy, Québec, Canada G1V 4G2. Phone: (418) 656-4141-6266. Fax: (418) 654-2280. E-mail: Bernard.Brodeur/at/crchul.ulaval.ca.
Present address: Intellivax International Inc., Ville St-Laurent, Quebec, Canada H4S 2A1.
Received May 19, 2000; Revisions requested June 29, 2000; Accepted July 17, 2000.
A protein of group B streptococci (GBS), named Sip for surface immunogenic protein, which is distinct from previously described surface proteins, was identified after immunological screening of a genomic library. Immunoblots using a Sip-specific monoclonal antibody indicated that a protein band with an approximate molecular mass of 53 kDa which did not vary in size was present in every GBS strain tested. Representatives of all nine GBS serotypes were included in the panel of strains. Cloning and sequencing of the sip gene revealed an open reading frame of 1,305 nucleotides coding for a polypeptide of 434 amino acid residues, with a calculated pI of 6.84 and molecular mass of 45.5 kDa. Comparison of the nucleotide sequences from six different strains confirmed with 98% identity that the sip gene is highly conserved among GBS isolates. N-terminal amino acid sequencing also indicated the presence of a 25-amino-acid signal peptide which is cleaved in the mature protein. More importantly, immunization with the recombinant Sip protein efficiently protected CD-1 mice against deadly challenges with six GBS strains of serotypes Ia/c, Ib, II/R, III, V, and VI. The data presented in this study suggest that this highly conserved protein induces cross-protective immunity against GBS infections and emphasize its potential as a universal vaccine candidate.
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 α and β 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.
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).
The GBS strains were grown overnight on tryptic soy agar plates containing 5% (vol/vol) sheep blood (Quelab Laboratories, Montreal, Canada) or in Todd-Hewitt broth (THB: Difco Laboratories, Detroit, Mich.) at 37°C in the presence of 8% CO2. Strains were stored at −70°C in brain heart infusion broth (Difco) containing 20% (vol/vol) glycerol (Sigma Chemical Co., St. Louis, Mo.). Escherichia coli XL1-Blue MRF′ [Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac (F′ proAB lacIqZΔM15 Tn10 [Tetr])], E. coli B strain BLR [F ompT hsdSB (rBmB) gal dcm Δ(srl-recA)306::Tn10 (Tetr)], and E. coli AD494(DE3) [ara leu-7697 lacX74 phoAPvuII phoR malF3 F′ (lac+ [lacIq) pro) trxB::kan(DE3)] (Novagen, Madison, Wis.) were used as recipients for cloning or production of recombinant proteins and were grown on Lennox Luria-Bertani (LB) agar or broth (Gibco-BRL, Gaithersburg, Md.) containing 40 μg of kanamycin (Sigma) per ml at 37°C for 18 h.
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 (λ = 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 λZAPII 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 λZAPII 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-β-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.
The sip genes from strain C388/90 (Ia/c) and five other GBS strains were amplified from purified chromosomal DNA by PCR with recombinant Taq DNA polymerase (Amersham Pharmacia Biotech) as described by the manufacturer. Primers OCRR-259 (5′-CCGGCAGATCTATGAAAATGAATAAAAAGGTACTATTG-3′) and OCRR-260 (5′-GGCCGTCTAGATTATTTGTTAAATGATACGTGAACA-3′), which contained BglII and XbaI restriction sites, respectively, were used to perform the amplifications. PCR was performed with 10 cycles of 30 s at 94°C, 20 s at 56°C, and 30 s at 72°C, followed by 25 cycles of 30 s at 94°C, 20 s at 70°C, and 30 s at 72°C, and a final elongation period of 5 min at 72°C. The products obtained after amplification were sequenced and, for the sip gene from strain C388/90 (Ia/c), also ligated into a modified p629 plasmid (13), named pURV22, in which the ampicillin resistance gene was replaced by a kanamycin resistance cassette obtained from plasmid pUC4K (Amersham Pharmacia Biotech). Recombinant plasmid was transformed into E. coli XLI-Blue MRF′ as described by Hanahan (16). The recombinant plasmid containing the sip gene was named pURV32. The sequence of the insert was also verified. To obtain a thioredoxin-Sip fusion protein with an N-terminal polyhistidine tag, the sip gene was also amplified by PCR using primers OCRR-304 (5′-GCCCAGATCTGATGAAAATGAATAAAAAGGTACTATTG-3′) and OCRR-305 (5′-CGGGAAGCTTATTATTTGTTAAATGATACGTGAACA-3′), which contained BglII and HindIII restriction sites, respectively. The amplification product was ligated into plasmid pET32 (Novagen Inc., Madison, Wis.), and after sequencing, the recombinant plasmid named pURV32.2 was transformed into E. coli XL1-Blue MRF′ and AD494(DE3) strains.
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.
Identification and sequencing of the sip gene.
A GBS chromosomal library was constructed from the GBS serotype Ia/c strain C388/90 in λZAPII. 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. Fig.1.1. 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).
FIG. 1
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.
It was determined that the 1,305 bp of ORF2 encode a 434-amino-acid-residue polypeptide with a predicted pI of 6.84 and a predicted molecular mass of 45.5 kDa. NH2-terminal amino acid sequencing of purified recombinant Sip indicated the existence of a 25-amino-acid signal peptide which is cleaved in the mature protein (Fig. (Fig.2).2). Analysis of ORF2 did not revealed the presence of repetitive structures, a cell wall-anchoring motif (LPXTG), or an IgA-binding motif (MLKKIE). Comparison of the nucleotide sequence with the sequences compiled in the available databases indicated that beside GBS, coding regions with homology to the sip gene were also present in two closely related streptococcal species. The first coding region with homology (62% identity) with the sip gene was identified in the genome of Streptococcus pneumoniae (The Institute for Genomic Research). An ORF which encodes a putative 42-kDa protein (GenBank accession number U09352) of Streptococcus pyogenes also showed homology (62% identity) with the GBS sip gene. This ORF is located upstream of an S. pyogenes 67-kDa myosin-cross-reactive antigen (22). However, since no known function was associated with these two ORFs, it is not possible at the moment to assign a potential role to the GBS Sip protein based on sequence homology.
FIG. 2
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 (more ...)
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. (Fig.22).
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. (Fig.3,3, lanes 1 and 2). This purified thioredoxin-Sip fusion recombinant protein was recognized by the pooled human sera (Fig. (Fig.3B)3B) used for the initial screening of the genomic library and by the Sip-specific MAb 5A12 (Fig. (Fig.3C).3C).
FIG. 3
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 (more ...)
The recombinant plasmid pURV32 was obtained following insertion of the sip gene into the pURV22 heat-inducible vector. The recombinant plasmid pURV32 was transformed into E. coli strain BLR. Immunoblot analysis of the resulting clone indicated that this recombinant E. coli BLR strain produced, upon heat induction, a protein with an approximate molecular mass of 53 kDa which reacted with the pool of normal human sera (Fig. (Fig.3B,3B, lanes 4 to 6). The recombinant Sip protein was identified in the cytoplasm of the E. coli cells but was also found in the culture supernatant. Therefore, the culture supernatant was the material used to purify the recombinant Sip. After ammonium sulfate precipitation followed by hydrophobic interaction and anion-exchange chromatographic steps, the purity of the recombinant Sip protein was estimated by SDS-PAGE to be >90%. This material was used to immunize the mice that were subsequently challenged with GBS strains and was also used as the coating antigen for the ELISA.
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. (Fig.3C).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. Fig.4.4. This MAb recognized this protein band in all 69 GBS WC preparations tested, and results with representative strains are presented in Fig. Fig.4.4. 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. (Fig.4,4, 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.
FIG. 4
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 (more ...)
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. (Fig.5),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. Fig.5.5. 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.
FIG. 5
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 (more ...)
Three weeks after the third immunization, groups of 10 mice were challenged i.p. with different GBS strains, and the protection results are presented in Fig. Fig.6.6. Eight of 10 mice immunized with purified recombinant Sip were protected against challenge with the homologous strain C388/90 (Ia/c), which is comparable to the protection results obtained in the group of mice immunized with WC prepared from the same strain. Most of the control mice died within 24 h following bacterial challenge. The observed protection was shown using Fisher's exact test to be significantly different (P = 0.0007) than the control mice, which only received PBS with QuilA adjuvant. Mice immunized three times with 20 μg of purified recombinant meningococcal protein or 20 μg of heat-killed E. coli WC preparations did not survived the GBS challenge with strain C388/90 (data not shown). Sip-immunized mice were also protected when they were challenged with GBS strains ATCC12401 (Ib), NCS 246 (II), NCS 954 (III), NCS 535 (V), and NCS 9842 (VI). For the groups challenged with NCS 954 (III), NCS 535 (V), and NCS 9842 (VI), the number of Sip-immunized mice that survived the heterologous infection was higher than the number of survivors in the groups of mice immunized with the GBS formaldehyde-killed WC prepared from the same strain that was used for challenge. When pooled together, the protection data indicate that 91% (55 of 60) of the mice immunized with purified recombinant Sip survived the lethal challenge, compared to only 20% (12 of 60) of mice which received the adjuvant, for a global protective efficacy of 89%. Immunoblots confirmed that the antibodies induced after immunization recognized a protein band corresponding to the Sip protein (data not shown).
FIG. 6
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 (more ...)
In addition to the polysaccharide antigens, protective immunity is also induced by GBS surface proteins such as the α and β proteins (6, 26, 47), Rib (41), α-like proteins purified from serotype III (24), and proteins from serotype V (1). Two of these, the α 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 α 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 α and β 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. (Fig.4).4). In addition, sequence analysis clearly confirmed that the Sip protein does not have any homology with the α 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.
ACKNOWLEDGMENTS
We gratefully acknowledge the contribution of Stéphane Tremblay in the development of the purification process for the Sip protein.
This research was financially supported by a grant from BioChem Pharma Inc.
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