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. 2000 Oct;68(10):5610-8.
doi: 10.1128/IAI.68.10.5610-5618.2000.

Identification of group B streptococcal Sip protein, which elicits cross-protective immunity

B R Brodeur  1 , M Boyer, I Charlebois, J Hamel, F Couture, C R Rioux, D Martin
Affiliations

Identification of group B streptococcal Sip protein, which elicits cross-protective immunity

B R Brodeur et al. Infect Immun. 2000 Oct.

Abstract

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 VSports手机版. 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. .

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Figures

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.
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 in the one-letter code, and identities are marked by a period. A 25-amino-acid-residue leader peptide is underlined.
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 (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.
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 (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).
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 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.
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 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.
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