Immunological Markers of the R4 Protein of Streptococcus agalactiae

Abstract

This study focuses on immunological markers of R4, an important Streptococcus group B (GBS) protein. The results obtained by using rabbit antisera and purified proteins for antigens in enzyme-linked immunosorbent assay-based experiments provided evidence that R4 possesses two antigenic determinants. One of the determinants is shared with the alpha-like protein 3 (Alp3) of GBS, was named R4/Alp3 common, and was expressed by GBS, which possessed the Alp3-encoding gene alp3 or the R4-encoding gene rib. The other antigenic determinant was detected only in rib-positive GBS organisms and was named R4 specific. This determinant probably is an immunological marker unique to the R4 protein. Neither of the antigenic R4 determinants showed serological cross-reactivity with the GBS proteins C␣, C␤, and R3 or with alpha-like protein 2. Of 60 clinical serotype III GBS strains, 56 (93%) isolates possessed the rib gene and 50 (89%) of the rib-positive isolates expressed levels of R4 detectable by antibody-based tests, consistent with R4 expression failure or low-level expression in ϳ10% of rib-positive GBS. alp3 was not detected in type III GBS but was possessed by six of eight type V strains and six of six type VIII strains. All alp3-positive strains were recognized by the R4/Alp3 common antibodies, but none of them were recognized by the R4-specific antibodies. NCTC 9828, a reference strain for R3 and R4, expressed the determinant R4/Alp3 common but not R4 specific. A monoclonal R4 antibody, previously considered to be R4 specific and used in GBS serotyping, targeted R4/Alp3 common and is thus not R4 specific. The results show that failure to discriminate between R4 specific and R4/Alp3 common by antisera designed for GBS serotyping can result in the false identification of Alp3 as R4 or vice versa, whereas anti-R4 antibodies targeting only the determinant R4 specific will detect only R4. Both R4 and Alp3 need further evaluation with respect to the immunobiological function of each distinct antigenic determinant, for instance, with regard to their potential as GBS vaccine components.Streptococcus agalactiae (group B streptococci [GBS]) is an important cause of infections in humans, notably in neonates. Serotyping based on the capsular polysaccharide antigens Ia, Ib, and II through VIII has been used extensively in epidemiological classification of GBS, sometimes supplemented by serosubtyping on the basis of strain-variable and surface-anchored protein antigens. These proteins include the C proteins C␣ (3) (encoded by bca [22]) and C␤ (3) (encoded by bac [10]) and the classical R proteins R1, R3, and R4 (8, 18, 35 P611, 2003). These proteins, except for C␤, belong to a protein family characterized among other things by similarity in primary structure, with up to 100% homology for some of the protein stretches (16,34), and by their generation of ladder-like patterns on Western blots, probably due to large and identical repeat units which vary in number from strain to strain (9,22,34). Horizontal transfer of genetic elements be...

Putative Novel Surface-Exposed Streptococcus agalactiae Protein Frequently Expressed by the Group B Streptococcus from Zimbabwe

“…Our impression is that R3 and Z resemble each other in their physicochemical characterisitcs and, possibly, may belong to a separate GBS protein family. However, the lack of immunological cross-reactivity between them, like the cross-reactivity which exists between different alpha-like proteins (17,18), indicates considerable structural dissimilarity. Sequencing of the encoding genes is required to establish if there is a structural relationship between Z and R3.…”

“…The reference and prototype strains used in this study have been described previously (12,18) and included strains of all CPS serotypes except serotype IX (27) and isolates which expressed one or more of the following GBS proteins: Cß, C␣, Alp1, Alp2, Alp3, Alp4, R4 (Rib), and R3. Type III/R4, sequence type 17, strains were from the collection of clinical isolates of the Laboratory of Medical Microbiology, St. Olav's Hospital, Trondheim, Norway.…”

“…The Alps are characterized, among other ways, by a repeat-containing region which has a causal relation to the ladder-like patterns formed by these proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting. The Alps possess chimeric sequences, show variable immunological crossreactivities, and induce increased resistance to GBS infections in experimental models (1,13,15,18,30). For this reason, these proteins can be considered vaccine candidates, either alone or as carrier proteins coupled to CPS (33, 34).…”

Novel Aspects of the Z and R3 Antigens of Streptococcus agalactiae Revealed by Immunological Testing

“…The original anti-R3 PAb used in the present study contained anti-Z2 antibodies due to the fact that for its preparation the antiserum had been crossabsorbed by the Z1-expressing strain CMFR30 but not by a Z2-expressing strain (11). To exclude the possibility that a putative R3-specific PAb should contain Z2 antibodies, another anti-R3 PAb was prepared, namely, by cross-absorption of anti-9828 (NT/ Alp4, R3) whole-cell serum, with absorption first by the clinical isolate 08-60 (II/R4) to remove anti-CPS type II antibodies if present (18,19) and then by strain 65604 (III/R4 [rib]) to secure removal of anti-Alp4 antibodies which cross-react with R4 (20). We know from an ongoing study that the resulting 9828 R3 PAb also contained antibodies specific for Alp4 (A. I. Kvam, personal communication).…”

“…However, the immunological uniqueness of the proteins Z1, Z2, and R3, indicated by the data of this and earlier studies (9,11), suggests that it may become possible to develop molecular typing methods for specific detection of each of the protein-encoding genes, perhaps by including primer sets for the genes in a multiplex PCR. This prediction is based on data which indicate that at least some strain-variable GBS genes which can be identified by gene-specific PCR often encode proteins which possess protein-specific epitopes (20,21,28,29). Knowledge of the usefulness of Z1, Z2, and R3 determination in GBS serotyping is sparse and unknown for GBS from most areas of the world (9)(10)(11)(12)30).…”