Epitope mapping of pneumococcal surface protein A of strain Rx1 using monoclonal antibodies and molecular structure modelling

Kolberg, Jan; Aase, Audun; Rødal, Gunnhild; Littlejohn, James; Jedrzejas, Mark J.

doi:10.1016/s0928-8244(03)00255-4

Cited by 17 publications

(19 citation statements)

References 40 publications

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“…The B cell epitope recognized by 18 of the 36 (50%) anti-PspA 3-286 mAbs localized to the region corresponding to aa 3-192 of PspA. The B cell epitopes recognized by the remaining 18 mAbs are spread across the surface-exposed portion of PspA (aa residues 1-288), which is consistent with previous studies (21,23,37). Anti-PspA mAbs that recognized PspA did not recognize its subfragments PspA and PspA 98 -192 (Table VI, groups 7 and 8).…”

Section: Pspa and B Cell Epitopessupporting

confidence: 90%

“…One region comprised the first 115 amino acids of PspA, and the other was from aa 192-260 (21). In another study, Kolberg et al located the B cell epitopes to two regions (aa 1-67 and aa 67-236) that essentially cover the extracellular domain (23). Recently, Miyaji and colleagues demonstrated that aa 32-212 (region A) and aa 213-312 (region B) of PspA are equally immunogenic and that immunization of mice with these subfragments induced high Ab titers.…”

Section: Pspa and B Cell Epitopesmentioning

confidence: 99%

“…Families 1 and 2 account for the majority (94 -99%) of the pneumococcal isolates (18 -20). Anti-PspA mAbs have been shown to be protective, and the protective epitopes have been mapped to the ␣-helical domain of PspA (21)(22)(23). However, the Ab response has not been analyzed at the molecular level.…”

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confidence: 99%

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Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse DH-Less Heavy Chain Gene Usage and Avidity Maturation

Rohatgi

Dutta²,

Tahir³

et al. 2009

The Journal of Immunology

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Immunization of human volunteers with a single dose of pneumococcal surface protein A (PspA) stimulates broad cross-reactive Abs to heterologous PspA molecules that, when transferred, protect mice from fatal infection with Streptococcus pneumoniae. In this study, we report the molecular characterization of 36 mouse mAbs generated against the extracellular domain of PspA (PspA 3-286 ) from strain R36A. Abs to PspA 3-286 were encoded by diverse V H and V families/genes. The H chain CDR3 and L chain CDR3 lengths were 3-13 (7.8 ؎ 0.5) and 8 -9 (8.7 ؎ 0.2) codons, respectively. Unexpectedly, seven hybridomas expressed H chains that lack D H gene-derived amino acids. Nontemplate-encoded addition(s) were observed in the H chain expressed in six of these seven hybridomas; Palindromic addition(s) were absent. Absence of D H gene-derived amino acids did not prevent antiPspA 3-286 mAbs from attaining average relative avidity. Avidity maturation occurred during primary IgG anti-PspA 3-286 polyclonal Ab response in PspA 3-286 -and R36A-immunized mice. Compared with PspA 3-286 -immunized mice, the relative avidity of the primary polyclonal IgG Abs was higher in R36A immunized mice on days 72, 86, and 100. Two pairs of clonally related hybridomas were observed. D H genes expressed in the majority (75.9%) of the hybridomas used reading frame 3. Analysis of replacement/silent mutation ratio in the CDR and framework regions provided evidence for Ag-driven selection in 11 mAbs. Based on epitope localization experiments, the mAbs were classified into 12 independent groups. ELISA additivity assay indicated that members within a group recognized topographically related epitopes. This study provides molecular insights into the biology of D H -less Abs.

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Section: Pspa and B Cell Epitopessupporting

confidence: 90%

Section: Pspa and B Cell Epitopesmentioning

confidence: 99%

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Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse DH-Less Heavy Chain Gene Usage and Avidity Maturation

Rohatgi

Dutta²,

Tahir³

et al. 2009

The Journal of Immunology

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“…The extent of protection against systemic pneumococcal infection has been shown to be influenced by target antigen accessibility to circulating host antibodies (Gor et al, 2005). Flow cytometry is a technique that permits epitope analyses on a large number of live bacteria Kolberg et al, 2000Kolberg et al, , 2001Kolberg et al, , 2003. In the present study we have used both polyclonal and mAbs recognizing the pneumococcal enolase protein and other surface determinants.…”

mentioning

confidence: 99%

“…Other mAbs used were also produced at NIPH, Oslo, Norway. mAb 145,F-2 (IgM) reacts with the phosphorylcholine determinant (Kolberg et al, 1997a), mAb 149,B-3 (IgG2a) and 159,D-7 (IgG2a) detect epitopes on PspA (Kolberg et al, , 2003 and 230,B-9 (IgG1) reacts with heat-shock protein 70 from S. pneumoniae and other streptococci (Kolberg et al, 2000).Rabbit antisera. Rabbit sera were obtained after immunization with recombinant enolase protein, purified PspA or purified PspC.…”

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confidence: 99%

Streptococcus pneumoniae enolase is important for plasminogen binding despite low abundance of enolase protein on the bacterial cell surface

et al. 2006

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Enolase represents one of the anchorless surface proteins of Streptococcus pneumoniae and has previously been identified as a plasminogen-binding protein, endowing this pathogen with host proteolytic activity. In this study the mAb 245,C-6 (IgG1) was produced in a BALB/c mouse after immunizing with a protein fraction from S. pneumoniae. The mAb reacted with recombinant pneumococcal enolase both under non-denaturing and denaturing conditions. The epitope for the mAb was mapped to residues 55 DKSRYGGLG 63 of pneumococcal enolase using a peptide array. By applying the previously reported structure of enolase, this epitope was localized in a surface-exposed loop in each of the monomers of the octameric enolase. Previous immunoelectron microscopic studies, using polyclonal rabbit antibodies against enolase, depicted enolase on the cell surface but did not quantify the amount of surface-exposed enolase on viable pneumococci. Here, flow cytometry revealed no binding of mAb 245,C-6 to viable pneumococci, including TIGR4 and its non-encapsulated isogenic mutant, and only a minor increase of fluorescence intensity was measured when the polyclonal anti-enolase antibodies were used. In contrast, control antibodies recognizing the choline-binding proteins (CBPs) PspA and PspC showed high reactivities. The non-encapsulated TIGR4 did not show increased levels of antibody binding for mAb 245,C-6 or polyclonal anti-enolase antibodies, but revealed increased binding of polyclonal antibodies reacting with PspA or PspC. These results suggest that, compared to other surface-exposed proteins such as CBPs, the amount of enolase under the selected conditions is low. Flow cytometry, however, with FITC-labelled plasminogen demonstrated that the amount of surface-exposed enolase is important for plasminogen binding and, therefore, is also important for pneumococcal pathogenesis. INTRODUCTIONStreptococcus pneumoniae is an important human pathogen causing a variety of diseases including pneumonia, meningitis, sepsis and otitis media. A prerequisite for invasive pneumococcal diseases (infections) is the ability of the bacteria to colonize the mucosal surface and penetrate the mucosal barriers. During these processes binding of bacterial factors such as adhesins are essential for the interactions with receptors on host cells. Among the bacterial factors that mediate direct adherence to human cells are phosphorylcholine (Cundell et al., 1995) and the choline-binding protein PspC, also known as SpsA and CbpA (Brooks-Walter et al., 1999;Hammerschmidt et al., 1997;Rosenow et al., 1997). The PspC protein binds to the secretory component of the polymeric Ig receptor or secretory IgA and this interaction mediates transmigration of pneumococci through mucosal epithelial cells (Elm et al., 2004;Zhang et al., 2000). In addition, PspC and the PspClike protein Hic have been shown to interact with complement factor H (Dave et al., 2004;Janulczyk et al., 2000). Many pathogenic bacteria also produce receptors for Abbreviations: GAPDH, glyceraldehyde-3-phos...

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Genome‐based identification and characterization of a putative mucin‐binding protein from the surface of Streptococcus pneumoniae

Bumbaca¹,

Littlejohn²,

Nayakanti³

et al. 2006

Proteins

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Streptococcus pneumoniae open reading frame SP1492 encodes a surface protein that contains a novel conserved domain similar to the repeated fragments of mucin-binding proteins from lactobacilli and lactococci. To investigate the functional role(s) of this protein and its potential adhesive properties, the surface-exposed region of SP1492 was expressed in Escherichia coli, purified to homogeneity, and partially characterized by biophysical and immunological methods. Circular dichroism and sedimentation measurements confirmed that SP1492 is an all-beta protein that exists in solution as a monomer. The SP1492 protein has been shown to be expressed by S. pneumoniae and was experimentally localized to its surface. The protein functional domain binds to mucins II and III from porcine stomach and to purified submaxillary bovine gland mucin. It appears to be one of the very few unambiguous pneumococcal adhesin molecules known to date. A hypothetical model constructed by ab initio techniques predicts a novel beta-sandwich protein structure.

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Epitope mapping of pneumococcal surface protein A of strain Rx1 using monoclonal antibodies and molecular structure modelling

Cited by 17 publications

References 40 publications

Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse DH-Less Heavy Chain Gene Usage and Avidity Maturation

Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse DH-Less Heavy Chain Gene Usage and Avidity Maturation

Streptococcus pneumoniae enolase is important for plasminogen binding despite low abundance of enolase protein on the bacterial cell surface

Genome‐based identification and characterization of a putative mucin‐binding protein from the surface of Streptococcus pneumoniae

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