Somatic Hypermutation and Diverse Immunoglobulin Gene Usage in the Human Antibody Response to the Capsular Polysaccharide of Streptococcus pneumoniaeType 6B

Abstract

Combinatorial cloning and expression library analysis were used to determine the expressed human antibody repertoire specific for the capsular polysaccharide (PS) of Streptococcus pneumoniae serotype 6B. Sequence analysis of 55 6B-specific antibody Fab fragments isolated from six vaccinated donors reveal that different individuals used a variety of heavy and light chain germ line variable (V) region genes to form pneumococcal capsular PS (PPS) 6B-specific paratopes. Within each donor, however, the response was more restricted, with five of the six donors using at most one or two gene pairs to form combining sites. Analysis also indicated that although the response in each donor was oligoclonal in terms of variable gene usage, the combination of extensive somatic hypermutation, deletion of germ line-encoded residues, insertion of non-germ line-encoded residues, and intraclonal isotype switching generated a surprising degree of paratope diversity within the individuals analyzed. In contrast to previously studied PS-specific responses, we find that the PPS 6B repertoire makes use of a diverse collection of heavy-chain and light-chain V region gene products to form specific paratopes, with no apparent tendency for conservation of immunoglobulin gene usage between individuals.Streptococcus pneumoniae is a significant human pathogen causing pneumonia, bacteremia, meningitis, and otitis media. The primary determinants of virulence for the many strains of S. pneumoniae are the pneumococcal capsular polysaccharides (PPS). The PPS are heterogeneous in structure, and at least 90 different serotypes occur within the species (10). PPS epitopes are immunogenic in adults, and immunization with the polysaccharides (PS) provides serotype-specific protection against infection (26). PPS-protein conjugates are immunogenic in infants and provide protection for this age group as well (31). Both plain PS and PS-conjugate vaccines are available and are currently recommended for the appropriate age groups.In addition to providing protection against disease, these vaccines offer an opportunity to explore several aspects of basic immunobiology in humans. The carbohydrate epitopes are structurally defined, the vaccines are routinely and safely administered to adults and children, and specific B cells circulate in the periphery following vaccination, thereby facilitating minimally invasive access to the cellular components of interest. Although the serology of the response to various PPS antigens has been studied in detail (13,19,20,24,27,30), the difficulty in constructing stable human heterohybridomas has limited the degree to which the PPS-specific antibody response could be studied at the level of immunoglobulin (Ig) gene usage.In this report we use repertoire cloning to examine the paratopic repertoire of human antibodies specific for the capsular PS of S. pneumoniae serotype 6B. Heavy (H)-and light (L)-chain variable (V) (V H and V L , respectively) region sequences are reported for 55 PPS 6B-specific Fab fragments isolated from six i...

Immune Response to Pneumococcal Polysaccharides 4 and 14 in Elderly and Young Adults: Analysis of the Variable Light Chain Repertoire

“…The repertoire, however, was dominated by six gene families and restricted within individuals, as previously described for the V L repertoire in the response to other pneumococcal polysaccharides (12,28). Several individuals utilized the same gene family and locus in response to both PPS4 and PPS14 although paired with different V H gene segments, illustrating the promiscuity of these V L gene families.…”

“…To date, studies examining the structure-function relationship of antipneumococcal antibodies have been performed using human monoclonal antibodies (1, 22, 26), combinatorial libraries (12,17,27,28), and monoclonal antibodies derived from a transgenic mouse strain with human immunoglobulin loci (2, 19). All studies, with the exception of those using the transgenic mouse, have analyzed the V repertoire using peripheral blood mononuclear cells obtained from young high responders.…”

Discovery of Immunodominant B Cell Epitopes within Surface Pneumococcal Virulence Proteins in Pediatric Patients with Invasive Pneumococcal Disease

“…Nevertheless, the identified epitopes were found to be disease-specific, most likely because they are exposed and recognized at the time of epithelial adhesion, bloodstream invasion, and massive inflammation, which is observed during IPD. Alternatively, the identified epitopes may represent targets of high-avidity antibodies that are generated through somatic hypermutation and/or gene conversion of the antibody-variable region following their affinity maturation in IPD (29). Notably, all IPD sera were found to be immunoreactive against at least one epitope, suggesting that a novel synthetic analogue combining all three peptides could broaden its immunogenic potential, as described previously (30 -32).…”

Domain specificity of the human antibody response to Bacillus anthracis protective antigen

“…Fab expression libraries were constructed from mononuclear cells (MNCs) enriched for PAspecific B cells in a manner similar to that previously described for PA and polysaccharidespecific antibody expression libraries [8,[12][13][14][15]. PA 83 , PA 20 , and PA 63 were purchased from List Biological Laboratories, Campbell, CA.…”