Combinatorial Library Cloning of Human Antibodies toStreptococcus pneumoniaeCapsular Polysaccharides: Variable Region Primary Structures and Evidence for Somatic Mutation of Fab Fragments Specific for Capsular Serotypes 6B, 14, and 23F

Abstract

Antibodies specific for capsular polysaccharides play a central role in immunity to encapsulated Streptococcus pneumoniae, but little is known about their genetics or the variable (V) region polymorphisms that affect their protective function. To begin to address these issues, we used combinatorial library cloning to isolate pneumococcal polysaccharide (PPS)-specific Fab fragments from two vaccinated adults. We determined complete V region primary structures and performed antigen binding analyses of seven Fab fragments specific for PPS serotype 6B, 14, or 23F. Fabs were of the immunoglobulin G2 or A isotype. Several V H III gene segments (HV 3-7, 3-15, 3-23, and 3-11) were identified. V L regions were encoded by several genes (KV 4-1, 3-15, 2-24, and 2D-29) and a gene (LV 1-51). Deviation of the V H and V L regions from their assigned germ line counterparts indicated that they were somatically mutated. Fabs of the same serotype specificity isolated from a single individual differed in affinity, and these differences could be accounted for either by the extent of mutation among clonal relatives or by usage of different V-region genes. Thus, functionally disparate anti-PPS antibodies can arise within individuals both by activation of independent clones and by intraclonal somatic mutation. For one pair of clonally related Fabs, the more extensively mutated V H was associated with lower affinity for PPS 14, a result suggesting that somatic mutation could lead to diminished protective efficacy. These findings indicate that the PPS repertoire in the adult derives from memory B-cell populations that have class switched and undergone extensive hypermutation.

Recurrent Variable Region Gene Usage and Somatic Mutation in the Human Antibody Response to the Capsular Polysaccharide ofStreptococcus pneumoniaeType 23F

“…cDNA was used as a template in the PCR to generate H-chain Fd fragments and total kappa L chains for insertion into the expression vector pComb 3H (3). The primer sets used to generate the H and L fragments were the same as those listed elsewhere (17), with the exclusion of the lambda L-chain primers and the IgM downstream primers. L-chain fragments were inserted into the SacI/XbaI site of the pComb 3H vector, and the resulting L-chain library was electroporated into XL1-Blue E. coli cells.…”

“…Representatives of each of the two dominant L-chain families were chosen for large-scale purification and binding analysis. The H-chain C H 1 domain of each of these Fabs were engineered to contain a C-terminal polyhistidine region as previously described (17). These were grown in 1-liter cultures, induced with IPTG (isopropyl-␤-D-thiogalactopyranoside), and periplasmic proteins extracted with B-Per (Pierce Chemical Co., St. Louis, Mo.).…”

“…The enrichment of PPS-specific B cells has been previously described in detail (17). Briefly, MNC were isolated from the 7 day postvaccination blood sample by using Ficoll-Hypaque.…”

“…We have previously reported two additional PPS 23F-specific Fabs, one utilizing V A23/ VH3-23 gene products, and one that utilizes V A2 paired with VH3-11 (17). V A23 Fab family.…”

Vaccination against encapsulated bacteria in humans: paradoxes

“…To account for the presence in humans of [11 12] mutated antibodies during a T-independent immune response that cannot normally trigger a cognate T-B dependent germinal center reaction, the classical explanation put forward by authors is that such responses are in fact taken care by memory B cells that bona fide have been primed by a previous encounter with the pathogen, either during an infection or by silent carriage . These memory B [13][14][15] cells would then eventually reside in the splenic marginal zone where they could acquire marginal zone B cell surface markers .[ [16][17][18] This explanation requires that during this natural priming which must have occurred in each case analyzed so far, the polysacharidic capsules from the pathogen are somehow linked to a protein moiety in order to drive the response into the classical germinal center-dependent memory B cell pathway. …”