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Infection and Immunity, February 2001, p. 853-864, Vol. 69, No. 2
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.2.853-864.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Alexander H. Lucas,* Karen D. Moulton, Vanessa R. Tang, and Donald C. Reason

Children's Hospital Oakland Research Institute, Oakland, California 94609

Received 7 June 2000/Returned for modification 18 September 2000/Accepted 8 November 2000

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 VHIII gene segments (HV 3-7, 3-15, 3-23, and 3-11) were identified. VL regions were encoded by several kappa  genes (KV 4-1, 3-15, 2-24, and 2D-29) and a lambda  gene (LV 1-51). Deviation of the VH and VL 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 VH 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.

* Corresponding author. Mailing address: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609. Phone: (510) 450-7635. Fax: (510) 601-3911. E-mail: alucas{at}chori.org.

Infection and Immunity, February 2001, p. 853-864, Vol. 69, No. 2
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.2.853-864.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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