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Infection and Immunity, June 2004, p. 3505-3514, Vol. 72, No. 6
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.6.3505-3514.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Jianhui Zhou,1 Kathleen R. Lottenbach,2 Stephen J. Barenkamp,3 and Donald C. Reason1*

Children's Hospital Oakland Research Institute, Oakland, California 94609,1 Department of Internal Medicine,2 Department of Pediatrics, St. Louis University School of Medicine, St. Louis, Missouri 631103

Received 30 December 2003/ Returned for modification 9 February 2004/ Accepted 2 March 2004


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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.


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INTRODUCTION
 
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) (VH and VL, respectively) region sequences are reported for 55 PPS 6B-specific Fab fragments isolated from six individuals. Sequence analysis indicates a response that has undergone extensive somatic modification in terms of hypermutation, residue insertion and deletion, and class switch. In contrast to previously studied PS-specific responses (25, 33), we find that the PPS 6B repertoire makes use of a diverse collection of VH and VL gene products to form specific paratopes, with no apparent tendency for conservation of Ig gene usage between individuals.


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MATERIALS AND METHODS
 
Subjects. Adult volunteers were randomly assigned to receive either the licensed 23-valent PS vaccine (Pnu-Immune, Wyeth-Lederle) or a 9-valent PS-protein conjugate vaccine consisting of PPS from serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F conjugated to the mutant diphtheria toxin CRM197 (Wyeth-Lederle). Blood was collected prior to vaccination and 30 days following vaccination to determine serum antibody response. In addition, a 100-ml blood sample was collected 7 days following vaccination for the isolation of mononuclear cells (MNCs). Human subject protocols were reviewed and approved by the Institutional Review Boards at both Children's Hospital Oakland and St. Louis University School of Medicine.

Affinity selection of cells. The enrichment of PPS-specific B cells has been previously described in detail (21, 33). Briefly, MNCs were isolated from the 7-day postvaccination blood sample by using Ficoll-Hypaque. An aliquot (106 cells) was placed into culture for 7 days in 1 ml of RPMI 1640 medium supplemented with 5% fetal calf serum, the supernatant was assayed for PPS 6B-specific antibody production, and the H-chain and L-chain isotypes of secreted antibody were determined. PPS 6B was biotinylated as previously described and used to arm avidin-coated paramagnetic beads (Immunotech Inc., Marseilles, France). These PPS 6B-coated beads were washed and added to 2 x 107 MNCs (preabsorbed with avidin-coated magnetic beads), and the mixture was incubated on ice for 30 min. C-PS (10 µg/ml) was included in the incubation buffer to inhibit the binding of C-PS-specific cells. PPS 6B-binding cells were then isolated with a magnet. Positively selected cells were washed twice with cold phosphate-buffered saline-0.5% bovine serum albumin and used for RNA extraction.

Construction of Fab expression libraries. The procedures for the construction of Fab libraries have been previously described in detail (21, 33). Briefly, total RNA was prepared from affinity-isolated cells (RNEasy; QIAGEN, Valencia, Calif.), and cDNA was prepared by using the Thermoscript reverse transcription-PCR System (GIBCO BRL, Carlsbad, Calif.) according to the manufacturer's instructions. cDNA was used as a template in the PCR to generate H-chain Fd fragments (VDJ-CH1) and total {kappa} and {lambda} L chains for insertion into the expression vector. Two expression vector systems were used in this study. For Fab fragments constructed in vector pComb 3H (2) (donors 001 and 010; {kappa} only), the primer sets used to generate the H-chain and L-chain fragments were the same as listed by Lucas et al. (16), with the exclusion of 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 Escherichia coli cells. An aliquot was plated to determine the transformation efficiency, and the balance was expanded for 8 h. Plasmid DNA was purified from the expanded culture, digested with XhoI and SpeI, and purified, and the H-chain Fd fragments were ligated into the XhoI/SpeI site of L-chain library plasmid DNA. The Fd fragment x L-chain (Fd x L) library was electroporated into XL1-Blue cells, plated at low density on Luria-Bertani-carbenicillin plates and grown overnight, and individual colonies were selected for analysis. Expression libraries for all other donors were constructed by using the pARC vector system developed in our laboratory during the course of this study. The details of this vector system are the subject of a separate report (unpublished data). The pARC vector system differs from the pComb 3H vector in the use of PCR primers incorporating eight-base restriction sites (FseI and NotI for the L chain; PacI and AscI for the H chain), redesigned upstream primers for the VH and VL regions, and the ability to express Fab fragments either as conventional two-chain disulfide-linked molecules or as single-chain Fab fragments where the H and L chains are joined by a flexible linker. The sequence of events involved in creating Fab expression libraries by using the pARC vectors was the same as described above for the pComb 3H vector except for the substitution of different restriction enzymes.

Identification of PPS 6B-specific Fab fragments. Individual transformed E. coli colonies were selected, mastered onto a Luria-Bertani-carbenicillin agar plate, and grown in 1-ml overnight cultures in deep-well 96-well plates under antibiotic selection. Bacteria were pelleted by centrifugation, resuspended in 140 µl of lysis buffer (phosphate-buffered saline plus protease inhibitor cocktail [Complete; Roche Molecular Biochemicals, Indianapolis, Ind.]), and rapidly frozen and thawed three times by using liquid nitrogen; the cellular debris was then pelleted by centrifugation. Fifty microliters of the lysate was added to assay plates that had been coated overnight with human L-chain-specific antibody (Biosource International, Camarillo, Calif.) and incubated for 2 h at 37°C to facilitate capture of the Fab fragments. Plates were then washed, and 50 µl of radiolabeled PPS 6B was added to each well. Following incubation at 37°C for 2 h, plates were washed and placed on a PhosphorImager detection plate (Molecular Dynamics, Sunnyvale, Calif.), and the plate was exposed for various lengths of time. Following exposure, the PhosphorImager plates were scanned, and the PPS-binding wells were identified. Residual lysate from corresponding clones was reassayed for binding by using a radio-antigen binding assay (RABA), described below. Positive cultures were identified on the master plates and streaked for isolation; individual colonies were picked and grown overnight, and Fab production was verified.

Sequencing and sequence analysis. Plasmids containing H- and L-chain genes were submitted to Davis Sequencing (Davis, Calif.), for VH and VL sequence determination. L-chain sequencing primer LSEQ (5'-GCTTCCGGCTCGTATGTTGTGTGG-3') and H-chain sequencing primer HSEQ (5'-GCAGCCGCTGGATTGTTATTACTC-3') both bind to the vector. Initial sequence analysis utilized the National Center for Biotechnology Information IgBlast server (http://www.ncbi.nlm.nih.gov/igblast/) to identify candidate germ line genes (1). Subsequent analysis, alignments, and translations were performed by using MacVector (Accelrys Inc., Princeton, N.J.). The {kappa} V (V{kappa}) region gene nomenclature is as described by Schable and Zachau (23). The {lambda} V (V{lambda}) region gene nomenclature is as described by Kawasaki et al. (11). VH gene nomenclature is as described in the ImMunoGeneTics database (12, 17). Complementarity determining regions (CDRs) are as defined by Kabat et al. (9).

Antigen binding and Fab concentration assays. The ability of Fab fragments and serum samples to bind PPS 6B was determined by a modified RABA. The preparation of radiolabeled PPS and the RABA have been described in a previous report (13). To analyze Fab samples, affinity-purified goat anti-human {kappa} antisera (5 µg/ml; Biosource International) was included in the reaction mixture to increase avidity and facilitate precipitation. Fab concentration was determined by a capture enzyme-linked immunosorbent assay in which goat anti-human Fd (The Binding Site, Birmingham, United Kingdom) or goat anti-IgA (Sigma, St. Louis, Mo.) immobilized on a microtiter plate captures Fab, which is then detected by alkaline-phosphatase labeled goat anti-human L chain (Biosource International). This assay is standardized with a purified Fab standard whose concentration was calculated from UV absorbance at 280 nm.

Accession numbers. All sequences are available from GenBank under the following accession numbers (Fab fragment [VL/VH]): 001.5A1 (AY423216/AY423163), 002.1F2 (AY423217/AY423164), 002.2H9 (AY423218/AY423165), 002.4B4 (AY423219/AY423166), 002.4E6 (AY423220/AY423167), 002.7B11 (AY423221/AY423168), 003.1H8 (AY423222/AY423169), 003.4C5 (AY423223/AY423170), 003.4D7 (AY423224/AY423171), 003.5H11 (AY423225/AY423172), 003.6A1 (AY423226/AY423173), 003.6A2 (AY423227/AY423174), 003.6B2 (AY423228/AY423175), 003.6F2 (AY423229/AY423176), 003.7D8 (AY423230/AY423177), 003.10G4 (AY487922/AY487923), 003.13H10 (AY487924, AY487925), 010.1D10 (AY423231/AY423178), 010.1D8 (AY423232/AY423179), 010.2C3 (AY423233/AY423180), 010.2C6 (AY423234/AY423181), 010.3H10 (AY423235/AY423182), 010.4B4 (AY423236/AY423183), 010.5B4 (AY423237/AY423184), 010.5C11 (AY423238/AY423185), 010.7H1 (AY423239/AY423186), 010.7H3 (AY423240/AY423187), 010.8D9 (AY423241/AY423188), 011.10E1 (AY423242/AY423189), 011.13A3 (AY423243/AY423190), 011.13A9 (AY423244/AY423191), 011.14A5 (AY423245/AY423192), 011.14E1 (AY423246/AY423193), 011.14F9 (AY423247/AY423194), 011.15H3 (AY423248/AY423195), 011.16A4 (AY423249/AY423196), 011.17F5 (AY423250/AY423197), 011.18B6 (AY423251/AY423198), 011.19D9 (AY423252/AY423199), 011.19E4 (AY423253/AY423200), 011.20H1 (AY423254/AY423201), 011.21E2 (AY423255/AY423202), 011.4H1 (AY423256/AY423203), 011.5D11 (AY423257/AY423204), 011.5E1 (AY423258/AY423205), 011.7B1 (AY423259/AY423206), 011.7B8 (AY423260/AY423207), 011.7E2 (AY423261/AY423208), 023.13A11 (AY423262/AY423209), 023.14E1 (AY423263/AY423210), 023.15A5 (AY423264/AY423211), 023.18C11 (AY423265/AY423212), 023.19E3 (AY423266/AY423213), 023.20H2 (AY423267/AY423214), and 023.4C8 (AY423268/AY423215).


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RESULTS
 
Isolation of recombinant PPS 6B-specific antibody Fab fragments. Donors (30 adults, aged 24 to 45 years) were immunized with either the PPS PS vaccine Pnu-Immune or a nine-valent conjugate vaccine consisting of serotypes 1, 4, 5, 6B, 9V, 14, 18C, 19F, and 23F PPS, individually conjugated to the mutant diphtheria toxin CRM197. MNCs were isolated from each individual 7 days following vaccination. An aliquot of cells was placed into culture to verify specific antibody secretion, and PPS 6B-specific cells were isolated from the remaining MNCs by using PPS 6B-armed magnetic beads. Based primarily on the production of 6B-specific antibody in vitro and the magnitude of serum antibody response following vaccination, eight donors were selected as cloning candidates (Table 1). From these, H-chain, L-chain, and H-chain x L-chain (H x L) Fab expression libraries were constructed as described in Materials and Methods. From each expression library 800 to 4,500 individual clones were assayed for the production of PPS 6B-specific Fab fragments. Positive clones were restreaked for single-colony isolation and recloned, and antigen binding and specificity were verified. VH and VL gene sequences were determined for each positive clone. A total of 85 PPS 6B-specific Fab fragments, 55 of which were unique in L-chain sequence, H-chain sequence, or both (Table 2), were isolated from six of the eight donors assayed. None of the isolated Fab fragments precipitated an irrelevant radiolabeled carbohydrate (PPS 23F), and none was inhibited by C-PS (10 µg/ml), indicating specificity for PPS 6B. Inspection of the sequences revealed that donors utilized diverse V{kappa}, V{lambda}, and VH genes to form PPS 6B-specific paratopes. Representative Fab fragments utilizing different VH and VL germ line genes were produced in quantity, and their relative affinities for PPS 6B were determined (Fig. 1). All Fab fragments precipitated radiolabeled PPS 6B in a specific and concentration-dependent manner.


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  		TABLE 1. Characteristics of MNC donors


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  		TABLE 2. Characteristics of the unique PPS 6B-specific Fab fragments



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  		FIG. 1. RABA analysis of PPS 6B binding by representative Fab fragments. L- and H-chain germ line genes are shown in parenthesis. Symbols: {blacksquare}, Fab 001.5A1 (B3 x VH3-23); {circ}, Fab 010.2C3 (A23 x VH3-23); •, Fab 010.7H1 (A2 x VH3-73); {blacklozenge} Fab 010.7H3 (B3 x VH3-23); {square}, Fab 010.8D9 (A17 x VH3-15).

V region gene utilization. Sequence analysis revealed that the donors in this study used a variety of VH and VL germ line genes to form PPS 6B-specific antibodies. Six V{kappa} genes, three V{lambda} genes, and five VH genes were identified in the repertoire of Fab fragments isolated from six individuals. The {kappa} L chains derived from three of the seven V{kappa} subgroups and include one member from the distal cluster of the locus, A2. All five {kappa} joining (J{kappa}) regions are utilized, and {kappa} chain CDR3 lengths vary from 5 to 10 amino acids. The three V{lambda} isolates represent two subgroups and two J-constant (J-C) regions and have CDR3 lengths of 10 or 11 amino acids. The five VH genes used were all members of the VH3 subgroup. These rearranged with four of the six functional H-chain J (JH) regions and at least six different diversity (D) region segments to generate CDR3 regions that varied from 6 to 20 amino acids in length. H-chain isotypes of the isolated Fab fragments were IgA1, IgA2, IgG1, IgG2, and IgG3. These findings suggest that the PPS-6B-specific response is diverse at the population level and indicate that a wide variety of antibody germ line genes are capable of forming a PPS 6B-specific paratope.

Within a single donor the response was more restricted. Most individuals utilized only one or two H- and L-chain pairs to form PPS 6B-specific paratopes. Moreover, in the majority of cases, J region usage, D region usage, and shared mutations from the germ line indicated that the individual H and L chains arose from a single initial rearrangement event. One individual (donor 010) was an exception to this generalization and used five different H- and L-chain pairs to form PPS 6B-specific paratopes. Overall, these findings are consistent with previous serological data showing the PPS 6B-specific response within the individual to be oligoclonal and restricted to a few distinct clonotypes (13).

In contrast to previously studied PS-specific responses (25, 33), we found little evidence for the recurrence of paratope usage between individuals in the response to PPS 6B. Only B3 x VH2-23 Fab fragments were isolated from multiple donors (001 and 010), and even in this case J{kappa} usage, VL CDR3 length, and VH CDR3 length were not conserved.

Generation of paratopic diversity. Although the PPS 6B-specific response is restricted within the individual donors in terms of V region gene usage, a substantial degree of paratope diversity has nevertheless been generated through somatic maturational events. Somatic hypermutation (SHM), deletion of germ line-encoded residues, insertion of non-germ line-encoded residues, and intraclonal isotype switching all contribute to the generation of a structurally diverse collection of binding domains in these individuals (Tables 3, 4, and 5) SHM appears to be the predominant process generating diversity in the binding domains we isolated. Both VH and VL regions were mutated, with the majority of mutations and replacements found in the CDRs. In individuals from whom several disparate Fab fragments were isolated, there is a tendency for the VH regions to be more mutated than VL regions (Table 6). In the 20 A17 x VH3-7 Fab fragments isolated from donor 011, for example, approximately 46% of the VH3-7 CDR1 and CDR2 residues have been substituted, versus 12% of the CDR residues in the L chain. A similar degree of asymmetry was seen in donors 003 and 010 and to a lesser degree in the {lambda} Fab fragments isolated from donor 023. Mutations were more equally distributed between VH and VL regions in the Fab fragments isolated from donors 001 and 002. Our germ line assignments are based on maximum similarity to reported V region germ line genes, and we cannot exclude that some of the differences we have ascribed to mutations may actually arise from as of yet unidentified alleles of the relevant germ line genes. However, the high number of substitutions, especially in the VH regions, leads us to conclude that the majority of the differences we see arise from mutations that occur during the course of SHM.


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  		TABLE 3. {kappa} L-chain CDRsa


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  		TABLE 4. {lambda} L-chain CDRsa


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  		TABLE 5. H-chain CDRsa


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  		TABLE 6. Percent substitutions in the V region CDRsa

In addition to SHM, the deletion of germ line-encoded residues and the insertion of non-germ line-encoded residues further diversifies the PPS 6B-specific repertoire in these individuals. A18b L chains isolated from donor 003 have deleted four of the seven V{kappa}-encoded residues in CDR3 (Table 3). This results in an unusually short five-amino-acid CDR3 for these antibodies. It is of interest that the V{kappa} gene A2 is also used in this response (donor 010). A2 is the distal homolog of A18b and differs from it by only three amino acids. One of these differences is in the deleted region of the A18b isolates. The A2 Fab isolated from donor 010 had a full-length nine-amino-acid CDR3. The L12 L chains used in the donor 002 isolates have deleted three residues in the CDR3 region of the V region gene as well as the first residue of the J region to again form a five-amino-acid CDR3. The single A17 Fab isolated from donor 010 had deleted a single residue, most probably the first of the J region, to form an eight-residue CDR3. In all cases our assumption is that these deletions occurred at the time of V-J joining prior to antigen stimulation.

Two types of amino acid insertions were seen in these Fab fragments. A17 L chains from donor 011 and B3 L chains from donor 010 had nontemplated additions at the V-J junction (Table 3). This type of insertion results from N or P additions, occurs commonly in antibodies specific for other PS (15, 25, 33), and is in fact required for the generation of the canonical paratope in the Haemophilus influenzae type b (Hib) PS repertoire (14). These insertions, like the deletions described above, are assumed to occur at the time of V-J joining during the initial rearrangement of the antigen-specific B-cell receptor and generate an extended L-chain CDR3 region.

The second type of insertion is seen in the Fab fragments isolated from donor 003 (Table 5). All but one of the VH3-15 Fab fragments isolated from this individual contained a two-residue insertion in H-chain CDR2. Fab fragments with the insertion share up to eight substitutions with the single Fab that lacks the insertion and have nearly identical CDR3s, thereby suggesting that the insertional event occurred as part of the somatic diversification of the response. A more extreme example of this type of insertion is seen in Fab 18C11 from donor 023, which appears to have a four-residue insertion in the CDR3 region of the H chain. At the DNA level, the VH region of this Fab shares 16 mutations from the germ line with the other VH3-23 H chains isolated from this donor. It is also identical at 29 of the 33 positions in CDR3. These facts together suggest that all of the VH3-23 Fab fragments isolated from this donor derived from a single initial rearrangement, and the large insertion into the CDR3 region occurred during the somatic differentiation of this clonotype. In addition to these insertions into the CDRs, Fab 023.20H2 had a single-residue insertion into the second framework region of the L chain, and Fab 003.1H8 had a single-residue deletion in the second framework region of the L chain (not shown).

Isotype switching occurs at multiple points during clonal diversification. The H chains of the 9 IgG2 and 11 IgA2 VH3-7 Fab fragments isolated from donor 011 (Table 5) are all rearranged to JH4b and share seven of the eight residues in CDR3, indicating that they arose from a single initial rearrangement event. In addition, all share four substitutions in CDR1 and CDR2, suggesting that SHM had begun prior to isotype switching. Hypermutation continues following class switch, as shown by the accumulation of shared substitutions within each isotype. There is further evidence that switching occurred several times during the maturation of the 6B-specific response in this individual. Clones 7B1 (IgG2) and 19D9 (IgA2) share more substitutions with each other than with other members of their respective isotypes. Likewise, clones 5E1 (IgG2) and 14A5 (IgA2) differ at a single residue in CDR1 and clearly shared a mutational history prior to isotype switch that differed from clones 7B1 and 19D9 described above. The single IgA2 VH3-15 isolate from donor 003 shares the majority of its substitutions with the IgG2 isolates from the same donor and clearly arose from the same differentiated B cell. The four VH3-23 x B3 isolates from donor 010 also share their history of somatic diversification and have given rise to IgG1, IgA1, and IgA2 isotypes. Taken together, these data indicate that class switch can occur at several points during the maturational history of a specific clonotype.


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DISCUSSION
 
Although the molecular mechanisms by which antibody diversity is generated are well understood, very little is known about how this diversity actually manifests itself in response to infection or vaccination. Antibody repertoires are particularly difficult to study in humans, due primarily to the difficulty in establishing stable and representative human heterohybridomas. The repertoire cloning methodologies we have developed permit us to examine many of the molecular and genetic aspects of antibody diversity in humans with a level of detail usually reserved for animal models. These techniques have been employed in the study of several paratopic repertoires, and it has been shown that this methodology is robust and that the results are representative of the antibody response actually generated by the host (16, 21, 33). The pneumococcal vaccines are particularly useful as model antigens in these studies. The PPS they contain are structurally defined, they are routinely and safely administered to adults and infants, and antigen-specific B cells are present in the periphery shortly after vaccination, thereby providing access to the cells of interest. PPS-specific antibody responses are oligoclonal within the individual, which facilitates Fab fragment isolation and analysis. By defining the antibody repertoires elicited by a variety of PS and protein antigens, we are beginning to establish a data set that will allow repertoire structure and diversification to be examined on a comparative basis.

Our findings concerning the diversity of the PPS 6B repertoire are most informative when considered in light of what is known about other PS-specific antibody responses. The Hib PS-specific repertoire, for example, is oligoclonal both within the individual and between different individuals (8). Most of the antibody in the majority of individuals vaccinated is composed of the same H- and L-chain pair (25). Likewise, the PPS 23F-specific repertoire is oligoclonal within the individual and highly restricted in the population, with the majority of individuals utilizing the same two H- and L-chain pairs to form 23F-specific paratopes (33). This latter finding has recently been refined to show that the two predominant paratopes in the PPS 23F-specific response recognize immunochemically disparate epitopes (22). For each PPS 23F epitope, therefore, the majority of the members of the population use the same H- and L-chain pair to form the corresponding paratope. Given the degree of diversity available to the immune system, this striking conservation of paratope usage suggests structural constraints on paratope assemblage for these antigens, i.e., only very specific V region and L-chain pairs can form antibody-combining sites of sufficient affinity. The PPS 6B repertoire differs from the two paratopes described above in that while it is also restricted within the individual, there appears to be little conservation of V region gene usage between different individuals. Our data, when combined with that previously reported (Table 7), suggest a widely divergent repertoire for this particular PPS antigen at the population level.


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  		TABLE 7. Summary of reported Ig V region gene pairings used in the response to PPS 6B and their method of isolation

The number of distinct antigenic epitopes associated with the PPS 6B polymer is unknown, however. The repeating subunit of PPS 6B is a linear polymer of D-galactose, D-glucose, L-rhamnose, and ribitol-5-phoshate and, therefore, longer and more complex in makeup than that of Hib PS (a linear ribose-ribitol-5-phosphate polymer). This increase in structural complexity would undoubtedly accommodate a greater degree of epitopic complexity as well, and the larger paratopic repertoire we observe for PPS 6B may be due in part to a larger number of distinct PPS 6B epitopes, each with its own corresponding paratope. As is the case for PPS 23F, each immunochemically distinct PPS 6B epitope might be limited by structural constraints to interact with a limited number of VH and VL region gene products. However, while the epitope structure of the antigen could account for the diversity of the response at the population level, it does not explain the oligoclonality of the response in the individual. Without additional information mapping individual PPS 6B-specific paratopes to the epitopic structure of PPS 6B, we cannot determine whether the oligoclonality of the PPS 6B-specific response arises from structural constraints, as may be the case for Hib PS and PPS 23F, or from some regulatory pathway that limits the complexity of the overall response to PS antigens.

Most of the 6B-specific paratopes we describe in this study have undergone extensive SHM compared to their germ line elements of origin. Substitutions are primarily confined to the CDRs and appear to have arisen during the course of antigen-driven somatic mutation. It has been demonstrated in mice that antibodies directed towards thymus-independent type 2 PS antigens mutate and undergo affinity maturation and class switch (4, 27). Human antibodies specific for other bacterial PS that appear to be mutated have been described (3, 16, 24, 25, 33), and it is probable that affinity maturation is a generalizable characteristic of the antibody response to these carbohydrate antigens as it is for protein-specific responses. Although PS antigens such as PPS 6B are generally considered to be T-cell independent, SHM is thought to require T-cell-derived cytokines, which suggests the involvement of T cells at some point during the development of the response in our donors. The appearance of such extensive modifications 7 days following vaccination leads us to believe that we are capturing a recall response in which vaccination activates memory B-cell clones that were expanded as a response to earlier exposure to the antigen, possibly though pneumococcal carriage. If this is the case, PPS 6B epitopes may have been first encountered by the host in a T-cell-dependent form (such as PS complexed with a bacterial surface protein), leading to affinity maturation of the response in an environment conducive to SHM and affinity maturation. The suggestion that vaccination reactivates memory B cells is also supported by the fact that Fab fragments isolated in this study from the single donor that had received the plain PS vaccine (i.e., T-cell independent) were also extensively mutated. It is also possible, however, that the PPS themselves have the ability to recruit all of the cellular components required for a fully matured antibody response. It is not feasible based on our present studies to determine if the observed SHM occurred at the time of original antigen exposure, or following vaccination, or both. It is of interest to note that the degree of SHM (especially in the H chains), when considered along with the necessity of generating H-chain CDR3 from non-V region gene elements during rearrangement, limits the extent to which germ line-encoded V region gene residues participate in PPS 6B binding. This fact may contribute to the lack of V region gene restriction that we have found for this repertoire.

Five of the six donors from which we successfully isolated PPS-6B specific Fab fragments had deleted or inserted nontemplated residues in the V region of the antibody combining site. The majority of these modifications occurred at the V-J junction of the L chain and most likely occurred at the time of V-J region joining prior to antigen stimulation. Similar modifications have been reported previously (3, 21, 33) and may be a common method for generating diversity prior to antigen-specific selection. A single-residue insertion at the V-J junction of the L chain is, in fact, required to generate the most commonly utilized combining site in the human Hib PS-specific repertoire (14). Multiple-residue deletions, however, such as those seen in the A18 and L12 L chains are uncommon. Less than 1% of randomly selected, productive V{kappa} rearrangements have such short (five-amino-acid) CDR3 regions (6). The selection of such a rare rearrangement event in two donors suggests a requirement for such a short L-chain CDR3, at least when these two germ line V{kappa} genes are utilized to form the PPS-6B-specific paratope.

The insertion or deletion of residues within the V regions themselves (i.e., not at a rearranging junction) has been relatively recently observed in randomly selected H and L chains and is thought to be an uncommon event (5, 32). The tendency for these modifications to occur in the CDRs and at the same hot spots of SHM suggest their association with SHM, but direct evidence has been difficult to obtain by the sequencing of randomly selected B cells. The VH3-15 Fab fragments from donor 003 we report here have all undergone extensive SHM in the H-chain CDRs, and eight of the nine have a two-residue insertion in CDR2. The sharing of identical CDR3s and several mutations in CDR1 and CDR2 make it highly probable that all of these chains arose during the course of the somatic maturation of a single original responding clone and that the insertion occurred at some point during this pathway. Likewise, the VH3-23 isolates from donor 023 share common mutations and homologous CDR3 regions and most likely a common ancestry. Clone 023.18C11, however, has an additional four residues in CDR3 that appear to have been inserted during the course of affinity maturation. Conversely, it is possible that the original rearrangement generated a 17-residue CDR3 from which 4 residues were subsequently deleted. The requirement to generate the entire H-chain CDR3 during rearrangement precludes the demonstration of insertions or deletions in this region by using randomly selected B cells. By analyzing clonal descendants in an antigen-specific response, we have demonstrated that extensive insertions and/or deletions can occur in this region following the initial rearrangement event. Examples of insertions such as these in Fab fragments specific for PPS 23F (33) and PPS 6B (16) have previously been reported, and it is likely that utilization of this mechanism for the generation of diversity is more predominant than previously believed.

In addition to SHM and related insertions and deletions, the sequences we report here have undergone class switch recombination (CSR). CSR and SHM are related but independent events. Both involve the modification of B-cell DNA and occur primarily, although not exclusively, in germinal centers following antigen stimulation (7). Activation-induced cytidine deaminase mediates both events and apparently differentiates the two through the interaction of cofactors (29). The analysis presented here of the shared mutations in the sequences suggests that SHM can occur both prior and subsequent to CSR. Our data also demonstrate that CSR can occur more than once during the course of clonal maturation. All Fab fragments reported here are presumed to have switched from IgM to IgG or IgA. In most donors, clonal derivatives of IgG Fab fragments switch again to downstream IgA C regions. In donor 011 (Table 5), switching of IgG to IgA CSR appears to have occurred at three different points during the branching of the maturational tree. The VH3-7 Fab fragments isolated from donor 003 have switched to IgG3 and IgG1. The higher number of mutations in the IgG1 Fab is consistent with a switch from the upstream IgG3 to the downstream IgG1 C region. In no case was the analysis of shared mutations consistent with CSR involving upstream (and presumably deleted) C regions. Our results are therefore consistent with a mechanism of sequential switching that involves the serial deletion of intervening H-chain C region genes (18).

The paratopes we describe in this report also emphasize the influence that elements other than the V region genes themselves have on specificity and affinity. Two of the PPS 6B Fab fragments reported in this study utilized the same VL and VH germ line genes but were isolated from different donors (001 and 010). They varied from each other in J{kappa} usage, VL CDR3 length, and VH CDR3 length and differed approximately 10-fold in their relative affinity for PPS 6B (Fig. 1). In addition, Fab fragments 2C3 and 5C11 from donor 010 utilized the same gene paring (A23 x VH3-23) that defines one of the predominant paratope families in the human response to PPS 23F (33). The PPS 6B-specific Fab fragments and PPS 23F-specific Fab fragments differ in SHM-induced sequence modifications (primarily in the VH CDRs) and in the length of the VH CDR3. Together, these observations illustrate the degree to which elements not encoded by the germ line V region genes themselves (such as CDR3 length and J region usage) and that arise randomly during the process of combinatorial V-J region and V-D-J region gene rearrangement not only influence affinity but determine specificity as well.

Our molecular and genetic analysis of the PPS 6B-specific antibody repertoire, when combined with previously published results, describes a response that is restricted in complexity within the individual but highly divergent across the population in terms of the genetic elements employed. Our findings also illustrate the extensive degree to which antibodies undergo somatic modification following exposure to an epitopically redundant antigen such as the 6B PPS. The extension of these studies to include other PS and protein antigens will allow us to determine in detail how the well-defined molecular mechanisms of diversity generation actually manifest themselves in the paratopic diversity of the human antibody response.


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ACKNOWLEDGMENTS
 
This work was supported by Public Health Service Grants AI47136 and AI 045250 from the National Institute of Allergy and Infectious Diseases.

We thank Hatice Sporer for technical assistance and Betty M. Ho for critically reading the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Children's Hospital Oakland, Research Institute, 5700 Martin Luther King, Jr., Way, Oakland, CA 94609. Phone: (510) 450-7638. Fax: (510) 601-3911. E-mail: dreason{at}chori.org. Back

Editor: D. L. Burns


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Infection and Immunity, June 2004, p. 3505-3514, Vol. 72, No. 6
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.6.3505-3514.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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