INTRODUCTION

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The capsular polysaccharide (PS) constitutes the major virulence factor of many pathogenic bacteria that cause invasive diseases. Antibodies (Abs) against these PSs are protective (27, 28). PSs are classified as thymus-independent-2 (TI-2) antigens (Ags) because they do not require mature T cells to elicit a humoral response in vivo. These PS Ags are immunogenic in adults but are only poorly or nonimmunogenic in infants and young children who are highly susceptible to infection caused by encapsulated bacteria (28, 31, 44, 65).

The response to capsular PS is markedly different from the response to most protein Ags (thymus-dependent [TD] Ags). The Ab response to TI-2 Ag develops late in ontogeny (25, 44, 50) and in mice utilizes a particular late-developing subset of B cells that is defined by the expression of Lyb5 and other cell markers (39, 60). TI Ags also generally fail to elicit a memory response or show affinity maturation. In contrast, the ability to respond to a TD Ag is present at birth and results in the formation of memory cells, and the Ab response undergoes subsequent affinity maturation upon reimmunization (61). For TI Ag, immunoglobulin G3 (IgG3) and IgM are the major isotypes expressed in mice, even after secondary immunization (45), whereas for TD Ag, the ratio of IgG to IgM increases after secondary immunization, with IgG1 being the major subclass (52, 59, 60).

The majority of the anti-PS responses are oligoclonal and encoded by a few variable regions of the heavy chain (V_H) gene families (10). The anti-(13) dextran Ab, for example, expresses mainly the V_HJ558 family (68); whereas anti-(21) fructosan Ab predominantly expresses the V_HJ606 gene family (11) and Abs to (26) fructosan and (16) galactan express the genes of the V_HX24 family (42, 67). The anti-group A streptococcal carbohydrate Ab reflects a germline repertoire that includes at least two V_H gene families, one of which belongs to the V_HJ606 family paired with several V gene families (46). The response to the glucuronoxylomannan component of the capsular PS of Cryptococcus neoformans serogroup D uses the V_HX24 family (13), and immunized mice respond with V_H7183 Ab specific for serogroup A (40), indicating the highly restricted usage of V_H gene families in anti-PS responses and differences depending on the structure. However, some Ab responses, for example, in the anti-(16) dextran response, were shown to be encoded by the V_H genes of the V_HJ606, V_HJ558, and V_H3660 families (3, 57).

The immunogenicity of TI Ag has been shown to be enhanced by covalently binding TI Ag to carrier proteins, thus converting the response to TD (5, 59, 60). Haemophilus influenzae type b (Hib) was once the most common cause of bacterial meningitis in children in the United States, but immunization with TD conjugate vaccines has been remarkably successful in decreasing the incidence of Hib disease (1, 15, 41). These conjugate vaccines have been particularly useful for prevention of Hib infection in high-risk infant populations (30, 53-55). The almost complete disappearance of Hib disease and the reduction in pharyngeal carriage of Hib (6) point out the importance of these conjugate vaccines (6, 15, 62).

Neisseria meningitidis remains one of the major causes of bacterial meningitis in children and young adults worldwide. N. meningitidis PS vaccines have been available for quite some time (29); however, the PS is a TI-2 Ag which is poorly immunogenic in infants and has a short duration of protection in young children (14, 24, 26, 35, 65). The capsular PS, N. meningitidis group C PS (MCPS), is a linear homopolymer of (29)-linked sialic acid residues that are O acetylated at carbons 7 and/or 8 (9, 19). Early murine studies of meningococcal conjugate vaccines showed mainly IgG1 antibodies to PS and carrier after one dose (8) and increased IgG titers after a second dose (17). Our previous studies with mice confirmed and extended these observations (52). Several oligosaccharide-protein conjugate vaccines that elicit a TD response to protect young children against invasive meningococcal disease (17, 32) have been developed and are currently being evaluated in clinical trials (4, 17, 20, 34, 36, 37, 49, 66).

In order to look at the influence of the form of the vaccine (TI versus TD) on the Ab repertoire, we generated two new monoclonal Ab (MAb) panels after primary immunization with MCPS-tetanus toxoid (MCPS-TT) followed by a boost with MCPS or MCPS-TT. Data show that, compared to MCPS, the response to MCPS-TT results in isotype shift, a shift in fine specificity, and increased affinity. The data also show that the secondary Ab repertoire is determined by the primary immunization in that the response to an MCPS booster after MCPS-TT priming resembled the response to MCPS-TT rather than that to MCPS. The results of the isotype, fine specificity, affinity, and V_H gene family analyses of the three MAb panels are presented here.

	MATERIALS AND METHODS

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Animals. Four-week-old female BALB/cAnN (BALB/c) and pregnant female BALB/cAnN mice were purchased from Charles River Laboratories through the National Institutes of Health Small Animal Section and maintained under pathogen-free conditions in our animal rooms. All animal protocols were approved by the Center for Biologics Evaluation Research Animal Care and Use Committee.

PS. The MCPS prepared from N. meningitidis C11 was obtained from Merck, Inc., West Point, Pa. (lot 1815T). The structures of the PSs used in these studies are as follows: native MCPS, a homopolymer of (29)-linked sialic acid residues that are O acetylated at carbons 7 and/or 8 (9, 19); OAc, a naturally occurring non-O-acetylated variant of MCPS; Escherichia coli K92, a homopolymer of alternating (29)- and (28)-linked sialic acid; and E. coli K1, a homopolymer of (28)-linked sialic acid.

Conjugate vaccines. A group C meningococcal oligosaccharide coupled to TT (also referred to as MCPS-TT) was prepared as previously described (32) and was used for all experiments as described in the work of Rubinstein et al. (52). The molecular mass of the MCPS in the conjugate is 10 kDa (52).

Immunization. The MCPS MAbs were produced by immunizing 8- to 12-week-old BALB/c mice as indicated previously (51). For the anticonjugate-primed and -boosted (C2) MAb, mice were immunized intraperitoneally with 10 µg of MCPS-TT in 5% Maalox as an adjuvant (modified from reference 48) and then rested for a minimum of 8 weeks, after which they were boosted intravenously with 10 µg of MCPS-TT. Similarly, for the anticonjugate-primed and PS-boosted (CP) MAb, mice were immunized intraperitoneally with 10 µg of MCPS-TT in 5% Maalox and boosted with MCPS in saline or 10⁸ CFU of fixed bacteria (see Table 2, footnote h, for identification).

Hybridoma production and MAb purification. Spleens were removed 3 days after the last injection. Fusions were performed with the nonsecreting myeloma cell line SP2/0, according to the protocol of Kennett (33) as modified by Rubinstein and Stein (51). Supernatants from wells containing growing hybrids were assayed by fluorescence enzyme-linked immunosorbent assay (FELISA) and selected on the basis of reactivity with MCPS and no reactivity on a chemically similar but non-cross-reactive PS, E. coli K1. Positive cells were expanded; tested on MCPS, OAc, K92, and K1 to confirm specificity; and cloned by limiting dilution. All IgM MAbs were purified on an anti-mouse -Sepharose column (187.1 rat anti-mouse  cell line [ATCC HB58]) from the nonbinding fraction of ascites that had been passed over a protein A-Sepharose column (Pharmacia Biotech Inc., Piscataway, N.J.). All IgG MAbs were purified from ascites fluid on a protein A-Sepharose column (Pharmacia) or a protein A/G or protein G column (Pierce, Rockford, Ill.). The IgA-secreting hybridomas were grown in Ultradoma protein-free medium (Biowhittaker, Walkersville, Md.) and purified from Amicon-filtered concentrates (Amicon, Beverly, Mass.) with a fast protein liquid chromatography Q-Sepharose column (Pharmacia, Uppsala, Sweden) followed by an anti-mouse -Sepharose column. All purified MAbs were shown to be >97% pure by immunoelectrophoresis. In addition, all IgG MAbs were clonally distinct by isoelectric focusing.

Characterization of the MAbs. The MAbs were characterized by Ouchterlony precipitation (43) at a concentration of 1 mg/ml for both MAb and PS, isoelectric focusing (58), and a direct binding in FELISA. Isotypes were determined by FELISA (50) with supernatants from the clones assayed on Ag-coated plates and developed with alkaline phosphatase-labeled anti-isotype reagents purchased from Southern Biotechnology (Birmingham, Ala.).

FELISA. The MCPS MAb FELISA has been described in detail elsewhere (50). Titers are expressed as reciprocal dilutions, determined by extrapolation to zero from the linear part of the titration curve. The proteins were tested by 12 threefold serial dilutions starting at an Ab concentration of 3 µg/ml. An  symbol is defined as approximately equal reactivity on MCPS, OAc, and K92. > is defined as an approximately 0.5- to 1-log-lower concentration for 50% binding, >> is defined as a 2-log-lower concentration for 50% binding, and >>> is defined as a 3-log-lower concentration for 50% binding. The FELISA is similar to a conventional ELISA; however, it has three unique features. The Microfluor "W" U plates (Dynatech Laboratories, Chantilly, Va.) are opaque, and PSs adhere well; the substrate, 4-methylumbelliferyl phosphate (Sigma Chemical Co., St. Louis, Mo.), is not hydrolyzed in water, resulting in a background that is stable over time; and the scale on the Microfluor reader (Dynatech) is 0 to 4 rather than 0 to 2.

RNA preparation, dot blot, and Northern blot analysis. Total RNA isolated from hybridoma cells was prepared by guanidinium thiocyanate lysis and cesium chloride purification essentially as described by Chirgwin et al. (16). mRNAs were isolated from hybridoma cells by using the Fast Track mRNA isolation kit (Invitrogen, Carlsbad, Calif.) following the manufacturer's directions. All RNA solutions were made in diethyl pyrocarbonate-treated H₂O (Research Genetics, Huntsville, Ala.). RNA dot blots of 40 µg of total RNA were performed as indicated by Boswell et al. (11) with nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, N.H.) on a Minifold I apparatus (Schleicher & Schuell). The filters were UV cross-linked while damp, two times, with 1,200 µJ in a Stratalinker (Stratagene, La Jolla, Calif.). Membranes were prehybridized for 4 h at 68°C followed by hybridization for 20 h at 68°C in fresh prehybridization buffer with ³²P-labeled DNA probes added to a final concentration of 2 × 10⁶ to 5 × 10⁶ cpm/ml as described by Boswell et al. (11).

Hybridization probes. The hybridization probes for V_H gene family assignments used in this study were agarose gel-purified DNA fragments passed through ELUTIP-d columns (Schleicher & Schuell) following the manufacturer's directions. C_H probes were isolated as indicated for V_H probes. All probes are the same as those used by S. H. Feng (22) and C. Boswell (11) in our laboratory. DNA probes were labeled (21) with [-³²P]dCTP with the Amersham Corp. random primer kit (Arlington Heights, Ill.) following the manufacturer's directions. Blots were stripped and rehybridized with C_H region DNA probes after V_H hybridization to confirm the presence of Ig RNA in all samples. The control myeloma and MAb cell lines for the different V_H gene family probes have been described previously (11, 12).

Primer extension of mRNA. mRNA was used as a template for synthesis of full-length cDNA from transcribed V_L genes as described by Shlomchik et al. (56). Briefly, the 5' ends of oligonucleotide Jk primers were labeled with ³²P by using polynucleotide kinase (New England Biolabs, Beverly, Mass.) and [-³²P]dATP (Amersham). The labeled primers were mixed with 1.5 µg of mRNA, and cDNA was synthesized by avian myeloblastosis virus reverse transcriptase (Stratagene) at 42°C for 45 min in the presence of excess deoxyribonucleic triphosphates (Pharmacia), followed by an incubation with terminal deoxynucleotidyl transferase (Stratagene) at 37°C for 30 min, and run in a 5% polyacrylamide gel.

Statistical analysis. Student's t test was used to compare the affinities of the three panels of MAbs to MCPS.

	RESULTS

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We previously described a panel of 15 MAbs generated against the TI form of MCPS (51). In order to investigate the influence of the form of the Ag (i.e., TI-2 versus TD) on the Ab response, we produced two additional panels of MAbs. The first new panel was generated by immunizing mice twice with MCPS-TT (referred to as C2 MAb, for conjugate twice), and the second panel, designed to mimic the situation in human infants who might encounter an encapsulated organism following a single immunization with a conjugate vaccine, was generated from mice immunized with MCPS-TT followed by immunization with MCPS (referred to as CP MAb for conjugate primed and PS or encapsulated fixed bacteria boosted).

Isotype distribution. The MCPS MAbs are primarily of the IgG3 and IgM isotypes with a small percentage of IgG1 and IgG2b (51) whereas both the C2 MAbs and CP MAbs are mainly IgG1, 87% (13 of 15) and 88% (7 of 8), respectively. Two IgA MAbs, 13% (2 of 15), and one IgG2b MAb, 12% (1 of 8), were observed in the C2 and CP MAb responses, respectively. No IgM or IgG3 MAbs were recovered from the response to conjugate vaccine. No IgG2a responses were observed with any form of immunization, and all MAbs from each panel used a kappa light chain.

Analysis of MCPS and MCPS-TT MAb fine specificity. IgA, IgG, and IgM MAbs were purified and tested for fine specificity by a quantitative measure of binding to purified MCPS, OAc, or K92 PS in a FELISA. Table 1 summarizes the differences in the patterns of fine specificity seen among the panels of MAbs. The titer of each MAb was determined on MCPS-, OAc-, and K92-coated plates, and the fine specificity listed is based on the relative titers with each of the Ags. Major fine specificity differences were seen in these panels. As reported earlier, the MCPS MAbs are predominantly of two specificities. Half of these are MCPS specific, with a majority of the others having a higher titer on OAc than on MCPS (OAc > MCPS) (51). The C2 MAbs do not exhibit a pronounced dominance of any one specificity, and interestingly, none are specific for native MCPS. Three of these also bound K92, suggesting that the C2 MAbs were primarily seeing the (29)-linked sialic acid backbone. A new specificity not seen in the MCPS MAb panel was found in the C2 MAb, with approximately the same reactivity on MCPS and OAc (MCPS  OAc). The CP MAb panel also lacked the specificity for native MCPS, but the MAbs were more limited in their fine specificity than the C2 panel, with six of eight showing equal reactivity with MCPS and OAc. Most of the CP MAbs were of this fine specificity, suggesting that this panel derived from a population of cells primed by conjugate.

Analysis of MAb affinity. The purified proteins were also tested for affinity by a quantitative measure of binding to purified PS in the FELISA. The distribution of fine specificities among the three panels of MAbs, expressed as the concentration for 50% binding ([50%]), is shown in Table 2. Reported relative affinities are the average of nine values from triplicate readings per assay for each MAb, tested in three separate assays. As seen in Table 2, MAbs fell into two categories: low-avidity binders (arbitrarily defined as the [50%] binding to PS of 0.1 µg/ml) and high-avidity binders (defined as the [50%] binding to PS of <0.1 µg/ml). To assess the overall influence of the Ag used for immunization on the affinity of the purified Abs, the mean values of binding to MCPS and OAc were determined for each panel. The mean [50%] binding by the MCPS MAb to MCPS was 1.29 µg/ml (range, 0.01 to >3 µg/ml) and to OAc was 0.527 µg/ml (range, 0.002 to >3 µg/ml). In contrast, the mean [50%] binding by C2 MAb to MCPS was 0.048 µg/ml (range, 0.002 to 0.3 µg/ml) and to OAc was 0.115 µg/ml (range, 0.002 to 1 µg/ml). The mean [50%] binding by CP MAb to MCPS was 0.104 µg/ml (range, 0.002 to 0.55 µg/ml) and to OAc was 0.018 µg/ml (range, 0.0025 to 0.12 µg/ml). The MAbs of both the C2 and CP panels had 1- to 2-orders-of-magnitude-lower [50%] binding to MCPS and OAc, indicating that the MAbs from mice primed with the TD Ag were of significantly higher affinity than the MAbs from mice primed with the TI Ag regardless of whether the booster was TD or TI (P < 0.002 for binding to MCPS for C2 versus MCPS MAb and P < 0.003 for binding to MCPS for CP versus MCPS MAb). Comparisons of C2 and CP MAb binding to OAc versus MCPS MAb binding to OAc were not significant.

V_H gene family usage. Because different fine specificities were generated against MCPS and MCPS-TT, it was of interest to explore the diversity of these Abs at the gene level. Panels of MAbs were analyzed first by dot blot or by Northern blot analysis, with 14 V_H family DNA probes. All MAbs were assigned to V_H families by independent testing with V_H family probes and verification of the presence of Ig RNA with a C region probe. A typical example of Northern blot analysis is shown in Fig. 1. Dot blot and Northern blot analyses suggest that the V_H gene family usage in three panels of MAbs stimulated by either MCPS or MCPS-TT is dominated by V_HJ558, the largest V_H gene family. Whether this reflects a random distribution or a restriction to V_HJ558 cannot be determined without knowledge of individual gene usage. It is of note, however, that among the 39 MAbs, 7 of 14 V_H families examined were not expressed. All families with four or more genes were expressed, except V_HJ606, which was not expressed in any of the panels (Table 3 and Fig. 2). V_HJ558 was found in 8 of 16 MCPS TI-2 MAbs and 15 of 23 MCPS-TT TD MAbs (both C2 and CP) (Table 3). V_HJ558 is represented in all fine specificity groups (Table 3), although in only one (C2/974.1) of four MAbs (2010.10, C2/706.12, C2/974.1, and C2/1076.10) cross-reactive with K92 (Table 3). The V_H3609 family was limited to the MCPS MAb panel and a single fine specificity while V_H7183 and V_HVGAM3-8 were limited to the C2 and CP MAb panels. Also presented in the table are the fusion number or letter and isoelectric point (pI) so that clonal relatedness can be ruled out, if possible. For example, two of the MAbs utilizing the V_H7183 gene family cross-react with K92, but these MAbs were derived from different fusions; therefore, they arose independently (Table 3). Although MAbs 1702.10 and 1922.2 are from the same fusion and have the same fine specificity, sequence data indicate that these MAbs utilize different members of the V_HQ52 gene family (P. A. García-Ojeda et al., unpublished data).

View larger version (86K): [in this window] [in a new window]   FIG. 1.   Examples of Northern blot hybridization of total RNA of the C2 MAb panel with VH probes for 2 of 15 murine VH gene families and CH probes for heavy chain constant region. Ethidium bromide staining of total RNA was included in the top scan followed by autoradiography of MAbs and positive controls under them.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   VH gene family usage in MAbs against MCPS and MCPS-TT in BALB/c mice. The expected frequencies were derived from the estimated number of VH genes per family (complexity) in the germ line (R. Riblet, presentation to the American Association of Immunologists, San Francisco, Calif., 1997, with permission).

V_L gene family usage. All MAbs in the three panels utilized  light chains. Examination of J region utilization indicates that, in 32 of 39 MAbs tested, all functional J genes are expressed (data not shown). Preliminary analysis of V_L genes from the MCPS MAbs revealed that the VOx1 light chain of the V4/5 family was the V_L most commonly paired with the V_HJ558 genes (P. A. García-Ojeda et al., unpublished data). Also, some V_H-V_L pairs are seen in this response in a restricted fashion. For example, a V_H3609-V23 combination (P. A. García-Ojeda et al., unpublished data) in MAbs 78.2, 2750.27, and 3624.22 correlates with reactivity to native MCPS. This fine specificity was seen only with the MCPS MAb panel, not with the C2 or CP MAb panels (Tables 1 and 3).

	DISCUSSION

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We have shown previously that the immune response to MCPS (a TI-2 Ag) in BALB/c mice provides a model system which closely parallels the response in humans (50). The primary response induced mostly Abs with IgG3 and IgM isotypes, and the secondary immunization with MCPS was similar to the primary immunization, typical of a TI-2 response (52, 61). In contrast, the response to MCPS-TT shifted the response to IgG1 Abs with bactericidal activity at least 10-fold higher than the response to MCPS. This response was maintained when boosted by either MCPS or MCPS-TT (52). IgG1 memory B cells against the capsular PS of meningococcal group C were observed in the MCPS-TT-primed mice as shown in adoptive transfer experiments, and this response could be boosted by either MCPS or MCPS-TT in the absence of T cells (52). The data demonstrated that the influence of the TD Ag during the primary immunization is to induce class switching and generate a memory B-cell population that can be boosted by either a TI-2 or a TD Ag (52).

Previously a panel of MAbs generated against the TI-2 form of the Ag, MCPS (51), was described. To expand these data to conjugate vaccines, we describe here two additional panels of MAbs generated against the TD form, MCPS-TT, and boosted with either MCPS-TT or MCPS. An analysis of these panels shows that increases in Ab diversity and affinity are additional features of the response to TD Ag priming.

Previously, we showed that MAbs generated in response to MCPS were largely restricted to two fine specificities (51) and were primarily of the IgG3 and IgM isotypes. Half of these MAbs are native MCPS specific, requiring the OAc groups, with most of the remaining MAbs reacting better with the OAc Ag than with MCPS. The MCPS-TT MAbs are mainly IgG1, consistent with the serum Ab data (52). The conjugate response (C2 and CP panels) generated a new fine specificity (MCPS  OAc) without a pronounced dominance of any one specificity, and none of the MCPS-TT MAbs were native MCPS specific. In the CP MAb panel, there was a predominance of the MCPS  OAc, a specificity seen in the C2 but not the MCPS panel, reinforcing the concept that the secondary Ab repertoire is determined by the primary immunization.

The native MCPS has very few non-O-acetylated sialic acid residues, with an average of 1.16 equivalents of O-acetyl per sialic acid (9). In the MCPS MAb panel, 7 of 16 MAbs bound to both MCPS and OAc, and of these, five bound 1 to 2 orders of magnitude better to OAc than to MCPS (Table 2) (51). All the C2 and CP MAbs also bound both Ags; however, most reacted equally well on both MCPS and OAc. These data are consistent with the finding of Glode et al. (23) that immunization with native MCPS elicits Abs that are bactericidal for both C11 (native O-acetylated) and MC19 (OAc) strains of N. meningitidis and that MC19 PS could absorb a large part of the bactericidal activity for strain C11.

The affinity of the MCPS-TT Abs is 10- to 100-fold higher than that of the MCPS Abs. Whether this increase in Ab affinity is due to somatic mutations (7) is currently under investigation (P. A. García-Ojeda et al., unpublished data). The data show, moreover, that, on the whole, the IgG3 Abs were of lower affinity than were the IgG1 Abs. Ab affinity may be an important determinant of host defense and should be considered as important as Ab concentration in evaluating Ab response to vaccination (63). Although we have not correlated the Ab affinity of our MAb with in vivo protection, Ahlstedt et al. (2) demonstrated that high-avidity Ab against E. coli O Ag was more protective against intraperitoneal infection in mice than was Ab of low avidity.

We examined the V_H gene families utilized by MAbs derived from the various immunization schemes in order to determine the relationship between the type of antigenic stimulation and fine specificities with V_H gene family usage. The primary Ab response in fetal mice is biased toward V_H family members that lie proximal to the D_H locus (47, 69), while in adult mice this bias disappears and the naive repertoire correlates with the size of the V_H gene family (18). The majority of anti-PS responses are encoded by a few V_H gene families, and neither chromosomal position nor family size seems important (10). The anti-(13) dextran Abs draw their specificity from restricted V_H gene family usage, mainly the V_HJ558 family (68), while the (16) dextran Abs are encoded by a variety of V_H and V_L genes (3, 57). Examining the diversity of the MAbs at the gene level indicates that the response to (29)-linked sialic acid is dominated by V_HJ558, the largest V_H gene family. Among 39 MAbs, more than half (58%) are V_HJ558. Nearly half of the MCPS MAbs used V_HQ52 and V_H3609. V_H3609 is the second largest V_H gene family, as recently reported by R. Riblet (presentation to the American Association of Immunologists, 1997; R. Riblet, personal communication).

The MCPS-TT MAb panel utilizes two V_H genes, V_H7183 and V_HVGAM3-8, not utilized by the MCPS MAb panel. The shift in gene usage could be a consequence of the recruitment of T cells by the MCPS-TT MAb-producing cells or by the TD Ag. Stein et al. (59) showed that IM6-keyhole limpet hemocyanin stimulated additional anti-(16) dextran clones that were not seen after immunization with dextran B512. Similar results have been observed by Matsuda and Kabat (38), where the immune response to the TI form of the Ag used different V genes than those responding to the TD form (38).

Two of the MAbs utilizing the V_H7183 gene family cross-react with K92. This fine specificity was also observed for one MCPS MAb. It is of interest that three different gene families (V_HX24, V_HJ558, and V_H7183) are used among four MAbs that cross-react with K92 (Table 3). These data suggest that there may be a variety of combining sites that can accommodate K92; however, definitive conclusions cannot be reached until the sequencing and molecular modeling studies of these MAbs are completed (P. A. García-Ojeda et al., unpublished data).

All MAbs in the three panels utilized members of kappa light chains. Although both the V_H and V families are made up of multiple gene families, anti-PS responses typically are characterized by a limited usage of V genes and particularly by restricted pairing of V_H genes with V or V genes (10). With the exception of V_H3609, no other correlation of V_H gene usage and fine specificity was observed in our panels. Akolkar et al. (3) reported that even when MAbs specific for (16) dextran having very similar properties (in terms of size, shape, fine structure, and binding constants in their combining sites) were examined, six different combinations of V_H and V_L genes were found.

In conclusion, the data show that the use of a TD form of MCPS, compared to the TI form, results in an isotype shift, a change in fine specificity, and an increase in affinity. The MCPS MAbs are primarily of the IgG3 and IgM isotypes, whereas the MCPS-TT MAbs are mainly IgG1, consistent with the serum Ab data (52). The responses to both the TI MCPS and the TD MCPS-TT are dominated by V_HJ558, and the affinity of the MCPS-TT Abs is 10- to 100-fold higher than that of the MCPS Abs. Sequencing studies are being conducted to examine the specific V_H genes within the V_HJ558 family and to determine whether specific gene usage correlates with fine specificity and affinity.