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Natural human antibodies to pneumococcus have distinctive molecular characteristics and protect against pneumococcal disease

Natural human antibodies to pneumococcus have distinctive molecular characteristics and protect... Summary The molecular and functional characteristics of natural antibody from the preimmune repertoire have not been explored in detail in man. We describe seven human IgM monoclonal antibodies selected on the basis of pneumococcal polysaccharide binding that share both molecular and functional characteristics with natural antibody, suggesting a common B cell lineage origin. Unlike class-switched antibodies, which are serotype-specific, the antibodies were polyreactive and bound all pneumococcal polysaccharide capsular serotypes tested. Some bound endogenous antigens, including blood group antigens and intermediate filament proteins. All the antibodies used unmutated heavy chain V (IGHV) that are expressed at an increased frequency in the elderly and in the preimmune repertoire. The CDR3 was characterized by long length (mean aa 18·4 (±4·2) and selective use of IGHD6 (P < 0·001) and IGHJ6 (P < 0·01) family genes. The clones expressing IGHV1-69 and IGHV 3-21 provided significant passive protection against invasive pneumococcal disease in vivo. function, human, molecular, natural antibody, pneumococcus Introduction Human and murine studies have demonstrated that natural IgM contributes to the early defence against invasive disease with encapsulated bacteria [1,2], clearing bacteria from the circulation and localizing them to the marginal zone of the spleen [2]. IgM has also been shown to facilitate the induction of adaptive immune responses to vaccine antigens in mice [3], regulate B cell differentiation and selection [4,5] clear apoptotic and dying cells from the circulation [6], thereby modulating the inflammatory response to tissue injury [7], and contribute to the induction and maintenance of autoimmune disease [8,9]. The origin of this type of antibody has been best defined in mice as the B-1a B cell population that resides in the pleural and peritoneal cavities and is committed to lineage at a very early stage in B cell development [10]. In man this population is less well defined, but best described as a long-lived B cell population with low cell turnover, that expresses predominantly CD5+ and unmutated IgM [11,12], and becomes relatively more clonally restricted with age [13,14]. This population forms the preimmune repertoire of natural antibody with low-grade polyspecificity for bacterial and self-antigens [11,12]. In this study, we examined seven human heterohybridomas that were generated from B cells isolated following pneumococcal polysaccharide immunization of healthy young adults, and selected on the basis of producing monoclonal antibody (mAb) binding to pneumococcal capsular polysaccharides (PncPS) that produced IgM mAbs with both the specificity and molecular characteristics of natural antibodies. Three of the clones provided significant protection against invasive pneumococcal disease. Materials and methods Isolation and selection of hybridomas from vaccinees Human heterohybridomas were prepared as described previously [15] from lymphocytes isolated from four healthy volunteers who had been vaccinated 7 days previously with either the pneumococcal polysaccharide or conjugate vaccine (Pneumovax™ and Prevanar™). Hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) for clones producing IgM that reacted with Streptococcus pneumoniae capsular polysaccharides on standard PncPS ELISA incorporating a heptavalent antigen mix comprising pneumococcal capsular polysaccharides (PPS) serotypes 4, 6B, 9V, 14, 18C, 19F and 23F [American Type Tissue Collection (ATTC), Rockville, MD, USA]. Specificity profiling Positive wells were then screened by limiting dilution using a multiplex bead assay including Pnc serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, a standard serum control: 89SF (C. Frasch, Rockville, MD, USA) and R-phycoerythrin (RPE)-conjugated rabbit anti-human IgM/A or G (Sigma, Poole, Dorset, UK), as described previously [16]. Antibodies were preincubated with cell wall polysaccharide (CWPS) and serotype 22F polysaccharide (10 μg and 5 μg/ml) (ATTC) to eliminate non-specific binding. A concentration-matched myeloma-derived IgM negative control was used in all assays (Immunopure human IgM; Perbio Science, Cramlington, UK). Specificity was evaluated further by competitive inhibition ELISA in which the PncPS-antibody binding intensity was compared with/without antibody preincubation with 10 μg/ml homologous polysaccharide or 10 μg/ml 22F with 5 μg/ml CWPS. A serotype-specific IgG mAb, CbE2 [17], was used in homologous polysaccharide competitive inhibition ELISAs as a positive control for PncPS serotype-specific binding and a phosphorylcholine (PC)-specific murine IgA (TEPC15; Sigma, St Louis, MO, USA) was used in the CWPS competitive inhibition ELISAs as a positive control for CWPS-specific binding. Monoclonal IgM positive controls were not available for these comparisons, so serum samples from immune individuals were included in each competitive inhibition assay to evaluate whether antigen-specific PC and CWPS binding could be demonstrated in polyclonal serum IgM. The IgM Mabs were quantified using a capture ELISA in which rabbit anti-human IgM was used to coat the plate and a non-cross-reactive goat anti-human IgM was used for detection. All assays were run in duplicate and serum standards (SPS-01) and negative controls were included in each plate. To evaluate cross-reactivity to other common bacterial antigens, mAb binding to phosphorylcholine (PC) and endotoxin was assessed by ELISA in accordance with previously described methods [18,19] using fixed starting concentrations of IgM, 10 μg/ml. PC–bovine serum albumin (BSA) was provided by Biosearch Technologies (Novato, CA, USA) (PC-1011-10) and precoated endotoxin plates (precoated with an equimolar mixture of incomplete core, rough, mutant endotoxins from each of four species of Gram-negative bacteria complexed with polymyxin B) were provided by Dr G. R. Barclay (Department of Medical Microbiology, University of Edinburgh Medical School, UK). Competitive inhibition ELISA was performed for PC binding mAbs using saturating doses of PC (200 μg/ml) as described for PncPS ELISAs. A serum control and a PC-specific mAb, TEPC 15, was included in the assay. Autoantigen binding As the antibodies were not serotype-specific, the binding activity against the natural antigen blood groups A and B and the xenotransplantation antigen Galα1–3Gal was assessed by ELISAs, performed in accordance with previously described methods [20,21], using fixed starting concentrations of IgM (10 μg/ml). A and B polyacrylamide-conjugated antigens (PAA-Bdi and PAA-Atri) were provided by Lectinity Inc. (Moscow, Russia). Galα1,3Gal polyacrylamide-conjugated antigens (PAA-Bdi, PAA-Atri and PAA-Btri) were provided by Lectinity Inc. Cross-reactivity to cellular endogenous antigens was evaluated by indirect immunofluorescence using HEp-2 (Biodiagnostics, Upton-upon-Severn, UK) and anti-nuclear antibody (ANA) [liver, kidney and stomach sections (LKS)] slides (Biodiagnostics), according to the manufacturer's instructions. In vivo passive protection IgM mAbs were used at a concentration of 100 μg/ml total IgM. Groups of 20 outbred CD1 mice were immunized by intraperitoneal (i.p.) injection of 100 μl (10 μg) of IgM mAb at 0 h, followed by i.p. challenge at 1 h with 103 colony-forming units (CFU) of S. pneumoniae serotype 2 (strain D39) and a boost of 100 ul IgM mAb 4 h later. The development of disease in mice was monitored as described previously [22], and mice were killed when they exhibited the following signs of severe disease: hunched posture, poor mobility and pilo-erection. The data were analysed using Kaplan–Meier survival curves and log rank tests. Variable region gene sequencing Heavy chain variable region genes were sequenced as described previously [17] using reverse transcription–polymerase chain reaction (RT–PCR). Each clone was sequenced a minimum of four times. Nucleotide sequence analysis was performed by imgt/v-quest[23] and imgt/JunctionAnalysis [24] (http://imgt.cines.fr). IMGT immunoglobulin gene and allele nomenclature were approved in 1999 by the Human Genome Organization (HUGO) Nomenclature Committee (HGNC) [25] and is available in imgt/gene-db[26] and in Entrez Gene at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA). Somatic mutations were analysed by comparison with the closest allele of the imgt/v-quest reference directory. Ethics Full ethical approval and informed consent was given from all participants prior to the onset of this study. Statistical analysis Correlation of antigen-binding activity between mAb panels was tested by Spearman's rho using the spss version 14 statistical software package. Fisher's exact two-tailed t-test and the χ2 test with one degree of freedom were used to compare the gene frequency of our sample with the frequency described in normal adult populations of circulating B cells [27–29]. Survival of groups of mice used for the passive protection experiments was compared using the log rank test. Population ranges for unmutated circulating IgM B cells were used where possible. Results Seven human heterohybridoma clones producing IgM mAbs binding capsular pneumococcal polysaccharides (PncPS) were generated. Their specificity and molecular characteristics were investigated. The antibodies bind to PncPS, PC and autoantigens Heterohybridomas were selected for expansion on the basis of producing IgM that bound to ELISA plates coated with seven different PncPS capsular serotypes. Following expansion, serospecificity and cross-reactivity was assessed using a combination of ELISA, luminex and immunohistochemistry assays. The antibodies (IgM mAbs) all demonstrated titrating non-serotype-restricted binding to PncPS as evaluated by ELISA and luminex assay. Representative luminex titration data are presented in Fig. 1a for one of the mAbs (DM17) in comparison to polyclonal immune sera (89SF) and a myeloma-negative control. The IgM mAbs bound to all the PncPS serotypes tested and there was a hierarchy of binding activity seen through most of the clones, with binding to PncPS type 14 being the most intense [mean fluorescence intensity (MFI): 2867 ± 1225] and binding to PncPS types 6B and 18C the weakest (mean MFI 199 ± 85·2 and mean MFI 175 ± 68·7, respectively) at a fixed total IgM concentration (Fig. 1b). This binding preference is unlikely to be attributed to differences in bead conjugation efficiency between the serotypes, because the same profile of serotype dominance was not seen in polyclonal serum IgM (89SF) at a constant serotype-specific IgM concentration, suggesting that this feature was specific to the IgM mAbs (Fig. 1c). Fig. 1 Open in new tabDownload slide Pneumococcal capsular polysaccharides (PncPS) binding studies and competitive inhibition assays. (a) Titration curves of mean fluorescent intensity (MFI) of IgM binding to seven different PncPS serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) by luminex assay. Data are shown for a representative IgM monoclonal antibody (mAb) (DM17), polyclonal standard serum (89SF) and an IgM negative control at specified total IgM concentrations [μg/ml by IgM capture enzyme-linked immunosorbent assay (ELISA)] for mAbs and at serum dilution for polyclonal serum 89SF*. (b) Hierarchy of PncPS binding of the IgM mAbs: mean PncPS serotype-specific binding activity (MFI) [±75% and ±95% confidence interval (CI)] of the seven mAbs (±75% and ±95% CI) is shown to each of seven different pneumococcal capsular polysaccharide (PncPS) serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) at a fixed concentration of total IgM (1·5 μg/ml) as assessed by luminex assay. (c) Detailed comparison of MFI of IgM binding to seven different pneumococcal capsular polysaccharide (PncPS) serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) by luminex at a fixed total IgM concentration for each IgM mAb (n = 7) and a negative control (M1) (total IgM concentration of 1·5 μg/ml) and immune serum 89SF (serotype-specific IgM concentration of 1·5 μg/ml). (d) Competitive inhibition of IgM binding to PncPS and phosphorylcholine (PC) by ELISA using saturating doses of homologous antigen: mean percentage inhibition of binding to PncPS following preincubation with saturating doses (10 μg/ml and 5 μg/ml, respectively) of homologous PncPS (▪) and cell wall polysaccharide (CWPS) (▴) relative to no preincubation is shown for IgM mAbs, polyclonal immune serum IgM (89SF) and PncPS and PC-specific class-switched mAbs (CbE2 [17] and TEPC15, respectively). Mean percentage inhibition of binding to PC (◊); following preincubation with saturating doses of PC relative to no preincubation is also shown for the IgM mAbs, polyclonal serum IgM and the PC-specific class-switched mAb (TEPC 15). All experiments were performed in duplicate a minimum of three times. (e) Titration curve showing mAb binding to PC for each of the seven mAbs as measure by ELISA OD (205 nm) with doubling dilutions of antibody at standardized total IgM concentration (10 μg/ml). Fig. 1 Open in new tabDownload slide Pneumococcal capsular polysaccharides (PncPS) binding studies and competitive inhibition assays. (a) Titration curves of mean fluorescent intensity (MFI) of IgM binding to seven different PncPS serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) by luminex assay. Data are shown for a representative IgM monoclonal antibody (mAb) (DM17), polyclonal standard serum (89SF) and an IgM negative control at specified total IgM concentrations [μg/ml by IgM capture enzyme-linked immunosorbent assay (ELISA)] for mAbs and at serum dilution for polyclonal serum 89SF*. (b) Hierarchy of PncPS binding of the IgM mAbs: mean PncPS serotype-specific binding activity (MFI) [±75% and ±95% confidence interval (CI)] of the seven mAbs (±75% and ±95% CI) is shown to each of seven different pneumococcal capsular polysaccharide (PncPS) serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) at a fixed concentration of total IgM (1·5 μg/ml) as assessed by luminex assay. (c) Detailed comparison of MFI of IgM binding to seven different pneumococcal capsular polysaccharide (PncPS) serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) by luminex at a fixed total IgM concentration for each IgM mAb (n = 7) and a negative control (M1) (total IgM concentration of 1·5 μg/ml) and immune serum 89SF (serotype-specific IgM concentration of 1·5 μg/ml). (d) Competitive inhibition of IgM binding to PncPS and phosphorylcholine (PC) by ELISA using saturating doses of homologous antigen: mean percentage inhibition of binding to PncPS following preincubation with saturating doses (10 μg/ml and 5 μg/ml, respectively) of homologous PncPS (▪) and cell wall polysaccharide (CWPS) (▴) relative to no preincubation is shown for IgM mAbs, polyclonal immune serum IgM (89SF) and PncPS and PC-specific class-switched mAbs (CbE2 [17] and TEPC15, respectively). Mean percentage inhibition of binding to PC (◊); following preincubation with saturating doses of PC relative to no preincubation is also shown for the IgM mAbs, polyclonal serum IgM and the PC-specific class-switched mAb (TEPC 15). All experiments were performed in duplicate a minimum of three times. (e) Titration curve showing mAb binding to PC for each of the seven mAbs as measure by ELISA OD (205 nm) with doubling dilutions of antibody at standardized total IgM concentration (10 μg/ml). As the mAbs were polyreactive, binding specificity to each antigen was evaluated by competitive inhibition ELISA through preincubation of the mAbs of interest with saturating doses of specific antigen. This assay is used characteristically to evaluate specificity of high-avidity class-switched antibodies. In contrast to serotype-specific IgG mAbs in which binding was inhibited almost 100% by homologous polysaccharide, preincubation of the mAbs with saturating doses of homologous PncPS did not abrogate PncPS binding significantly (Fig. 1d). Antibodies to phosphorylcholine (PC) are known to produce non-serotype-restricted binding in PncPS ELISAs due to contamination of the coating PncPS with PC containing cell wall polysaccharide (CWPS) [30]. To evaluate whether the PncPS binding could be accounted for purely on the basis of CWPS binding, PC binding was assessed directly by ELISA and indirectly by competitive inhibition assay using saturating doses of PC on PC ELISA and CWPS on PncPS ELISA. Five of the IgM mAbs bound to PC when assessed by PC ELISA (Fig. 1e), but preincubation with saturating doses of PC (200 μg/ml) or CWPS (10 μg/ml) did not abrogate binding to PC or PncPS, despite inhibiting PC-specific IgA (TEPC 15) binding to PncPS and PC (Fig. 1d). These data suggest that while PC binding may contribute to the non-serotype-restricted pneumococcal binding profiles of the IgM mAbs, the antibodies were not specific to either PC or PncPS. To characterize the breadth of polyreactivity of these IgM mAbs, the binding activity of the mAbs was then assessed by ELISA against a number of common carbohydrate antigens, including a pool of endotoxins derived from common commensal gut pathogens, the evolutionarily conserved ABO blood group antigens and the xenotransplantation antigen, Gal1,3 alpha Gal. Cell- and tissue-specific autoreactivity was also assessed by immunohistochemistry. The pattern of binding activity was analysed to determine the likelihood of common epitope recognition between clones. More distinct profiles were seen in the tissue-specific antigen-binding of the mAbs compared to the PncPS binding profiles at a fixed concentration of IgM (Fig. 2a). DM17 and Bcl showed a similar pattern of higher-intensity binding to all the antigens tested by ELISA, 6b1A7 was intermediate and AbA6, 6b5D7 and CbH4 showed relatively low-intensity binding. Only three of the mAbs (DM17, Bcl and AbA6) showed titration of binding activity to endotoxins, while six of the seven mAbs demonstrated titration of binding to the tissue-specific antigens. Two of the mAbs (AbA6 and 6b5D7), which showed almost identical binding profiles against the natural antigens when assessed by ELISA (R2 0·957, P < 0·02), also bound HEp-2 cells in a binding pattern consistent with intermediate filament protein-binding (Fig. 2b). However, no ANA binding was seen, and tissue-specific antigen binding was also negative (data not shown). Fig. 2 Open in new tabDownload slide Antibody binding to blood group antigens A and B, Gal 1,3 Gal and endotoxin as assessed by enzyme-linked immunosorbent assay (ELISA) and to cytoplasmic and nuclear antigens as assessed by immunohistochemistry. (a) IgM monoclonal antibody (mAb) binding activity to blood group antigens A and B, the xenotransplantation antigen Galα1-3Gal and a pool of four endotoxins [19] as measured by ELISA (OD 405), with doubling dilutions of antibody at standardized total IgM concentration. Titration curves for antigen binding are shown for each of the seven mAbs. (b) Anti-nuclear antibody (ANA) staining profiles for each of the seven IgM mAbs at a concentration of 10 μg/ml and ANA-positive and -negative controls on HEp-2 cells. Detection mAb: fluorescein isothiocyanate-labelled anti-Fcμ. Magnification ×400. Fig. 2 Open in new tabDownload slide Antibody binding to blood group antigens A and B, Gal 1,3 Gal and endotoxin as assessed by enzyme-linked immunosorbent assay (ELISA) and to cytoplasmic and nuclear antigens as assessed by immunohistochemistry. (a) IgM monoclonal antibody (mAb) binding activity to blood group antigens A and B, the xenotransplantation antigen Galα1-3Gal and a pool of four endotoxins [19] as measured by ELISA (OD 405), with doubling dilutions of antibody at standardized total IgM concentration. Titration curves for antigen binding are shown for each of the seven mAbs. (b) Anti-nuclear antibody (ANA) staining profiles for each of the seven IgM mAbs at a concentration of 10 μg/ml and ANA-positive and -negative controls on HEp-2 cells. Detection mAb: fluorescein isothiocyanate-labelled anti-Fcμ. Magnification ×400. Molecular analysis demonstrated that the IgM mAbs were most probably derived from B-1-like CD5+ B cell lineage While the specificity profiles we described were consistent with the IgM mAbs being natural antibodies, molecular analysis of the variable regions provided the most robust evidence that these clones were derived from the B-1-like B cell lineage which expresses CD5. The mAbs had molecular homology in terms of IGHV and IGHJ gene use and CDR3 length, with B cell clonal expansions in the healthy elderly [31] and with published CD5+ UM CLL clones bearing stereotypic BcRs [32]. [GenBank/EMBL/DDBJ Accession numbers: AF408742 (CbH4); AF408717 (6b1A7); AF408721 (6b5D7); AF408722 (6b5B12); AbA6 (EF555586); DM17 (EF555587); Bcl (EF555588)]. Apart from single nucleotide changes in the IGHD genes of DM17 and 6b5B12 and a triplet mutation in the IGHD gene of 6b5D7, there was 100% alignment of IGHV, D and J genes to the germline, indicating that the clones were expressing antibody that was essentially unmutated (Fig. 3) [25]. The antibodies used IGHV gene families IGHV3, IGHV4 and IGHV1, which are used commonly in the human repertoire (Table 1). However, the actual IGHV genes used (IGHV1-02, IGHV1-69, IGHV3-21, IGHV3-53, IGHV3-64 and IGHV4-39) are neither common in the PncPS binding repertoire of class switched antibodies [17,33] nor the unselected circulating repertoire of CD5+ or CD5– populations from healthy young adults (P < 0·05) [34]. While the V genes used were all diverse, the CDR3 regions were distinctive, showing a degree of restriction that may account for the specificity profiles presented (Fig. 3). The homology in the CDR3 regions was due largely to the dominant use of the IGHJ6 gene, which was expressed in six of the seven IgM clones but found in only one of the 11 PncPS serotype-specific class-switched clones we described previously [17], and does not dominate in the repertoire of healthy young adults [29,35] (P < 0·05). Selective use of an uncommon D gene family IGHD6 was also demonstrated, with three of the seven clones expressing IGHD6 genes, and again this was a significant bias in relation to the circulating repertoire of young adults (P < 0·001) [29,35]. Fig. 3 Open in new tabDownload slide IgM CDR3-IMGT sequences. Nucleotide sequence, alignment to germline genes, locations of N addition and amino acid translations are shown for the seven IgM clones. CDR3-IMGT encompasses positions 105–117, and the junction includes positions 104 (second CYS) and 118 (J-TRP for the IGH) according to the IMGT unique numbering [63]. Fig. 3 Open in new tabDownload slide IgM CDR3-IMGT sequences. Nucleotide sequence, alignment to germline genes, locations of N addition and amino acid translations are shown for the seven IgM clones. CDR3-IMGT encompasses positions 105–117, and the junction includes positions 104 (second CYS) and 118 (J-TRP for the IGH) according to the IMGT unique numbering [63]. Table 1 IgM hybridoma molecular characterization. IGHV, IGHD and IGHJ gene and allele use, % IGHV gene homology to germline V gene (closest allele), CDR3-IMGT length and numbers of N addition are shown. Subject . mAb . Vh gene . % gene . D gene . Jh gene . CDR3 length . N addition . A AbA6 3–53 100 6–19 6b 19 15 B DM17 3–21 100 5–5 6b 18 4 Bcl 3–21 100 3–09 6c 27 31 C* CbH4 3–64 100 6–13 6b 25 24 E* 6b1A7 4–39 100 3–22 4b 21 40 6b5D7 1–69 100 6–6 6b 14 2 6b5B12 1–02 100 1–7 6b 19 8 Subject . mAb . Vh gene . % gene . D gene . Jh gene . CDR3 length . N addition . A AbA6 3–53 100 6–19 6b 19 15 B DM17 3–21 100 5–5 6b 18 4 Bcl 3–21 100 3–09 6c 27 31 C* CbH4 3–64 100 6–13 6b 25 24 E* 6b1A7 4–39 100 3–22 4b 21 40 6b5D7 1–69 100 6–6 6b 14 2 6b5B12 1–02 100 1–7 6b 19 8 * Partial description previously [17]. mAb: monoclonal antibodies. Open in new tab Table 1 IgM hybridoma molecular characterization. IGHV, IGHD and IGHJ gene and allele use, % IGHV gene homology to germline V gene (closest allele), CDR3-IMGT length and numbers of N addition are shown. Subject . mAb . Vh gene . % gene . D gene . Jh gene . CDR3 length . N addition . A AbA6 3–53 100 6–19 6b 19 15 B DM17 3–21 100 5–5 6b 18 4 Bcl 3–21 100 3–09 6c 27 31 C* CbH4 3–64 100 6–13 6b 25 24 E* 6b1A7 4–39 100 3–22 4b 21 40 6b5D7 1–69 100 6–6 6b 14 2 6b5B12 1–02 100 1–7 6b 19 8 Subject . mAb . Vh gene . % gene . D gene . Jh gene . CDR3 length . N addition . A AbA6 3–53 100 6–19 6b 19 15 B DM17 3–21 100 5–5 6b 18 4 Bcl 3–21 100 3–09 6c 27 31 C* CbH4 3–64 100 6–13 6b 25 24 E* 6b1A7 4–39 100 3–22 4b 21 40 6b5D7 1–69 100 6–6 6b 14 2 6b5B12 1–02 100 1–7 6b 19 8 * Partial description previously [17]. mAb: monoclonal antibodies. Open in new tab The IgM mAbs all had long CDR3s (Fig. 3) (mean 20·4 ± 1·7 amino acids), significantly longer than that of PncPS serotype-specific class-switched antibodies (mean 12·7 ± 1·2 P < 0·005) [17]. The increase in CDR3 length was attributable to a combination of high levels of N addition and selective use of the IGHJ6 gene, which is 3–5 amino acids longer than other heavy chain junctional genes. Six of the seven clones used almost the entire IGHJ6 gene, with conservation of a six-amino acid stretch (YYGMDV) comprised largely of non-polar hydrophobic amino acids, making a considerable contribution to the overall characteristically strong hydrophilic composition of the CDR3 in these clones. These are the first data, as far as we are aware, to demonstrate that IGHJ6 gene use and a long CDR3 is associated significantly with PncPS binding in antibodies of IgM isotype. Natural antibody protects against invasive pneumococcal disease To evaluate the biological role of these antibodies in the context of invasive pneumococcal disease, we assessed the potential of the mAbs to provide passive protection in a murine model of Pnc septicaemia [36]. Of the seven IgMs investigated, three provided significant protection against a lethal challenge with a type 2 (D39) PncPS strain, with delayed progression of fatal infection (Fig. 4). DM17 gave the most protection, with a median time before the development of fatal infection of 52 h versus 32 h for mice given phosphate-buffered saline (PBS) alone (P < 0·005) followed by the two clones Bcl and 6b5D7 (median time of death 48 h and 40 h, respectively, P ≤ 0·005). CbH4 (Fig. 4) and AbA6 (data not shown) both showed a trend towards protection, but this did not reach statistical significance (P = 0·07 and P = 0·17, respectively). 6b5B12 and 6b1A7 did not show any evidence of protection (data not shown). Although infection was delayed, long-term survival was unaltered in all groups, suggesting that the protective effect of these IgMs against a lethal dose of systemic Pnc infection was relatively weak. Fig. 4 Open in new tabDownload slide Functional activity of monoclonal antibodies (mAbs): in vivo protection. Kaplan–Meyer survival curves following intraperitoneal challenge with 103 colony-forming units/mouse (n = 20 per group) with and without passive immunization with one of four IgM mAbs DM17, Bcl, CbH4, 6b5D7 and phosphate-buffered saline control. Fig. 4 Open in new tabDownload slide Functional activity of monoclonal antibodies (mAbs): in vivo protection. Kaplan–Meyer survival curves following intraperitoneal challenge with 103 colony-forming units/mouse (n = 20 per group) with and without passive immunization with one of four IgM mAbs DM17, Bcl, CbH4, 6b5D7 and phosphate-buffered saline control. Discussion The IgM mAbs that we have described, although generated following PncPS immunization and selected on the basis of PncPS binding, had both structural and functional features that were distinct from class-switched PncPS binding mAbs [17,37], and were characteristic of natural germline encoded polyreactive antibody derived from the poorly characterized B-1-like B cell lineage which recognizes common conformational motifs on both self- and microbial structures [38]. The broad specificity of the IgM mAbs produced by the heterohybridomas suggests that the individual antigenic determinants that these antibodies recognize was likely to be different to those for previously identified PC-specific and PncPS serotype-specific mAbs and are likely to be conformational epitopes rather than a continuous primary sequence [39,40]. The failure of the competitive inhibition ELISAs to compete out antigen binding with saturating doses of free antigen is consistent with the antibodies being of relatively low affinity, achieving high avidity through polyvalent binding to antigen. The autoreactivity profiles showing blood group antigen and intermediate filament protein binding are characteristic of the low-affinity cross-reactive natural antibodies using unmutated IgM genes that are produced by leukaemic B cell clones [41,42]. This type of antibody is seen at increased frequency in autoimmune diseases and in the elderly [43]. Unmutated, they are not considered to be pathogenic. Cross-reactivity of IgM mAbs for PncPS and blood group antigens was demonstrated nearly 40 years ago, when immunization studies demonstrated that both invasive pneumococcal disease and immunization with PncPS vaccines boosted blood group A and B antibodies [44–47]. This cross-reactivity was assumed to be attributable to molecular mimicry between Pnc carbohydrate antigens and blood group antigens [48]. Such molecular mimicry has been described most extensively for the lipopolysaccharides of Gram-negative gut pathogens such as Helicobacter pylori, which is considered to be a bacterial strategy to avoid host defence [49,50], although lectins have been described which function in much the same way as natural IgM, recognizing conformatory structures, specifically binding both blood group antigens and certain bacterial lipopolysaccharide O-antigens [51,52]. The functional advantage of the autoreactivity demonstrated by the IgM mAbs produced by these B cell clones and previously described natural antibodies is unclear. Endogenous antigen binding may be required to provide the essential survival signal in health, adequate to prevent clonal deletion [53] but not be strong enough to induce antibody production and cell cycling [54], resulting in a long-lived relatively stable population. The rise in titre of these types of antibody following immunization with PncPS and during Pnc infection [48] suggests that when these clones bind to bacterial antigens such as PncPS, antibody production and possibly cell cycling is stimulated. How that response is then regulated is likely to vary, depending on antigenic dose, the age and immune competence of the individual and the location and context in which the B-1-like B cell sees the antigen [55]. Although the functional activity of B-1-derived antibody is not a good correlate of protective immunity relative to B-2-derived antibody, we have addressed the question of whether IgM antibody of such broad specificity provides protection against invasive pneumococcal disease. A number of groups have demonstrated recently that natural antibody is important in the early control of invasive bacterial infection in mice [36,56], but there have been few studies evaluating human antibody. Three of the IgM mAbs we analysed delayed the development of invasive Pnc disease in a mouse model of septicaemia; however, in contrast to marginal zone memory-derived mutated serotype-specific IgM, which is reported to provide protective efficacy against a lethal challenge of live Pnc of up to 100% [57], long-term survival was unaltered. It is unclear whether this difference in protective efficacy is due to differences in the murine models used and bacterial virulence or whether serotype-specific mutated IgM provides superior protection against invasive Pnc disease. A head-to-head comparison would be required to address this issue. We compared the molecular phenotypes of these mAbs with class-switched mAbs showing PncPS binding [17,33] and the B cell receptor of B-1 clones reported in the literature [58–60]. The mAbs were distinct from PncPS serotype-specific mAbs at all levels, including the use of distinctive unmutated IGHV genes and the selective use of IGHJ6 genes, which were partly responsible for the tyrosine-rich long CDR3 region that is characteristic of polyreactive antibodies [61]. These data, the specificity data, suggest that the clones from which the IgM mAbs were generated were not destined to move down the B-2 developmental pathway to become PncPS serotype-specific class-switched Abs but were more similar to the B cell clones of B-1 phenotype that have been characterized most clearly in unmutated chronic lymphocytic leukaemia [62] and autoimmunity [58,59]. In summary, we have presented a group of human IgM mAbs derived from healthy young adults and selected on the basis of PncPS binding that have both the specificity and molecular features characteristic of natural antibodies binding carbohydrate antigens on endogenous and bacterial structures. Three of the antibodies provided weak but significant protection against invasive pneumococcal disease. Acknowledgements This work was supported by the Special Trustees of Great Ormond Street NHS Trust, the Wellcome Trust (Clinical Training Fellowship to H. E. B. and Project Grant to J. S. 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Natural human antibodies to pneumococcus have distinctive molecular characteristics and protect against pneumococcal disease

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Oxford University Press
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Copyright © 2022 British Society for Immunology
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0009-9104
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1365-2249
DOI
10.1111/j.1365-2249.2007.03535.x
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Abstract

Summary The molecular and functional characteristics of natural antibody from the preimmune repertoire have not been explored in detail in man. We describe seven human IgM monoclonal antibodies selected on the basis of pneumococcal polysaccharide binding that share both molecular and functional characteristics with natural antibody, suggesting a common B cell lineage origin. Unlike class-switched antibodies, which are serotype-specific, the antibodies were polyreactive and bound all pneumococcal polysaccharide capsular serotypes tested. Some bound endogenous antigens, including blood group antigens and intermediate filament proteins. All the antibodies used unmutated heavy chain V (IGHV) that are expressed at an increased frequency in the elderly and in the preimmune repertoire. The CDR3 was characterized by long length (mean aa 18·4 (±4·2) and selective use of IGHD6 (P < 0·001) and IGHJ6 (P < 0·01) family genes. The clones expressing IGHV1-69 and IGHV 3-21 provided significant passive protection against invasive pneumococcal disease in vivo. function, human, molecular, natural antibody, pneumococcus Introduction Human and murine studies have demonstrated that natural IgM contributes to the early defence against invasive disease with encapsulated bacteria [1,2], clearing bacteria from the circulation and localizing them to the marginal zone of the spleen [2]. IgM has also been shown to facilitate the induction of adaptive immune responses to vaccine antigens in mice [3], regulate B cell differentiation and selection [4,5] clear apoptotic and dying cells from the circulation [6], thereby modulating the inflammatory response to tissue injury [7], and contribute to the induction and maintenance of autoimmune disease [8,9]. The origin of this type of antibody has been best defined in mice as the B-1a B cell population that resides in the pleural and peritoneal cavities and is committed to lineage at a very early stage in B cell development [10]. In man this population is less well defined, but best described as a long-lived B cell population with low cell turnover, that expresses predominantly CD5+ and unmutated IgM [11,12], and becomes relatively more clonally restricted with age [13,14]. This population forms the preimmune repertoire of natural antibody with low-grade polyspecificity for bacterial and self-antigens [11,12]. In this study, we examined seven human heterohybridomas that were generated from B cells isolated following pneumococcal polysaccharide immunization of healthy young adults, and selected on the basis of producing monoclonal antibody (mAb) binding to pneumococcal capsular polysaccharides (PncPS) that produced IgM mAbs with both the specificity and molecular characteristics of natural antibodies. Three of the clones provided significant protection against invasive pneumococcal disease. Materials and methods Isolation and selection of hybridomas from vaccinees Human heterohybridomas were prepared as described previously [15] from lymphocytes isolated from four healthy volunteers who had been vaccinated 7 days previously with either the pneumococcal polysaccharide or conjugate vaccine (Pneumovax™ and Prevanar™). Hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) for clones producing IgM that reacted with Streptococcus pneumoniae capsular polysaccharides on standard PncPS ELISA incorporating a heptavalent antigen mix comprising pneumococcal capsular polysaccharides (PPS) serotypes 4, 6B, 9V, 14, 18C, 19F and 23F [American Type Tissue Collection (ATTC), Rockville, MD, USA]. Specificity profiling Positive wells were then screened by limiting dilution using a multiplex bead assay including Pnc serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, a standard serum control: 89SF (C. Frasch, Rockville, MD, USA) and R-phycoerythrin (RPE)-conjugated rabbit anti-human IgM/A or G (Sigma, Poole, Dorset, UK), as described previously [16]. Antibodies were preincubated with cell wall polysaccharide (CWPS) and serotype 22F polysaccharide (10 μg and 5 μg/ml) (ATTC) to eliminate non-specific binding. A concentration-matched myeloma-derived IgM negative control was used in all assays (Immunopure human IgM; Perbio Science, Cramlington, UK). Specificity was evaluated further by competitive inhibition ELISA in which the PncPS-antibody binding intensity was compared with/without antibody preincubation with 10 μg/ml homologous polysaccharide or 10 μg/ml 22F with 5 μg/ml CWPS. A serotype-specific IgG mAb, CbE2 [17], was used in homologous polysaccharide competitive inhibition ELISAs as a positive control for PncPS serotype-specific binding and a phosphorylcholine (PC)-specific murine IgA (TEPC15; Sigma, St Louis, MO, USA) was used in the CWPS competitive inhibition ELISAs as a positive control for CWPS-specific binding. Monoclonal IgM positive controls were not available for these comparisons, so serum samples from immune individuals were included in each competitive inhibition assay to evaluate whether antigen-specific PC and CWPS binding could be demonstrated in polyclonal serum IgM. The IgM Mabs were quantified using a capture ELISA in which rabbit anti-human IgM was used to coat the plate and a non-cross-reactive goat anti-human IgM was used for detection. All assays were run in duplicate and serum standards (SPS-01) and negative controls were included in each plate. To evaluate cross-reactivity to other common bacterial antigens, mAb binding to phosphorylcholine (PC) and endotoxin was assessed by ELISA in accordance with previously described methods [18,19] using fixed starting concentrations of IgM, 10 μg/ml. PC–bovine serum albumin (BSA) was provided by Biosearch Technologies (Novato, CA, USA) (PC-1011-10) and precoated endotoxin plates (precoated with an equimolar mixture of incomplete core, rough, mutant endotoxins from each of four species of Gram-negative bacteria complexed with polymyxin B) were provided by Dr G. R. Barclay (Department of Medical Microbiology, University of Edinburgh Medical School, UK). Competitive inhibition ELISA was performed for PC binding mAbs using saturating doses of PC (200 μg/ml) as described for PncPS ELISAs. A serum control and a PC-specific mAb, TEPC 15, was included in the assay. Autoantigen binding As the antibodies were not serotype-specific, the binding activity against the natural antigen blood groups A and B and the xenotransplantation antigen Galα1–3Gal was assessed by ELISAs, performed in accordance with previously described methods [20,21], using fixed starting concentrations of IgM (10 μg/ml). A and B polyacrylamide-conjugated antigens (PAA-Bdi and PAA-Atri) were provided by Lectinity Inc. (Moscow, Russia). Galα1,3Gal polyacrylamide-conjugated antigens (PAA-Bdi, PAA-Atri and PAA-Btri) were provided by Lectinity Inc. Cross-reactivity to cellular endogenous antigens was evaluated by indirect immunofluorescence using HEp-2 (Biodiagnostics, Upton-upon-Severn, UK) and anti-nuclear antibody (ANA) [liver, kidney and stomach sections (LKS)] slides (Biodiagnostics), according to the manufacturer's instructions. In vivo passive protection IgM mAbs were used at a concentration of 100 μg/ml total IgM. Groups of 20 outbred CD1 mice were immunized by intraperitoneal (i.p.) injection of 100 μl (10 μg) of IgM mAb at 0 h, followed by i.p. challenge at 1 h with 103 colony-forming units (CFU) of S. pneumoniae serotype 2 (strain D39) and a boost of 100 ul IgM mAb 4 h later. The development of disease in mice was monitored as described previously [22], and mice were killed when they exhibited the following signs of severe disease: hunched posture, poor mobility and pilo-erection. The data were analysed using Kaplan–Meier survival curves and log rank tests. Variable region gene sequencing Heavy chain variable region genes were sequenced as described previously [17] using reverse transcription–polymerase chain reaction (RT–PCR). Each clone was sequenced a minimum of four times. Nucleotide sequence analysis was performed by imgt/v-quest[23] and imgt/JunctionAnalysis [24] (http://imgt.cines.fr). IMGT immunoglobulin gene and allele nomenclature were approved in 1999 by the Human Genome Organization (HUGO) Nomenclature Committee (HGNC) [25] and is available in imgt/gene-db[26] and in Entrez Gene at the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA). Somatic mutations were analysed by comparison with the closest allele of the imgt/v-quest reference directory. Ethics Full ethical approval and informed consent was given from all participants prior to the onset of this study. Statistical analysis Correlation of antigen-binding activity between mAb panels was tested by Spearman's rho using the spss version 14 statistical software package. Fisher's exact two-tailed t-test and the χ2 test with one degree of freedom were used to compare the gene frequency of our sample with the frequency described in normal adult populations of circulating B cells [27–29]. Survival of groups of mice used for the passive protection experiments was compared using the log rank test. Population ranges for unmutated circulating IgM B cells were used where possible. Results Seven human heterohybridoma clones producing IgM mAbs binding capsular pneumococcal polysaccharides (PncPS) were generated. Their specificity and molecular characteristics were investigated. The antibodies bind to PncPS, PC and autoantigens Heterohybridomas were selected for expansion on the basis of producing IgM that bound to ELISA plates coated with seven different PncPS capsular serotypes. Following expansion, serospecificity and cross-reactivity was assessed using a combination of ELISA, luminex and immunohistochemistry assays. The antibodies (IgM mAbs) all demonstrated titrating non-serotype-restricted binding to PncPS as evaluated by ELISA and luminex assay. Representative luminex titration data are presented in Fig. 1a for one of the mAbs (DM17) in comparison to polyclonal immune sera (89SF) and a myeloma-negative control. The IgM mAbs bound to all the PncPS serotypes tested and there was a hierarchy of binding activity seen through most of the clones, with binding to PncPS type 14 being the most intense [mean fluorescence intensity (MFI): 2867 ± 1225] and binding to PncPS types 6B and 18C the weakest (mean MFI 199 ± 85·2 and mean MFI 175 ± 68·7, respectively) at a fixed total IgM concentration (Fig. 1b). This binding preference is unlikely to be attributed to differences in bead conjugation efficiency between the serotypes, because the same profile of serotype dominance was not seen in polyclonal serum IgM (89SF) at a constant serotype-specific IgM concentration, suggesting that this feature was specific to the IgM mAbs (Fig. 1c). Fig. 1 Open in new tabDownload slide Pneumococcal capsular polysaccharides (PncPS) binding studies and competitive inhibition assays. (a) Titration curves of mean fluorescent intensity (MFI) of IgM binding to seven different PncPS serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) by luminex assay. Data are shown for a representative IgM monoclonal antibody (mAb) (DM17), polyclonal standard serum (89SF) and an IgM negative control at specified total IgM concentrations [μg/ml by IgM capture enzyme-linked immunosorbent assay (ELISA)] for mAbs and at serum dilution for polyclonal serum 89SF*. (b) Hierarchy of PncPS binding of the IgM mAbs: mean PncPS serotype-specific binding activity (MFI) [±75% and ±95% confidence interval (CI)] of the seven mAbs (±75% and ±95% CI) is shown to each of seven different pneumococcal capsular polysaccharide (PncPS) serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) at a fixed concentration of total IgM (1·5 μg/ml) as assessed by luminex assay. (c) Detailed comparison of MFI of IgM binding to seven different pneumococcal capsular polysaccharide (PncPS) serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) by luminex at a fixed total IgM concentration for each IgM mAb (n = 7) and a negative control (M1) (total IgM concentration of 1·5 μg/ml) and immune serum 89SF (serotype-specific IgM concentration of 1·5 μg/ml). (d) Competitive inhibition of IgM binding to PncPS and phosphorylcholine (PC) by ELISA using saturating doses of homologous antigen: mean percentage inhibition of binding to PncPS following preincubation with saturating doses (10 μg/ml and 5 μg/ml, respectively) of homologous PncPS (▪) and cell wall polysaccharide (CWPS) (▴) relative to no preincubation is shown for IgM mAbs, polyclonal immune serum IgM (89SF) and PncPS and PC-specific class-switched mAbs (CbE2 [17] and TEPC15, respectively). Mean percentage inhibition of binding to PC (◊); following preincubation with saturating doses of PC relative to no preincubation is also shown for the IgM mAbs, polyclonal serum IgM and the PC-specific class-switched mAb (TEPC 15). All experiments were performed in duplicate a minimum of three times. (e) Titration curve showing mAb binding to PC for each of the seven mAbs as measure by ELISA OD (205 nm) with doubling dilutions of antibody at standardized total IgM concentration (10 μg/ml). Fig. 1 Open in new tabDownload slide Pneumococcal capsular polysaccharides (PncPS) binding studies and competitive inhibition assays. (a) Titration curves of mean fluorescent intensity (MFI) of IgM binding to seven different PncPS serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) by luminex assay. Data are shown for a representative IgM monoclonal antibody (mAb) (DM17), polyclonal standard serum (89SF) and an IgM negative control at specified total IgM concentrations [μg/ml by IgM capture enzyme-linked immunosorbent assay (ELISA)] for mAbs and at serum dilution for polyclonal serum 89SF*. (b) Hierarchy of PncPS binding of the IgM mAbs: mean PncPS serotype-specific binding activity (MFI) [±75% and ±95% confidence interval (CI)] of the seven mAbs (±75% and ±95% CI) is shown to each of seven different pneumococcal capsular polysaccharide (PncPS) serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) at a fixed concentration of total IgM (1·5 μg/ml) as assessed by luminex assay. (c) Detailed comparison of MFI of IgM binding to seven different pneumococcal capsular polysaccharide (PncPS) serotypes (4; 6B; 9V; 14; 18C; 19F; 23F) by luminex at a fixed total IgM concentration for each IgM mAb (n = 7) and a negative control (M1) (total IgM concentration of 1·5 μg/ml) and immune serum 89SF (serotype-specific IgM concentration of 1·5 μg/ml). (d) Competitive inhibition of IgM binding to PncPS and phosphorylcholine (PC) by ELISA using saturating doses of homologous antigen: mean percentage inhibition of binding to PncPS following preincubation with saturating doses (10 μg/ml and 5 μg/ml, respectively) of homologous PncPS (▪) and cell wall polysaccharide (CWPS) (▴) relative to no preincubation is shown for IgM mAbs, polyclonal immune serum IgM (89SF) and PncPS and PC-specific class-switched mAbs (CbE2 [17] and TEPC15, respectively). Mean percentage inhibition of binding to PC (◊); following preincubation with saturating doses of PC relative to no preincubation is also shown for the IgM mAbs, polyclonal serum IgM and the PC-specific class-switched mAb (TEPC 15). All experiments were performed in duplicate a minimum of three times. (e) Titration curve showing mAb binding to PC for each of the seven mAbs as measure by ELISA OD (205 nm) with doubling dilutions of antibody at standardized total IgM concentration (10 μg/ml). As the mAbs were polyreactive, binding specificity to each antigen was evaluated by competitive inhibition ELISA through preincubation of the mAbs of interest with saturating doses of specific antigen. This assay is used characteristically to evaluate specificity of high-avidity class-switched antibodies. In contrast to serotype-specific IgG mAbs in which binding was inhibited almost 100% by homologous polysaccharide, preincubation of the mAbs with saturating doses of homologous PncPS did not abrogate PncPS binding significantly (Fig. 1d). Antibodies to phosphorylcholine (PC) are known to produce non-serotype-restricted binding in PncPS ELISAs due to contamination of the coating PncPS with PC containing cell wall polysaccharide (CWPS) [30]. To evaluate whether the PncPS binding could be accounted for purely on the basis of CWPS binding, PC binding was assessed directly by ELISA and indirectly by competitive inhibition assay using saturating doses of PC on PC ELISA and CWPS on PncPS ELISA. Five of the IgM mAbs bound to PC when assessed by PC ELISA (Fig. 1e), but preincubation with saturating doses of PC (200 μg/ml) or CWPS (10 μg/ml) did not abrogate binding to PC or PncPS, despite inhibiting PC-specific IgA (TEPC 15) binding to PncPS and PC (Fig. 1d). These data suggest that while PC binding may contribute to the non-serotype-restricted pneumococcal binding profiles of the IgM mAbs, the antibodies were not specific to either PC or PncPS. To characterize the breadth of polyreactivity of these IgM mAbs, the binding activity of the mAbs was then assessed by ELISA against a number of common carbohydrate antigens, including a pool of endotoxins derived from common commensal gut pathogens, the evolutionarily conserved ABO blood group antigens and the xenotransplantation antigen, Gal1,3 alpha Gal. Cell- and tissue-specific autoreactivity was also assessed by immunohistochemistry. The pattern of binding activity was analysed to determine the likelihood of common epitope recognition between clones. More distinct profiles were seen in the tissue-specific antigen-binding of the mAbs compared to the PncPS binding profiles at a fixed concentration of IgM (Fig. 2a). DM17 and Bcl showed a similar pattern of higher-intensity binding to all the antigens tested by ELISA, 6b1A7 was intermediate and AbA6, 6b5D7 and CbH4 showed relatively low-intensity binding. Only three of the mAbs (DM17, Bcl and AbA6) showed titration of binding activity to endotoxins, while six of the seven mAbs demonstrated titration of binding to the tissue-specific antigens. Two of the mAbs (AbA6 and 6b5D7), which showed almost identical binding profiles against the natural antigens when assessed by ELISA (R2 0·957, P < 0·02), also bound HEp-2 cells in a binding pattern consistent with intermediate filament protein-binding (Fig. 2b). However, no ANA binding was seen, and tissue-specific antigen binding was also negative (data not shown). Fig. 2 Open in new tabDownload slide Antibody binding to blood group antigens A and B, Gal 1,3 Gal and endotoxin as assessed by enzyme-linked immunosorbent assay (ELISA) and to cytoplasmic and nuclear antigens as assessed by immunohistochemistry. (a) IgM monoclonal antibody (mAb) binding activity to blood group antigens A and B, the xenotransplantation antigen Galα1-3Gal and a pool of four endotoxins [19] as measured by ELISA (OD 405), with doubling dilutions of antibody at standardized total IgM concentration. Titration curves for antigen binding are shown for each of the seven mAbs. (b) Anti-nuclear antibody (ANA) staining profiles for each of the seven IgM mAbs at a concentration of 10 μg/ml and ANA-positive and -negative controls on HEp-2 cells. Detection mAb: fluorescein isothiocyanate-labelled anti-Fcμ. Magnification ×400. Fig. 2 Open in new tabDownload slide Antibody binding to blood group antigens A and B, Gal 1,3 Gal and endotoxin as assessed by enzyme-linked immunosorbent assay (ELISA) and to cytoplasmic and nuclear antigens as assessed by immunohistochemistry. (a) IgM monoclonal antibody (mAb) binding activity to blood group antigens A and B, the xenotransplantation antigen Galα1-3Gal and a pool of four endotoxins [19] as measured by ELISA (OD 405), with doubling dilutions of antibody at standardized total IgM concentration. Titration curves for antigen binding are shown for each of the seven mAbs. (b) Anti-nuclear antibody (ANA) staining profiles for each of the seven IgM mAbs at a concentration of 10 μg/ml and ANA-positive and -negative controls on HEp-2 cells. Detection mAb: fluorescein isothiocyanate-labelled anti-Fcμ. Magnification ×400. Molecular analysis demonstrated that the IgM mAbs were most probably derived from B-1-like CD5+ B cell lineage While the specificity profiles we described were consistent with the IgM mAbs being natural antibodies, molecular analysis of the variable regions provided the most robust evidence that these clones were derived from the B-1-like B cell lineage which expresses CD5. The mAbs had molecular homology in terms of IGHV and IGHJ gene use and CDR3 length, with B cell clonal expansions in the healthy elderly [31] and with published CD5+ UM CLL clones bearing stereotypic BcRs [32]. [GenBank/EMBL/DDBJ Accession numbers: AF408742 (CbH4); AF408717 (6b1A7); AF408721 (6b5D7); AF408722 (6b5B12); AbA6 (EF555586); DM17 (EF555587); Bcl (EF555588)]. Apart from single nucleotide changes in the IGHD genes of DM17 and 6b5B12 and a triplet mutation in the IGHD gene of 6b5D7, there was 100% alignment of IGHV, D and J genes to the germline, indicating that the clones were expressing antibody that was essentially unmutated (Fig. 3) [25]. The antibodies used IGHV gene families IGHV3, IGHV4 and IGHV1, which are used commonly in the human repertoire (Table 1). However, the actual IGHV genes used (IGHV1-02, IGHV1-69, IGHV3-21, IGHV3-53, IGHV3-64 and IGHV4-39) are neither common in the PncPS binding repertoire of class switched antibodies [17,33] nor the unselected circulating repertoire of CD5+ or CD5– populations from healthy young adults (P < 0·05) [34]. While the V genes used were all diverse, the CDR3 regions were distinctive, showing a degree of restriction that may account for the specificity profiles presented (Fig. 3). The homology in the CDR3 regions was due largely to the dominant use of the IGHJ6 gene, which was expressed in six of the seven IgM clones but found in only one of the 11 PncPS serotype-specific class-switched clones we described previously [17], and does not dominate in the repertoire of healthy young adults [29,35] (P < 0·05). Selective use of an uncommon D gene family IGHD6 was also demonstrated, with three of the seven clones expressing IGHD6 genes, and again this was a significant bias in relation to the circulating repertoire of young adults (P < 0·001) [29,35]. Fig. 3 Open in new tabDownload slide IgM CDR3-IMGT sequences. Nucleotide sequence, alignment to germline genes, locations of N addition and amino acid translations are shown for the seven IgM clones. CDR3-IMGT encompasses positions 105–117, and the junction includes positions 104 (second CYS) and 118 (J-TRP for the IGH) according to the IMGT unique numbering [63]. Fig. 3 Open in new tabDownload slide IgM CDR3-IMGT sequences. Nucleotide sequence, alignment to germline genes, locations of N addition and amino acid translations are shown for the seven IgM clones. CDR3-IMGT encompasses positions 105–117, and the junction includes positions 104 (second CYS) and 118 (J-TRP for the IGH) according to the IMGT unique numbering [63]. Table 1 IgM hybridoma molecular characterization. IGHV, IGHD and IGHJ gene and allele use, % IGHV gene homology to germline V gene (closest allele), CDR3-IMGT length and numbers of N addition are shown. Subject . mAb . Vh gene . % gene . D gene . Jh gene . CDR3 length . N addition . A AbA6 3–53 100 6–19 6b 19 15 B DM17 3–21 100 5–5 6b 18 4 Bcl 3–21 100 3–09 6c 27 31 C* CbH4 3–64 100 6–13 6b 25 24 E* 6b1A7 4–39 100 3–22 4b 21 40 6b5D7 1–69 100 6–6 6b 14 2 6b5B12 1–02 100 1–7 6b 19 8 Subject . mAb . Vh gene . % gene . D gene . Jh gene . CDR3 length . N addition . A AbA6 3–53 100 6–19 6b 19 15 B DM17 3–21 100 5–5 6b 18 4 Bcl 3–21 100 3–09 6c 27 31 C* CbH4 3–64 100 6–13 6b 25 24 E* 6b1A7 4–39 100 3–22 4b 21 40 6b5D7 1–69 100 6–6 6b 14 2 6b5B12 1–02 100 1–7 6b 19 8 * Partial description previously [17]. mAb: monoclonal antibodies. Open in new tab Table 1 IgM hybridoma molecular characterization. IGHV, IGHD and IGHJ gene and allele use, % IGHV gene homology to germline V gene (closest allele), CDR3-IMGT length and numbers of N addition are shown. Subject . mAb . Vh gene . % gene . D gene . Jh gene . CDR3 length . N addition . A AbA6 3–53 100 6–19 6b 19 15 B DM17 3–21 100 5–5 6b 18 4 Bcl 3–21 100 3–09 6c 27 31 C* CbH4 3–64 100 6–13 6b 25 24 E* 6b1A7 4–39 100 3–22 4b 21 40 6b5D7 1–69 100 6–6 6b 14 2 6b5B12 1–02 100 1–7 6b 19 8 Subject . mAb . Vh gene . % gene . D gene . Jh gene . CDR3 length . N addition . A AbA6 3–53 100 6–19 6b 19 15 B DM17 3–21 100 5–5 6b 18 4 Bcl 3–21 100 3–09 6c 27 31 C* CbH4 3–64 100 6–13 6b 25 24 E* 6b1A7 4–39 100 3–22 4b 21 40 6b5D7 1–69 100 6–6 6b 14 2 6b5B12 1–02 100 1–7 6b 19 8 * Partial description previously [17]. mAb: monoclonal antibodies. Open in new tab The IgM mAbs all had long CDR3s (Fig. 3) (mean 20·4 ± 1·7 amino acids), significantly longer than that of PncPS serotype-specific class-switched antibodies (mean 12·7 ± 1·2 P < 0·005) [17]. The increase in CDR3 length was attributable to a combination of high levels of N addition and selective use of the IGHJ6 gene, which is 3–5 amino acids longer than other heavy chain junctional genes. Six of the seven clones used almost the entire IGHJ6 gene, with conservation of a six-amino acid stretch (YYGMDV) comprised largely of non-polar hydrophobic amino acids, making a considerable contribution to the overall characteristically strong hydrophilic composition of the CDR3 in these clones. These are the first data, as far as we are aware, to demonstrate that IGHJ6 gene use and a long CDR3 is associated significantly with PncPS binding in antibodies of IgM isotype. Natural antibody protects against invasive pneumococcal disease To evaluate the biological role of these antibodies in the context of invasive pneumococcal disease, we assessed the potential of the mAbs to provide passive protection in a murine model of Pnc septicaemia [36]. Of the seven IgMs investigated, three provided significant protection against a lethal challenge with a type 2 (D39) PncPS strain, with delayed progression of fatal infection (Fig. 4). DM17 gave the most protection, with a median time before the development of fatal infection of 52 h versus 32 h for mice given phosphate-buffered saline (PBS) alone (P < 0·005) followed by the two clones Bcl and 6b5D7 (median time of death 48 h and 40 h, respectively, P ≤ 0·005). CbH4 (Fig. 4) and AbA6 (data not shown) both showed a trend towards protection, but this did not reach statistical significance (P = 0·07 and P = 0·17, respectively). 6b5B12 and 6b1A7 did not show any evidence of protection (data not shown). Although infection was delayed, long-term survival was unaltered in all groups, suggesting that the protective effect of these IgMs against a lethal dose of systemic Pnc infection was relatively weak. Fig. 4 Open in new tabDownload slide Functional activity of monoclonal antibodies (mAbs): in vivo protection. Kaplan–Meyer survival curves following intraperitoneal challenge with 103 colony-forming units/mouse (n = 20 per group) with and without passive immunization with one of four IgM mAbs DM17, Bcl, CbH4, 6b5D7 and phosphate-buffered saline control. Fig. 4 Open in new tabDownload slide Functional activity of monoclonal antibodies (mAbs): in vivo protection. Kaplan–Meyer survival curves following intraperitoneal challenge with 103 colony-forming units/mouse (n = 20 per group) with and without passive immunization with one of four IgM mAbs DM17, Bcl, CbH4, 6b5D7 and phosphate-buffered saline control. Discussion The IgM mAbs that we have described, although generated following PncPS immunization and selected on the basis of PncPS binding, had both structural and functional features that were distinct from class-switched PncPS binding mAbs [17,37], and were characteristic of natural germline encoded polyreactive antibody derived from the poorly characterized B-1-like B cell lineage which recognizes common conformational motifs on both self- and microbial structures [38]. The broad specificity of the IgM mAbs produced by the heterohybridomas suggests that the individual antigenic determinants that these antibodies recognize was likely to be different to those for previously identified PC-specific and PncPS serotype-specific mAbs and are likely to be conformational epitopes rather than a continuous primary sequence [39,40]. The failure of the competitive inhibition ELISAs to compete out antigen binding with saturating doses of free antigen is consistent with the antibodies being of relatively low affinity, achieving high avidity through polyvalent binding to antigen. The autoreactivity profiles showing blood group antigen and intermediate filament protein binding are characteristic of the low-affinity cross-reactive natural antibodies using unmutated IgM genes that are produced by leukaemic B cell clones [41,42]. This type of antibody is seen at increased frequency in autoimmune diseases and in the elderly [43]. Unmutated, they are not considered to be pathogenic. Cross-reactivity of IgM mAbs for PncPS and blood group antigens was demonstrated nearly 40 years ago, when immunization studies demonstrated that both invasive pneumococcal disease and immunization with PncPS vaccines boosted blood group A and B antibodies [44–47]. This cross-reactivity was assumed to be attributable to molecular mimicry between Pnc carbohydrate antigens and blood group antigens [48]. Such molecular mimicry has been described most extensively for the lipopolysaccharides of Gram-negative gut pathogens such as Helicobacter pylori, which is considered to be a bacterial strategy to avoid host defence [49,50], although lectins have been described which function in much the same way as natural IgM, recognizing conformatory structures, specifically binding both blood group antigens and certain bacterial lipopolysaccharide O-antigens [51,52]. The functional advantage of the autoreactivity demonstrated by the IgM mAbs produced by these B cell clones and previously described natural antibodies is unclear. Endogenous antigen binding may be required to provide the essential survival signal in health, adequate to prevent clonal deletion [53] but not be strong enough to induce antibody production and cell cycling [54], resulting in a long-lived relatively stable population. The rise in titre of these types of antibody following immunization with PncPS and during Pnc infection [48] suggests that when these clones bind to bacterial antigens such as PncPS, antibody production and possibly cell cycling is stimulated. How that response is then regulated is likely to vary, depending on antigenic dose, the age and immune competence of the individual and the location and context in which the B-1-like B cell sees the antigen [55]. Although the functional activity of B-1-derived antibody is not a good correlate of protective immunity relative to B-2-derived antibody, we have addressed the question of whether IgM antibody of such broad specificity provides protection against invasive pneumococcal disease. A number of groups have demonstrated recently that natural antibody is important in the early control of invasive bacterial infection in mice [36,56], but there have been few studies evaluating human antibody. Three of the IgM mAbs we analysed delayed the development of invasive Pnc disease in a mouse model of septicaemia; however, in contrast to marginal zone memory-derived mutated serotype-specific IgM, which is reported to provide protective efficacy against a lethal challenge of live Pnc of up to 100% [57], long-term survival was unaltered. It is unclear whether this difference in protective efficacy is due to differences in the murine models used and bacterial virulence or whether serotype-specific mutated IgM provides superior protection against invasive Pnc disease. A head-to-head comparison would be required to address this issue. We compared the molecular phenotypes of these mAbs with class-switched mAbs showing PncPS binding [17,33] and the B cell receptor of B-1 clones reported in the literature [58–60]. The mAbs were distinct from PncPS serotype-specific mAbs at all levels, including the use of distinctive unmutated IGHV genes and the selective use of IGHJ6 genes, which were partly responsible for the tyrosine-rich long CDR3 region that is characteristic of polyreactive antibodies [61]. These data, the specificity data, suggest that the clones from which the IgM mAbs were generated were not destined to move down the B-2 developmental pathway to become PncPS serotype-specific class-switched Abs but were more similar to the B cell clones of B-1 phenotype that have been characterized most clearly in unmutated chronic lymphocytic leukaemia [62] and autoimmunity [58,59]. In summary, we have presented a group of human IgM mAbs derived from healthy young adults and selected on the basis of PncPS binding that have both the specificity and molecular features characteristic of natural antibodies binding carbohydrate antigens on endogenous and bacterial structures. Three of the antibodies provided weak but significant protection against invasive pneumococcal disease. Acknowledgements This work was supported by the Special Trustees of Great Ormond Street NHS Trust, the Wellcome Trust (Clinical Training Fellowship to H. E. B. and Project Grant to J. S. 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Journal

Clinical & Experimental ImmunologyOxford University Press

Published: Dec 7, 2007

Keywords: streptococcus pneumoniae; immunoglobulin m; antibodies; clone cells; genes; serotype; b-lymphocytes; antigens; invasive pneumococcal disease

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