Somatic diversity in CDR3 loops allows single V-genes to encode innate immunological memories for multiple pathogens.

The human Ab response to many common pathogens is oligoclonal, with restricted usage of Ig V-genes. Intriguingly, the IGVK3-11 and IGVH3-30 V-genes are repeatedly paired in protective Abs against the 23F polysaccharide of Streptococcus pneumoniae, as well as against the gB envelope protein of human CMV, where germline-encoded amino acids make key contacts with the gB protein. We constructed IgGs encoded by the germline IGVK3-11 and IGVH3-30 V-genes together with DNA encoding the respective CDR3 regions of the L chain and H chain found in a hypermutated anti-23F Ab. These IgGs encoded by germline V-genes bound specifically to 23F pneumococcal capsular polysaccharides with no reactivity to other serotypes of pneumococcal capsular polysaccharides or arrayed glycans and recognized L-rhamnose, a component of the 23F repeating subunit. IgGs encoded by this pair of germline V-genes mediated complement-dependent phagocytosis of encapsulated 23F S. pneumoniae by human neutrophils. Mutations in CDRL3 and CDRH3 had significant effects on binding. Thus, IGKV3-11 and IGHV3-30, depending on with which distinct DNA sequences encoding CDR3 they are recombined, can encode binding sites for protective Abs against chemically distinct Ags and thus, may encode innate immunological memory against human CMV and S. pneumoniae.

Combined roles of human IgG subclass, alternative complement pathway activation, and epitope density in the bactericidal activity of antibodies to meningococcal factor h binding protein.

Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.

Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

Paratope diversity in the human antibody response to Bacillus anthracis protective antigen.

The active component of the licensed human anthrax vaccine (BioThrax, or AVA) is a Bacillus anthracis toxin known as protective antigen (PA). Second generation anthrax vaccines currently under development are also based on a recombinant form of PA. Since the current and future anthrax vaccines are based on this toxin, it is important that the immunobiology of this protein in vaccinated humans be understood in detail. We have isolated and analyzed the PA-specific antibody repertoire from an AVA-vaccinated individual. When examined at the clonal level, we find an antibody response that is complex in terms of the combinatorial elements and immunoglobulin variable genes employed. All PA-specific antibodies had undergone somatic hypermutation and class switch recombination, both signs of affinity maturation. Although the antigenic epitopes recognized by the response were distributed throughout the PA monomer, the majority of antibodies arising in this individual following vaccination recognize determinants located on the amino-terminal (PA20) sub-domain of the molecule. This latter finding may have implications for the rational design of future PA-based anthrax vaccines.

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity.

BACKGROUND: Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity. METHODS AND PRINCIPAL FINDINGS: Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity. CONCLUSIONS: The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.

A broadly cross-reactive monoclonal antibody against an epitope on the n-terminus of meningococcal fHbp.

Meningococcal factor H binding protein (fHbp) is an important vaccine antigen for prevention of disease caused by capsular group B strains. The protein has been sub-classified into three variant groups. Most anti-fHbp antibodies are variant group-specific and recognize epitopes on the C-terminal domain. We report a murine IgG1 mAb, JAR 41, which broadly cross-reacted with fHbp sequence variants from all variant groups. The mAb bound to the surface of live meningococci with fHbp from each of the three variant groups. In combination with second non-bactericidal anti-fHbp mAbs, JAR 41 elicited complement-mediated bactericidal activity in vitro, and augmented passive protection against meningococcal bacteremia in human fH transgenic rats. The epitope was located on a conserved region of the N-terminal portion of the fHbp molecule opposite that of fH contact residues. The data underscore the importance of broadly cross-reactive, surface-exposed epitopes on the N-terminal domain in the design of protective fHbp vaccines.

Domain specificity of the human antibody response to Bacillus anthracis protective antigen.

Protective antigen (PA) is the cell surface recognition moiety of the Bacillus anthracis A-B toxin system, and the active immunogenic component in the currently licensed human anthrax vaccine (BioThrax, or AVA). The serum antibody response to the PA protein is polyclonal and complex both in terms of the antibody combining sites utilized to bind PA and the PA-associated epitopes recognized. We have cloned, sequenced, and expressed a large panel of PA-specific human monoclonal antibodies from seven AVA-immunized donors. Dot blots, Western blots, and radiolabeled antigen capture assays employing both proteolytic fragments of PA and engineered PA sub-domain fusion proteins were used to determine the region (domain) of the PA monomer to which each of the cloned human antibodies bound. The domain specificity of the isolated monoclonals was highly biased towards the amino-terminal 20kDa fragment of PA (PA(20)), with the majority (62%) of independently arising antibody clones reacting with determinants located on this PA fragment. A similar bias in domain specificity was also demonstrated in the serum response of AVA-vaccinated donors. Since PA(20) is cleaved from the remainder of the monomer rapidly following cell surface binding and has no known role in the intoxication process, the immunodominance of PA(20)-associated epitopes may directly affect the efficacy of PA-based anthrax vaccines.

Codon insertion and deletion functions as a somatic diversification mechanism in human antibody repertoires.

UNLABELLED: It has been suggested that codon insertion and/or deletion may represent a mechanism that, along with hypermutation, contributes to the affinity maturation of antibodies. We used repertoire cloning to examine human antibodies directed against 3 carbohydrate antigens and 1 protein antigen for the presence of such modifications. We find that both the insertion and deletion of codons occur frequently in antigen-specific responses following vaccination. Codon insertions and deletions were observed most often in the complementarity determining regions, and less frequently in the framework regions, of VH, Vkappa, and Vlambda gene segments, and involved motifs known to be preferred targets of somatic hypermutation. Clonal lineage analysis shows that these events occur through out the course of the somatic maturation of individual antibody clones. We also determined that these alterations of paratope structure have varying effects on the relative affinity of the binding site for its cognate antigen. REVIEWERS: This article was reviewed by Mark Shlomchik, Deborah Dunn-Walters (nominated by Dr. Andrew Macpherson), and Rachel M. Gerstein. OPEN PEER REVIEW: Reviewed by Mark Shlomchik, Deborah Dunn-Walters (nominated by Dr. Andrew Macpherson), and Rachel M. Gerstein. For the full reviews, please go to the Reviewers' comments section.

Correlation of antigenic epitope and antibody gene usage in the human immune response to Streptococcus pneumoniae type 23F capsular polysaccharide.

The human antibody response to the capsular polysaccharide of Streptococcus pneumoniae type 23F is predominated by antibodies which express either VkappaL6 or VkappaA23 light chain genes. In this report, we demonstrate that the antigenic epitopes recognized by these two families can be differentiated based on their specificity for the hapten l-rhamnose, a constituent sugar of the bacterial capsule polymer. The VkappaL6 family utilizes light chains with an unusually long third complementarity-determining region. These VkappaL6-encoded Fabs are 100-fold more sensitive to inhibition with l-rhamnose than VkappaA23 Fabs. VkappaL6 Fabs preferentially recognize the l-isomer of the hapten and can distinguish l-rhamnose from the structurally similar sugar l-mannose. We also demonstrate that the VkappaL6 family component of the antibody response recognizes the polysaccharide antigens of Group B streptococci (GBS), thereby accounting for the previously reported cross-reaction between PPS 23F-specific antisera and Group B streptococci.

Somatic hypermutation and diverse immunoglobulin gene usage in the human antibody response to the capsular polysaccharide of Streptococcus pneumoniae Type 6B.

Combinatorial cloning and expression library analysis were used to determine the expressed human antibody repertoire specific for the capsular polysaccharide (PS) of Streptococcus pneumoniae serotype 6B. Sequence analysis of 55 6B-specific antibody Fab fragments isolated from six vaccinated donors reveal that different individuals used a variety of heavy and light chain germ line variable (V) region genes to form pneumococcal capsular PS (PPS) 6B-specific paratopes. Within each donor, however, the response was more restricted, with five of the six donors using at most one or two gene pairs to form combining sites. Analysis also indicated that although the response in each donor was oligoclonal in terms of variable gene usage, the combination of extensive somatic hypermutation, deletion of germ line-encoded residues, insertion of non-germ line-encoded residues, and intraclonal isotype switching generated a surprising degree of paratope diversity within the individuals analyzed. In contrast to previously studied PS-specific responses, we find that the PPS 6B repertoire makes use of a diverse collection of heavy-chain and light-chain V region gene products to form specific paratopes, with no apparent tendency for conservation of immunoglobulin gene usage between individuals.

Recurrent variable region gene usage and somatic mutation in the human antibody response to the capsular polysaccharide of Streptococcus pneumoniae type 23F.

Combinatorial cloning and expression library analysis were used to isolate human antibody Fab fragments specific for the capsular polysaccharide of Streptococcus pneumoniae serotype 23F. Thirty 23F-specific Fabs were isolated from seven vaccinated donors, and the sequences of the heavy (H)- and light (L)-chain variable regions were determined. All individuals utilized either the Vkappa A23 L chain, the Vkappa L6 L chain, or both chains in forming the 23F-specific combining site. Vkappa A23 L chains paired primarily with VH3-23 H chains. Vkappa L6 L chains were more promiscuous in heavy-chain usage between individuals. Both H and L chains were mutated, primarily in the complementarity-determining regions, compared to their closest germ line counterpart, suggesting a recall response that has undergone affinity maturation. H-chain isotypes were reflective of those found in the serum. Shared somatic modifications demonstrated that immunoglobulin G2 (IgG2) and IgA antibodies arose from the same somatically matured B cell. Our results indicate that the response to the serotype 23F pneumococcal capsular polysaccharide is oligoclonal within the individual, with one or two paratope families accounting for the majority of expressed antibody. We also determined that, in spite of the combinatorial diversity available to the immune system, the 23F-specific response is highly restricted at the population level, with the same two L-chain-determined paratope families recurring in all individuals. Lastly, analysis of the isolated Fabs indicate all have undergone extensive somatic mutation, as well as class switch, maturational events that presumably require the participation of T cells.

Molecular ontogeny of the human antibody repertoire to the Haemophilus influenzae type B polysaccharide: expression of canonical variable regions and their variants in vaccinated infants.

A structurally conserved antibody combining site, encoded by the IGH V3-23 and kappa A2 variable (V) region gene segments, predominates the adult immune response to the Haemophilus influenzae type b (Hib) capsular polysaccharide (PS). This site has been elevated to canonical status based upon its relative molecular uniformity and prevalence in adults. To date, no studies have examined the primary structure of Hib PS-specific antibodies in young infants, who are the primary targets of Hib vaccination. In this study we show that canonical Hib PS-specific heavy (H) and light (L) chain V regions are present in 4-month-old infants following two vaccinations with Hib PS-protein conjugates. The infant V regions contain sequence polymorphisms that resemble those found in adult antibodies, as well as polymorphisms at position 95a of the A2 L chain not previously observed in adults. In vitro studies of Fab fragments and recombinant IgG2 antibodies using these V regions identify sequence polymorphisms that impact Hib PS binding affinity and bactericidal activity. These results demonstrate the establishment of canonical V regions in early ontogeny and provide a structural explanation of how canonical antibodies in the infant can vary in their affinity and protective activity against Hib.