Differential idiotype utilization for the in vivo type 14 capsular polysaccharide-specific Ig responses to intact Streptococcus pneumoniae versus a pneumococcal conjugate vaccine.

Murine IgG responses specific for the capsular polysaccharide (pneumococcal capsular polysaccharide serotype 14; PPS14) of Streptococcus pneumoniae type 14 (Pn14), induced in response to intact Pn14 or a PPS14-protein conjugate, are both dependent on CD4(+) T cell help but appear to use marginal zone versus follicular B cells, respectively. In this study, we identify an idiotype (44.1-Id) that dominates the PPS14-specific IgG, but not IgM, responses to intact Pn14, isolated PPS14, and Group B Streptococcus (strain COH1-11) expressing capsular polysaccharide structurally identical to PPS14. The 44.1-Id, however, is not expressed in the repertoire of natural PPS14-specific Abs. In distinct contrast, PPS14-specific IgG responses to a soluble PPS14-protein conjugate exhibit minimal usage of the 44.1-Id, although significant 44.1-Id expression is elicited in response to conjugate attached to particles. The 44.1-Id elicited in response to intact Pn14 was expressed in similar proportions among all four IgG subclasses during both the primary and secondary responses. The 44.1-Id usage was linked to the Igh(a), but not Igh(b), allotype and was associated with induction of relatively high total PPS14-specific IgG responses. In contrast to PPS14-protein conjugate, avidity maturation of the 44.1-Id-dominant PPS14-specific IgG responses was limited, even during the highly boosted T cell-dependent PPS14-specific secondary responses to COH1-11. These results indicate that different antigenic forms of the same capsular polysaccharide can recruit distinct B cell clones expressing characteristic idiotypes under genetic control and suggest that the 44.1-Id is derived from marginal zone B cells.

Crystal structure reveals vaccine elicited bactericidal human antibody targeting a conserved epitope on meningococcal fHbp.

Data obtained recently in the United Kingdom following a nationwide infant immunization program against serogroup B Neisseria meningitidis (MenB) reported >80% 4CMenB vaccine-mediated protection. Factor H-binding protein (fHbp) is a meningococcal virulence factor and a component of two new MenB vaccines. Here, we investigated the structural bases underlying the fHbp-dependent protective antibody response in humans, which might inform future antigen design efforts. We present the co-crystal structure of a human antibody Fab targeting fHbp. The vaccine-elicited Fab 1A12 is cross-reactive and targets an epitope highly conserved across the repertoire of three naturally occurring fHbp variants. The free Fab structure highlights conformational rearrangements occurring upon antigen binding. Importantly, 1A12 is bactericidal against MenB strains expressing fHbp from all three variants. Our results reveal important immunological features potentially contributing to the broad protection conferred by fHbp vaccination. Our studies fuel the rationale of presenting conserved protein epitopes when developing broadly protective vaccines.

Genome-based identification and characterization of a putative mucin-binding protein from the surface of Streptococcus pneumoniae.

Streptococcus pneumoniae open reading frame SP1492 encodes a surface protein that contains a novel conserved domain similar to the repeated fragments of mucin-binding proteins from lactobacilli and lactococci. To investigate the functional role(s) of this protein and its potential adhesive properties, the surface-exposed region of SP1492 was expressed in Escherichia coli, purified to homogeneity, and partially characterized by biophysical and immunological methods. Circular dichroism and sedimentation measurements confirmed that SP1492 is an all-beta protein that exists in solution as a monomer. The SP1492 protein has been shown to be expressed by S. pneumoniae and was experimentally localized to its surface. The protein functional domain binds to mucins II and III from porcine stomach and to purified submaxillary bovine gland mucin. It appears to be one of the very few unambiguous pneumococcal adhesin molecules known to date. A hypothetical model constructed by ab initio techniques predicts a novel beta-sandwich protein structure.

Carbohydrate moieties as vaccine candidates.

Carbohydrate epitopes or glycotopes are structurally diverse, occur in a variety of chemical contexts, and are present on the surfaces of cells in the body and on the surfaces of pathogens. These various structures and modes of presentation affect how they are perceived and processed by the body and dictate the outcome of the immune response directed against them. This review focuses on mechanisms of carbohydrate immunity, with an emphasis on carbohydrate vaccines that have been or are being developed for protection against encapsulated bacterial pathogens. We discuss the cellular basis of carbohydrate immunity, newly identified glycotope processing pathways and recognition capabilities, and the synthetic and microarray technologies that are being developed that will permit new experimental approaches to carbohydrate vaccine development and the exploration of the interaction of the immune system with self and nonself glycans.

Functional Analysis of the Human Antibody Response to Meningococcal Factor H Binding Protein.

UNLABELLED: Two licensed serogroup B meningococcal vaccines contain factor H binding protein (FHbp). The antigen specifically binds human FH, which downregulates complement. In wild-type mice whose mouse FH does not bind to FHbp vaccines, the serum anti-FHbp antibody response inhibited binding of human FH to FHbp. The inhibition was important for eliciting broad anti-FHbp serum bactericidal activity. In human FH transgenic mice and some nonhuman primates, FHbp was able to form a complex with FH and FHbp vaccination elicited anti-FHbp antibodies that did not inhibit FH binding. To investigate the human anti-FHbp repertoire, we cloned immunoglobulin heavy- and light-chain-variable-region genes of individual B cells from three adults immunized with FHbp vaccines and generated 10 sequence-distinct native anti-FHbp antibody fragments (Fabs). All 10 Fabs bound to live meningococci; only 1 slightly inhibited binding of human FH, while 4 enhanced FH binding. Affinity-purified anti-FHbp antibody from serum of a fourth immunized adult also enhanced binding of human FH to live meningococcal bacteria. Despite the bound FH, the affinity-purified serum anti-FHbp antibodies elicited human complement-mediated bactericidal activity that was amplified by the alternative pathway. The lack of FH inhibition by the human anti-FHbp Fabs and serum antibodies suggests that binding of human FH to the vaccine antigen skews the anti-FHbp antibody repertoire to epitopes outside the FH-binding site. Mutant FHbp vaccines with decreased FH binding may represent a means to redirect the human antibody repertoire to epitopes within the FH binding site, which can inhibit FH binding and, potentially, increase safety and protective activity. IMPORTANCE: Two meningococcal vaccines contain factor H binding protein (FHbp). Immunized mice whose mouse factor H (FH) does not bind to FHbp develop serum anti-FHbp antibodies that block binding of human FH to the bacteria. With less bound FH, the bacteria become more susceptible to complement killing. To investigate human responses, we isolated 10 recombinant anti-FHbp antibody fragments (Fabs) from immune cells of three immunized adults. One slightly inhibited binding of FH to the bacteria, and four enhanced FH binding. Purified serum anti-FHbp antibodies from a fourth immunized adult also enhanced FH binding. Although bound FH would be expected to block the alternative pathway, the human anti-FHbp antibodies retained bactericidal activity and the ability to activate the alternative pathway. Mutant FHbp vaccines with decreased binding to human FH may redirect the human antibody repertoire to epitopes within the FH binding site that inhibit FH binding, which are expected to increase protective activity.

Induction of opsonic antibodies to the gamma-D-glutamic acid capsule of Bacillus anthracis by immunization with a synthetic peptide-carrier protein conjugate.

The capsule of Bacillus anthracis, a polymer of gamma-D-glutamic acid, functions as a virulence determinant and is a poor immunogen. In this study we show that antibodies reactive with the B. anthracis capsule can be elicited in mice by immunization with a conjugate consisting of a synthetic gamma-D-glutamic acid nonamer peptide (gamma-D-glu9) covalently coupled to keyhole limpet hemocyanin. The serum response to gamma-D-glu9 was comprised primarily of IgG antibodies that recognized an epitope requiring a minimum of four gamma-linked D-glutamic acid residues. Antibodies to (gamma-D-glu9) bound to the surface of encapsulated B. anthracis cells and mediated opsonophagoctosis. These findings suggest that anti-capsular antibodies could mediate the clearance of vegetative B. anthracis cells in vivo. Thus, inclusion of an immunogenic capsular component as well as protective antigen in new anthrax vaccines would generate immune responses targeting both the bacteremic and toxigenic aspects of anthrax infection and thus may increase protective efficacy.

Potential for autoimmune pathogenesis of Rift Valley Fever virus retinitis.

Rift Valley Fever (RVF) is a significant threat to human health because it can progress to retinitis, encephalitis, and hemorrhagic fever. The timing of onset of Rift Valley Fever virus (RVFV) retinitis suggests an autoimmune origin. To determine whether RVFV retinitis is associated with increased levels of IgG against retinal tissue, we measured and compared levels of IgG against healthy human eye tissue by immunohistochemical analysis. We found that serum samples from RVFV-exposed Kenyans with retinitis (n = 8) were slightly more likely to have antibodies against retinal tissue than control populations, but the correlation was not statistically significant. Further investigation into the possible immune pathogenesis of RVFV retinitis could lead to improved therapies to prevent or treat this severe complication.

IGH V3-23*01 and its allele V3-23*03 differ in their capacity to form the canonical human antibody combining site specific for the capsular polysaccharide of Haemophilus influenzae type b.

The IGH V3-23*01 gene is used in the formation of the canonical combining site which dominates the human antibody repertoire to the Haemophilus influenzae type b (Hib) polysaccharide (PS). An allele of the human IGH V3-23*01 gene, known as V3-23*03, differs from V3-23*01 in nine bases, eight of which are located in the second complementarity determining region. These eight differences encode five amino acid substitutions. In this study we investigated whether the V3-23*03 sequence polymorphism affected Hib PS binding. We constructed two Fab fragments that had the canonical Hib PS combining site VH-VL configuration but that had either V3-23*01 or V3-23*03. Radioantigen binding assay showed that on a concentration basis the V3-23*03 Fab was 20-fold more effective in binding Hib PS than the V3-23*01 Fab. The V3-23*03 Fab was 4-fold more effective than the V3-23*01 Fab in mediating facilitated bactericidal activity against Hib organisms. These findings identify a functional consequence of V3-23 allelism, and suggest that utilization of the V3-23*03 gene in the human Hib PS repertoire would generate canonical antibodies with higher affinity and protective efficacy than canonical antibodies utilizing V3-23*01. Thus, IGH V gene allelic variation has the potential to impact the generation of protective immunity to Hib.

The capsule of Bacillus anthracis behaves as a thymus-independent type 2 antigen.

Bacillus anthracis elaborates a homopolymeric capsule composed of gamma-D-glutamic acid residues. Mice were immunized with formalin-fixed encapsulated B. anthracis bacilli, and the serum antibody response to a gamma-D-glutamyl capsular epitope was measured. Antiglutamyl antibodies were elicited in athymic BALB/c Nu/Nu, BALB/c Nu/+, and CBA/J mice but not in CBA/N xid mice. These response patterns define the capsule of B. anthracis as a thymus-independent type 2 antigen.

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.