Genetic variation influences the B-cell response to immunization with a pneumococcal polysaccharide conjugate vaccine.

CBA/J mice immunized with pneumococcal 23F-CRM(197) vaccine produce significantly lower titers of 23F-specific antibodies and fewer 23F-specific antibody-secreting cells (ASC) than did BALB/c or (CBA/J x BALB/c)F(1) (CCBAF(1)) mice. The reduced 23F-specific titers of CBA/J versus BALB/c or CCBAF(1) mice are presumably related to lower frequencies of 23F-specific ASC influenced by genetic variation.

Mapping the antigenicity of copper-treated cellular prion protein with the scrapie isoform.

When recombinant and cellular prion protein (PrP(C)) binds copper, it acquires properties resembling the scrapie isoform (PrP(Sc)), namely protease resistance, detergent insolubility and increased beta sheet content. However, whether the conformations of PrP(C) induced by copper and PrP(Sc) are similar has not been studied in great detail. Here, we use a panel of seven monoclonal antibodies to decipher the epitopes on full-length mouse PrP(C) that are affected by exogenous copper, and to compare the antigenicity of the copper-treated full-length PrP(C) with the full-length PrP(Sc) present in scrapie-infected mouse brains. In the presence of copper, we found that epitopes along residues 115-130 and 153-165 become more accessible on PrP(C). These regions correspond to the two beta sheet strands in recombinant PrP and they were proposed to be important for prion conversion. However, when we compared the antibody-binding patterns between full-length PrP(C) with full-length PrP(Sc) and between copper-treated full-length PrP(C) with full-length PrP(Sc), antibody binding to residues 143-155 and 175-185 was consistently increased on PrP(Sc). Collectively, our results suggest that copper-treated full-length PrP(C) does not resemble full-length PrP(Sc), despite acquiring PrP(Sc)-like properties. In addition, since each full-length protein reacts distinctively to some of the antibodies, this binding pattern could discriminate between PrP(C) and PrP(Sc).

Immunization with Haemophilus influenzae type b-CRM(197) conjugate vaccine elicits a mixed Th1 and Th2 CD(4+) T cell cytokine response that correlates with the isotype of antipolysaccharide antibody.

Haemophilus influenzae type b (Hib) capsular polysaccharide (PS) induces protective antibodies but is T independent and poorly immunogenic in infants. Conjugate vaccines of Hib PS linked to proteins, such as CRM(197), increase the PS antibody titer and elicit immunologic memory. To define the conjugate-induced memory T cell response, 19 adults were immunized with Hib-CRM(197), and antibody titers, carrier protein-specific CD4(+) T cell proliferation, and cytokine production were measured. Hib-CRM(197) induced PS and CRM(197) antibodies, vigorous T cell recall responses, and production of cytokines, including interleukin (IL)-2, IL-5, IL-10, and interferon-gamma. There was marked variability in PS antibody titer, despite consistent CRM(197)-specific recall responsiveness, which correlated with peak IgM and IgA PS antibody titers. Correlations were also found between IL-2 and IL-5 and IgA PS antibody levels. Hib-CRM(197) induced a rapid increase in CRM(197)-specific memory T cells and mixed Th1/Th2 cytokines, which may regulate the isotype and quantity of PS antibody.

Dimensions of antigen recognition and levels of immunological specificity.

Although recognition and specificity are among the most fundamental concepts in immunology, there is a common tendency to equate these notions with the fit, especially in terms of molecular shape, between interacting molecules. Even in the case of monovalent recognition, there are factors that contribute to the energetics of the interaction that are not readily accounted for by detailed structural analysis of the interacting (epitopic and paratopic) molecular surfaces. Consequently, recognition involves more than just the three spatial dimensions and time. Factors such as solute-solvent interactions, molecular crowding, and confinement, not directly related to the details of the intermolecular interface, can play crucial roles in determining both intrinsic affinity and differential intrinsic affinity. Furthermore, stating that a given structural subunit (e.g., amino acid) is recognized in a given noncovalent interaction does not clarify whether the structural subunit in question participates in the interaction through van der Waals contact, contribution to intrinsic affinity, or differential contribution to relative intrinsic affinities for two or more different ligands. Additional factors become relevant in considering the specificity exhibited in multivalent interactions, cell activation, and activation of the whole immune system. Therefore, specificity as defined for a monovalent binding event can diverge from specificity as it is defined for higher-order interactions. A corollary of this conclusion is that the composition of epitopes and paratopes, defined in terms of the structural elements for which substitutions have an effect on the specificity-defining measurement, can differ in different contexts despite complete conservation of the structures that physically make direct contact. An analysis of specificity at the organismal level suggests that the immune system does not recognize or respond to substances that correspond precisely to either nonself substances or to dangerous substances. An alternative notion for the molecular origins of immunological discrimination does not require that there be any single reason for immune responsiveness. This concept of what the immune system recognizes and responds to derives from the recognition that the ultimate function of the immune system is to contribute to survival and reproductive success through any available means.

CpG oligodeoxynucleotides act as adjuvants for pneumococcal polysaccharide-protein conjugate vaccines and enhance antipolysaccharide immunoglobulin G2a (IgG2a) and IgG3 antibodies.

Pneumococcal polysaccharide-protein conjugate vaccines elicit antipolysaccharide antibodies, but multiple doses are required to achieve protective antibody levels in children. In addition, the immunogenicity of experimental multivalent pneumococcal conjugate vaccines varies with different polysaccharide serotypes. One strategy to improve these vaccines is to incorporate an adjuvant to enhance their immunogenicity. Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are adjuvants that promote T-cell and T-dependent antibody responses to protein antigens, but it has been unclear whether CpG ODN can enhance polysaccharide-specific antibody responses. The present studies demonstrate significant adjuvant activity of CpG ODN for antibody responses against Streptococcus pneumoniae polysaccharide types 19F and 6B induced by conjugates of 19F and 6B with the protein carrier CRM(197). BALB/c ByJ mice were injected with 19F-CRM(197) or 6B-CRM(197) with or without CpG ODN, and sera were tested for anti-19F or anti-6B antibodies by enzyme-linked immunosorbent assay. The polysaccharide-specific antibody response to 19F-CRM(197) alone was predominantly of the immunoglobulin G1 (IgG1) and IgM isotypes, but addition of CpG ODN markedly increased geometric mean titers of total anti-19F antibody (23-fold), anti-19F IgG2a (26-fold), and anti-19F IgG3 (>246-fold). The polysaccharide-specific antibody response to 6B-CRM(197) alone consisted only of IgM, but addition of CpG ODN induced high titers of anti-6B IgG1 (>78-fold increase), anti-6B IgG2a (>54-fold increase), and anti-6B IgG3 (>3,162-fold increase). CpG ODN also increased anti-CRM(197) IgG2a and IgG3. Adjuvant effects were not observed with control non-CpG ODN. Thus, CpG ODN significantly enhance antipolysaccharide IgG responses (especially IgG2a and IgG3) induced by these glycoconjugate vaccines.

Pretransplant frequency of donor-specific, IFN-gamma-producing lymphocytes is a manifestation of immunologic memory and correlates with the risk of posttransplant rejection episodes.

While matching for MHC Ags improves renal allograft survival, closely matched grafts sometimes fail due to rejection, and poorly matched allografts are often well tolerated by the recipient. The severity of the rejection process may partially depend on the presence of environmentally primed T cells in the recipient that cross-react with donor Ags. To test for the presence of primed, donor-specific T cells in humans before transplantation, we used an enzyme-linked immunospot assay for detection of allospecific cytokines produced by individual human PBLs. We demonstrate that this approach detects cytokine production at single cell resolution and detects production of IFN-gamma only when there is defined immunologic priming, thus representing a measure of primed donor-specific immunity. Because the environmental Ag exposure of the recipient is not a function of the HLA mismatch between donor and potential recipient, the number of HLA mismatches may not correlate with the frequency of pretransplant, donor-specific IFN-gamma-producing PBLs. Studies of donor-specific IFN-gamma-producing lymphocytes in a cohort of patients being evaluated for renal transplantation corroborated this hypothesis. Moreover, for recipients of both living and cadaver renal allografts, the pretransplant frequency of donor-specific memory cells correlated with the posttransplant risk of developing acute rejection episodes. This improved ability to define the strength of the allospecific immune response by enzyme-linked immunospot assay may allow improved pairing of recipients with donors and identification of kidney allograft donor-recipient pairs at high risk for acute rejection, thus permitting targeted interventions aimed at prolonging graft survival.

B- and T-cell immune responses to pneumococcal conjugate vaccines: divergence between carrier- and polysaccharide-specific immunogenicity.

Conjugation of various serotypes of pneumococcal polysaccharide (PnPS) to carrier protein enhances the magnitude of the polysaccharide-specific antibody response, presumably by eliciting T-cell help. However, variability in PnPS serotype-specific immunogenicity has been observed. CBA/J mice immunized with either 6B or 19F PnPS conjugated to the protein carrier Cross Reactive Material(197) (CRM(197)) produce a strong anti-PnPS antibody response; however, when mice are immunized with 23F PnPS conjugated to CRM(197), they fail to produce a significant anti-PnPS response. In order to determine whether this difference was related to alterations in antigen processing of the carrier protein and the subsequent T-cell responses, we studied proliferation of lymphocytes from CBA/J mice immunized with CRM(197) alone or conjugated to 6B, 19F, or 23F PnPS. T-cell proliferative responses to synthetic peptides demonstrated that lymph node cells elicited by the poorly immunogenic conjugate 23F-CRM(197) recognized many, but not all, of the epitopes recognized by lymph node cells elicited by 6B- and 19F-CRM(197) as well as additional epitopes. Despite marked differences in PnPS-specific immunogenicity, all mice made high titers of CRM(197) antibodies of the immunoglobulin G(1) isotype. Cells from mice immunized with any of the conjugates yielded vigorous T-cell responses to whole antigen. We conclude that the serotype of PnPS can alter the peptide specificities of T-cell responses, but even a poorly immunogenic PnPS conjugate can elicit a significant T-cell response. Thus, conjugation of PnPS to a carrier protein that elicits carrier-specific T- and B-cell responses does not necessarily enhance PnPS immunogenicity.

Evolutionary consequences of selected locus-specific variations in epistasis and fitness contribution in Kauffman's NK model.

Mathematical analysis and computer simulations are used to evaluate three modifications to Kauffman's NK model in an attempt to incorporate unexplored aspects of epistatic interaction between loci in genome evolution. Two modifications--one to the amount and the other to the distribution of epistatic interaction--further support Kauffman's conclusion that high levels of epistatic interaction lead to a decrease in overall fitness of the genome. The third model, however, provides a condition under which increased epistatic interaction at certain loci results in higher genome fitness.

All-D peptides recognized by an anti-carbohydrate antibody identified from a positional scanning library.

Monoclonal antibodies recognize antigens with high affinity and specificity, but the structural basis for molecular mimicry remains unclear. It is often assumed that cross-reactive antigens share some structural similarity that is specifically recognized by a monoclonal antibody. Recent studies using combinatorial libraries, which are composed of millions of sequences, have examined antibody cross-reactivity in a manner entirely different from traditional epitope mapping approaches. Here, peptide libraries were screened against an anti-carbohydrate monoclonal antibody for the identification of peptide mimics. Positional scanning libraries composed of all-l or all-d hexapeptides were screened for inhibition of monoclonal antibody HGAC 39.G3 binding to an antigen displaying N-acetyl-d-glucosamine (GlcNAc) residues on a polyrhamnose backbone. Inhibitory activity by mixtures from the all-d hexapeptide library was greater than the activity from the all-l libraries. The most active d-amino acid residues defined in each of the six positions of the library were selected to prepare 27 different individual hexapeptides. The sequence Ac-yryygl-NH2 was specifically recognized by mAb HGAC 39.G3 with a relative affinity of 300 nM when measured in a competitive binding assay. The contributions to overall specificity of the residues of the all-d peptide (Ac-yryygl-NH2) in binding to mAb HGAC 39.G3 were examined with a series of truncation, l and d-amino acid substitution, and retro analogs. Dimeric forms of the all-d peptide were recognized with tenfold to 100-fold greater affinities relative to the monomer. The all-d peptide was found to inhibit mAb HGAC 39.G3 binding to an anti-idiotype antibody with approximately 1000-fold greater affinity than GlcNAc. As demonstrated here, the study of immune recognition using combinatorial chemistry may offer new insights into the molecular basis of cross-reactivity.

Gamma 3 gene-disrupted mice selectively deficient in the dominant IgG subclass made to bacterial polysaccharides undergo normal isotype switching after immunization with polysaccharide-protein conjugate vaccines.

Bacterial polysaccharides (PS) are T-independent type 2 Ags that elicit restricted Ab responses of IgM and IgG3 in mice and IgM and predominantly IgG2 in humans. Immunodeficiency in the dominant IgG subclass made to PS is associated with chronic sinus and pulmonary infections with PS-encapsulated bacteria. To elucidate the biologic role of the dominant IgG subclass in the immune response to PS and to make an animal model of human IgG subclass deficiency, we generated mice with a targeted disruption of the exon encoding the CH1 domain of the gamma 3 heavy-chain constant region gene. Homozygotes had no detectable serum IgG3, and their splenocytes did not produce IgG3 after LPS stimulation. IgG3(-/-) mice immunized with PS from Pseudomonas aeruginosa LPS O-side chain or Streptococcus pneumoniae type 19F capsule did not produce any IgG3 anti-PS Abs, in contrast to wild-type mice in which IgG3 was the major IgG subclass. Immunizing both wild-type and IgG3(-/-) mice with 19F PS-protein conjugate elicited IgG1 Abs. We conclude that IgG3(-/-) mice have a selective deficiency in the dominant murine IgG subclass made to T-independent type 2 Ags and may be a useful animal model of IgG subclass deficiency. In addition, we show that the anti-PS Ab class switching to IgG1 that occurs when mice are immunized with a PS-protein conjugate vaccine does not require sequential Ig expression or an intact, upstream gamma 3 heavy-chain gene.

Mitogenic synthetic polynucleotides suppress the antibody response to a bacterial polysaccharide.

Unmethylated bacterial DNA containing a high frequency of the CpG motif, is mitogenic and induces T-cell independent, murine B-cell proliferation. These stimulatory effects are also induced by synthetic oligonucleotides that contain one or more unmethylated CpG dinucleotides (CpG oligo). Such mitogenicity is not seen with highly methylated vertebrate DNA, which has a lower prevalence of the CpG motif than bacterial DNA. Due to their stimulatory effects, CpG oligo have been proposed for use as vaccine adjuvants. In order to determine if a synthetic CpG oligo that was stimulatory for B-cell proliferation could augment the murine antibody response to protective bacterial polysaccharide epitopes (Pseudomonas aeruginosa LPS-O polysaccharide side chain; high-molecular-weight polysaccharide or high-MW PS), BALB/c mice were injected with mitogenic doses of CpG oligo simultaneously with high-MW PS, and antibody titers were measured by ELISA weekly for 4 weeks. Controls received PBS, a nonstimulatory control oligo plus PS, CpG alone, or PS alone. Despite evidence of B-cell mitogenicity and an increase in total IgM in CpG oligo-treated mice, CpG oligo treatment plus PS significantly decreased the high-MW PS antibody response compared to PS alone. The blunting of the anti-PS antibody response could be eliminated by vaccinating the animals with PS prior to CpG oligo. We conclude that despite in vitro and in vivo evidence of B-cell proliferation, this CpG oligo reduces PS-specific antibody responses in an animal model when given simultaneously with a bacterial polysaccharide. Based on results in this model, oligonucleotides containing stimulatory unmethylated CpG dinucleotides may not be useful adjuvants when given simultaneously with bacterial PS vaccines.

Bispecific antibodies overcome the opsonin-receptor mismatch of cystic fibrosis in vitro: restoration of neutrophil-mediated phagocytosis and killing of Pseudomonas aeruginosa.

Inflammation and infection associated with bacterial pathogens, primarily Pseudomonas aeruginosa (Pa), are the primary causes of morbidity and mortality for cystic fibrosis (CF) patients. CF patients may be predisposed to these bacterial infections by a defect in phagocytosis due to "opsonin-receptor mismatch," in which a complement receptor (CR1) and an important opsonin (iC3b) are destroyed by proteolytic enzymes. We show that opsonin-receptor mismatch can be mitigated in vitro using a bispecific Ab (bsAb) to cross-link neutrophils via the beta-chain of leukocyte integrins (CD18) to bacterial epitopes or C3d on opsonized Pa. Two chemically cross-linked bsAb were constructed with mAb specific for C3d (or the O-specific side chain of Fisher Devlin Immunotype 1 Pa) and CD18. Using an in vitro model of elastase-mediated opsonin-receptor mismatch, these bsAb specifically enhanced Pa phagocytosis and killing, with the anti-C3d-containing bsAb restoring the levels of phagocytosis to approximately those for the non-elastase-treated opsonic control. These results encourage the further investigation of bsAb as therapeutic agents for bacterial infection in the lungs of CF patients.

Functional dissociation of CD8 alpha's Ig homologue and connecting peptide domains.

The contribution of the CD8 alpha.IgV homologue domain to class I MHC binding was evaluated using a series of chimeric human CD8 alpha:Fc polypeptides incorporating alternative CD8 alpha extracellular domain components. Using a nonisotopic cellfree physical binding assay, those Fc chimeras encompassing the CD8 alpha.IgV homologue domain only (dissociated from the 48-amino acid CD8 alpha connecting peptide) were shown to retain the capacity of the complete CD8 alpha extracellular domain to bind to a recombinant soluble class I MHC alpha 3 domain unit or to intact class I MHC. The specificity of the CD8 alpha:class I MHC alpha 3 domain interaction was verified by mAb and soluble polypeptide blocking experiments. Furthermore, co-precipitation of an Fc chimera incorporating only the CD8 alpha.IgV homologue domain and a recombinant soluble class I MHC alpha 3 domain unit was accomplished. In addition, a glycosylphosphatidylinositol (GPI)-modified variant of the CD8 alpha.IgV homologue domain was generated via chimerization with the GPI signal sequence from decay-accelerating factor. GPI anchorage for this truncated CD8 alpha polypeptide was verified, and its capacity to promote intercellular adhesion through class I MHC binding was shown in a cell:cell binding assay. The findings indicate that the CD8 alpha.IgV homologue domain acts as an independent structural unit when dissociated from the CD8 alpha connecting peptide, and in so doing retains class I MHC binding capacity. This further establishes the principle that Ig superfamily domains from receptor:counter-receptor pairs can interact with each other as isolated units, providing an experimental path for tailoring therapeutically useful IgSF protein derivatives.

Complementarity, specificity and the nature of epitopes and paratopes in multivalent interactions.

Structural elements of an antibody (Ab) or antigen (Ag) distant from the actual sites mediating contact between Ab and Ag can exert substantial influence on binding to, and discrimination among, multivalent targets. Consequently, multivalent molecules that express the same number of identical binding sites, but that differ in other structural features, can exhibit differences in their ability to discriminate between multivalent ligands. Here, Neil Greenspan and Laurence Cooper review evidence for these effects, and explore implications of the conclusion that effective specificity in multivalent interactions is not completely determined by the degree of complementarity between epitopes and paratopes.

Roles of immunoglobulin valency and the heavy-chain constant domain in antibody-mediated downregulation of Sindbis virus replication in persistently infected neurons.

Clearance of infectious Sindbis virus from neurons is mediated by antibody to the E2 glycoprotein. Properties of the antibody important for downregulation of Sindbis virus replication are unknown. Immunoglobulin isotypes and valency determine many biological properties of antibodies. An immunoglobulin G1 (IgG1) isotype switch mutant and F(ab')2 and Fab fragments of IgG3 monoclonal antibody 209 were prepared and tested for clearance of infectious virus from persistently infected rat dorsal root ganglion neurons in vitro. IgG1, IgG3, and IgG3-derived F(ab')2 fragments were similarly efficacious, while IgG3-derived Fab fragments had no effect on virus replication. Cross-linking of Fab with secondary antibodies restored antiviral activity. Therefore, we found no evidence that IgG subclass plays a role in control of intracellular Sindbis virus replication. However, bivalency appears to be crucial for the ability of E2-specific IgG molecules to mediate clearance of infectious virus from neuron cells, suggesting that cross-linking of E2 molecules is essential.