Venkatasamy Manivel

Maturation of an Antibody Response Is Governed by Modulations in Flexibility of the Antigen-Combining Site

Although affinity maturation constitutes an integral part of T-dependent humoral responses, its structural basis is less well understood. We compared the physicochemical properties of antigen binding of several independent antibody panels derived from both germline and secondary responses. We found that antibody maturation essentially reflects modulations in entropy-control of the association, but not dissociation, step of the binding. This influence stems from variations in conformational heterogeneity of the antigen-combining site, which in turn regulates both the affinity and specificity for antigen. Thus, the simple device of manipulating conformational flexibility of paratope provides a mechanism wherein the transition from a degenerate recognition capability to a high-fidelity effector response is readily achieved, with the minimum of somatic mutations.

The primary antibody repertoire represents a linked network of degenerate antigen specificities

In this study, germline Abs were used to select clones from a random dodecapeptide phage-display library. This revealed a much greater heterogeneity of binders than could be obtained with mutated daughter Abs that presumably had been selected in vivo by nominal Ag during active immune responses. We demonstrate that the pluripotency of germline Abs can subsequently be optimized by binding interactions that correlate with thermodynamic changes indicative of structural adaptations at the interface. This singular feature confers on each Ab a distinct window of Ag specificities, where the entropic space explored constitutes a thermodynamic signature of that particular Ab. Combining site plasticity may facilitate overlaps in such windows, with independent Abs converging onto common determinants with near identical binding affinities. In addition to providing for an amplified recognition potential, this networking of individual spectra of Ag specificities simultaneously facilitates the rapid recognition of Ag. Importantly, it also ensures that the primary response is composed of Abs with a high degree of “evolvability.”

B Cell Responses to a Peptide Epitope. X. Epitope Selection in a Primary Response Is Thermodynamically Regulated

We examine the etiological basis of hierarchical immunodominance of B cell epitopes on a multideterminant Ag. A model T-dependant immunogen, containing a single immunodominant B cell epitope, was used. The primary IgM response to this peptide included Abs directed against diverse determinants presented by the peptide. Interestingly, affinity of individual monomeric IgM Abs segregated around epitope recognized and was independent of their clonal origins. Furthermore, affinity of Abs directed against the immunodominant epitope were markedly higher than that of the alternate specificities. These studies suggested that the affinity of an epitope-specific primary response, and variations therein, may be determined by the chemical composition of epitope. This inference was supported by thermodynamic analyses of monomer IgM binding to Ag, which revealed that this interaction occurs at the expense of unfavorable entropy changes. Permissible binding required compensation by net enthalpic changes. Finally, the correlation between chemical composition of an epitope, the resultant affinity of the early primary humoral response, and its eventual influence on relative immunogenicity could be experimentally verified. This was achieved by examining the effect of various amino-terminal substitutions on immunogenicity of a, hitherto cryptic, amino-terminal determinant. Such experiments permitted delineation of a hierarchy of individual amino acid residues based on their influence; which correlated well with calculated Gibbs-free energy changes that individual residue side chains were expected to contribute in a binding interaction. Thus, maturation of a T-dependant humoral response is initiated by a step that is under thermodynamic control.

B Cell Responses to a Peptide Epitope. IX. The Kinetics of Antigen Binding Differentially Regulates Costimulatory Capacity of Activated B Cells

We explore the possible mechanism by which association rates of Ag with activated B cells influences the ability of the latter to selectively recruit Th subsets. Our system used cocultures of Ag-activated B and T cells, where the Ag was a synthetic peptide, G41CT3. Restimulation was with either peptide G41CT3 or its analogue, G28CT3. Peptide G28CT3 has been previously shown to display a higher on rate, relative to the homologous peptide G41CT3, of binding to G41CT3-activated B cells. This difference in on rates was eventually exerted at the level of IFN-γ, but not of IL-10, induction from T cells, with peptide G28CT3 proving more effective. However, various treatment regimens rendered peptide G41CT3 as potent as peptide G28CT3 at eliciting IFN-γ responses from the above cultures. This included simultaneous treatment of B cells with peptide G41CT3 and the protein tyrosine kinase inhibitor tyrphostin. Alternatively, pretreatment of B cells with a peptide representing only the B cell epitope constituent of peptide G28CT3 (G28) was also equally effective. Subsequent experiments revealed that IFN-γ production from activated T cells resulted from an engagement of CD28 by B7-1 on the B cell surface. Finally, the extent of cell surface B7-1 up-regulation on activated B cells was dependent on the on rate of Ag binding to the membrane-bound Ig receptor. Thus, cumulative results suggest that the kinetics of Ag binding to activated B cells can differentially regulate intracellular signaling. This influences selective costimulatory molecule expression, with its consequent effects on relative Th subset activation.

Interleukin-1 derived synthetic peptide as an added co-adjuvant in vaccine formulations

A synthetic peptide containing the immunostimulatory and receptor binding sequences of human IL-1 beta was synthesized and tested for its immunoadjuvant properties. Using a commercially available hepatitis B vaccine as model antigen we found that added peptide enhanced both total and protective antibody responses in high and low responder strains of mice but was unable to overcome non-responsiveness in a third strain. Increased antibody response to antigen in the responder strains was not accompanied by any significant alteration in IgG isotype composition. These results suggest that this peptide may prove useful as a co-adjuvant in vaccines.

A self-associating hepatitis B surface antigen-derived peptide that is immunogenic in alum

We previously described an oligomeric synthetic peptide derived from the hepatitis B surface antigen that displayed a limited tendency to form self-associating macromolecular structures in solution. Here it is demonstrated that amino-terminal myristylation of this peptide results in near quantitative aggregation of the oligomeric peptide. The myristylated peptide is highly immunogenic when used in conjunction with alum as adjuvant in both the rabbit and rhesus monkey models. The antibody response generated by peptide also cross-reacted with native antigen and was long-lasting. Collectively the results described in this and previous reports offer an attractive new approach for generating immunogenic peptide mimetics of conformational epitopes that may find application as vaccines.

Comparison of immune responses to a native viral antigen and a synthetic peptide derived from it: implications for vaccine development

Murine immune responses to the hepatitis B surface antigen (HBsAg) and a synthetic peptide derived from it were compared at the humoral level. Six of nine strains used responded to either peptide or HBsAg, though restriction profiles were not superimposable. Two of three strains non-responsive to HBsAg produced an antibody response on immunization with peptide which was cross-reactive with both peptide and HBsAg. In in vitro lymphocyte stimulation assays, lymphocyte from all six peptide-immunized mouse strains could be induced to proliferate on challenge with HBsAg. However, of the HBsAg-immunized groups, lymphocytes from only three of six responder strains proliferated on in vitro HBsAg challenge. Cumulatively, these results suggest that a vaccine formulation that includes both protein antigens and synthetic peptides derived from these proteins may be more effective at eliciting an immune response in a broader cross-section of target population.