Human Immune Response to Cationized Proteins: I. Characterization of the in Vitro Response to Cationized Diphteria Toxoid

Abstract

Cationization of proteins, i.e., increasing net positive charge by the substitution of carboxyl groups with positively charged residues, has been reported to enhance protein immunogenicity in animal model systems. In the present study, we have investigated the effect of cationization on the in vitro cell-mediated immune response of human mononuclear cells to diphtheria toxoid. A series of cationized DT preparations were generated by covalent modification with ethylene-diamine, with pIs ranging from 4.6 to ≥9.3, and tested for their ability to induce proliferation of normal human peripheral blood mononuclear cells. Cationized DT (cDT) was found to induce an antigen-specific, augmented proliferative response, relative to native antigen, which was directly proportional to the degree of cationization. Further characterization of the response to cDT demonstrated that (1) proliferative responses could be detected considerably earlier, and typically at much lower antigen concentrations, than the response to native DT; (2) the response was dependent on HLA-DR: (3) production of a number of cytokines, sp. IL-1β, IL-2, and IFN-γ was also elevated in cDT-stimulated cultures; and (4) the enhanced proliferative response to cDT could be attributed to CD4 + helper T cells. These results demonstrate that cationization of proteins enhances the ability to generate a cell-mediated immune response in humans and suggest that cationization may have utility in the design of more effective carrier proteins for human vaccines.