Grafting of a hepatitis B S-preS(2) T-cell epitope on lysozyme enhances the immunogenicity of lysozyme in responder mice primed with the T-cell epitope

Abstract Subunit immunogens composed of well-defined T-and B-cell epitopes might represent a valuable approach to design vaccines. The reduction of the size of the T-cell epitope is clearly in the line of this strategy. In this study we evaluated the capacity of a hepatitis B S-preS(2) surface antigen-derived T-cell epitope (i.e., S2b) to enhance the humoral immune response towards lysozyme when covalently linked to this antigen. We hereby anticipated that new problems, related to processing of a subunit immunogen, may emerge when grafting minimalized T-cell epitopes on protein antigens. Indeed, insertion of a T-cell epitope containing peptide (i.e., S2b) in a new protein context does not warrant a correct processing of the T-cell epitope. To avoid such potential processing problems an acid labile linker between T-cell and B-cell epitopes was devised in order to provide a processing-independent cleavage site. Using a T-cell hybridoma specific for the S2b T-cell epitope the S2bC-lysozyme conjugate was found to be presented by functional antigen-presenting cells. However, fixed APC did not present the conjugate in vitro indicating that processing is required for the release and presentation of S2b. The ability of the conjugate to generate an enhanced immune response was investigated in vivo. In S2b-primed mice the S2bC-lysozyme conjugate was found to elicit a faster and higher anti-lysozyme humoral response, as compared to uncoupled mixtures of lysozyme and S2b. Moreover, more IgG was produced when the mice were immunised with the conjugate as compared to the mixture of lysozyme and S2b, indicating the induction of a ‘secondary-type’ of immune response against lysozyme. In contrast, in non-responder mice for the S2b T-cell epitope, the same immunization protocol did not induce anti-lysozyme antibodies. These results demonstrate that S2b-induced memory T cells provide help to lysozyme-specific B cells during primary immunizations with the S2bC-lysozyme conjugate, resulting in a secondary IgG-type anti-lysozyme humoral response.