Enumeration of hepatitis B surface antigen-specific B lymphocytes in responder and non-responder normal individuals vaccinated with recombinant hepatitis B surface antigen.

Abstract

Determination of the frequency of specific B lymphocytes has important implications for investigation of the immune response to different antigens and pathogens. Unresponsiveness to some viruses and antigens, such as hepatitis B virus (HBV) and its surface antigen (HBsAg), has been attributed to lack or insufficient production of the specific B-cell repertoire. In this study, peripheral blood B lymphocytes of 45 adult normal individuals vaccinated with recombinant hepatitis B vaccine were transformed with Epstein-Barr virus (EBV) and cultured at different dilutions on human fetal fibroblasts as a feeder layer. The vaccinees were classified into good, poor and non-responder groups. Following 2 to 3 weeks of incubation, culture supernatants were collected from wells containing transformed and proliferating B lymphocytes. The supernatants were subsequently screened for the presence of total immunoglobulin and antibody to HBsAg (anti-HBs) by enzyme-linked immunosorbent assay (ELISA). Accordingly, positive and negative wells were enumerated in each plate and the frequency of B lymphocytes producing anti-HBs antibody was estimated based on the Poisson statistical analysis. The total number of CD19+ B lymphocytes were counted in the peripheral blood of all subjects by flow cytometry. Our results demonstrated a similar precursor frequency of specific B lymphocytes in all subject groups before vaccine administration (< 2 x 10(-5)). Following vaccination, however, a significant increase in the number of specific B lymphocytes was observed in good-responder (1.5 x 10(-4)) and to a lesser extent poor-responder (3.5 x 10(-5)) individuals, but not in non-responders. These findings suggest a defect in either the primary B-cell repertoire or helper T-cell function in non-responder individuals.