Detection of memory B lymphocytes specific to hepatitis B virus (HBV) surface antigen (HBsAg) from HBsAg-vaccinated or HBV-immunized subjects by ELISPOT assay

To improve the investigation of the role of human memory B lymphocytes following hepatitis B virus (HBV) infection or vaccination, we developed a method to characterize circulating memory B cells specific to hepatitis B surface antigen (HBsAg). Our approach combined: (1) purification of CD19+ cells, (2) CD40–CD40L polyclonal stimulation, and (3) enumeration of memory B cells differentiated into anti-HBs antibody (Ab)-secreting cells (HBs-SCs) by a HBs-ELISPOT assay. In this way, HBs-SCs were detected in 17 HBsAg-vaccinated and nine HBV-immunized subjects including four individuals with serum anti-HBs Ab levels < 10 mIU/ml, but not in six controls. IgG+, IgA+ plus IgM+ HBs-SCs, representing 5–1736 cells/106 circulating B cells and 0.02–0.58% of total immunoglobulin-SCs generated by the B cell polyclonal stimulation, were counted by an Ig two-colour ELISPOT assay. In addition, anti-HBs Abs were found in 8/15 supernatants recovered from B cell cultures which contained HBs-SCs, suggesting that the HBs-ELISPOT assay is more reliable in tracking HBsAg-specific memory B cells than ELISA measurement of anti-HBs Abs secreted in supernatants. This new approach could be useful to explore the presence and the longevity of HBsAg-specific memory B cells in vaccinated and immunized subjects, in chronic HBV infection and after liver transplantation for HBV-related disease.