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/10(6) 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.
Tuaillon
Edouard
E
Laboratoire de Virologie, Hôpital Lapeyronie, 291 av. du Doyen G. Giraud, 34295 Montpellier, France.
Tabaa
Yassine Al
YA
Petitjean
Gaël
G
Huguet
Marie-France
MF
Pajeaux
Georges
G
Fondere
Jean-Michel
JM
Ponseille
Benoît
B
Ducos
Jacques
J
Blanc
Pierre
P
Vendrell
Jean Pierre
JP
D000921:Antibody-Producing Cells / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology*; D019013:CD40 Antigens / Q000378:metabolism; D016022:Case-Control Studies; D002454:Cell Differentiation; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006509:Hepatitis B / Q000276:immunology*; D006514:Hepatitis B Surface Antigens / Q000097:blood / Q000276:immunology*; D017325:Hepatitis B Vaccines / Q000276:immunology*; D006801:Humans; D007136:Immunoglobulins / Q000097:blood; D007156:Immunologic Memory
D019013:CD40 Antigens; D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines; D007136:Immunoglobulins