MIMICKING THE HUMORAL IMMUNE-RESPONSE IN-VITRO RESULTS IN ANTIGEN-SPECIFIC ISOTYPE SWITCHING SUPPORTED BY SPECIFIC AUTOLOGOUS T-HELPER CELLS - GENERATION OF HUMAN HIV-1-NEUTRALIZING IGG MONOCLONAL-ANTIBODIES FROM NAIVE DONORS

Molecular and cellular requirements for antigen-specific isotype switc h of human B cells have been investigated by mimicking signaling occur ring in germinal centers. Peripheral blood mononuclear cells from heal thy seronegative blood donors were first primary immunized in vitro, u sing a synthetic immunogen containing both a T and B cell epitope, whi ch generated specific IgM-secreting B cells. We used the apex of the V 3 loop of gp120 as B cell epitope linked to a promiscuous T helper epi tope from tetanus toxin. In parallel, CD4(+) T helper cell clones spec ific for the T epitope of the immunogen were established. In a seconda ry in vitro stimulation period, we co-cultured the antigen-specific T and B cells on CD32-transfected fibroblasts, together with an anti-CD4 0 monoclonal antibody. This resulted in isotype switching and human an tigen-specific, IgG-secreting B cells were detected. This response was strictly dependent upon the presence of autologous T helper cells and the immunogen. Antigen-specific human B cells derived from this prima ry and secondary in vitro immunization were subsequently subjected to electrofield-induced somatic cell hybridization and hybridomas secreti ng human anti-V3 IgG monoclonal antibodies were isolated. One human an tibody was further characterized and shown to be specific for the immu nizing antigen with an affinity constant of 24 nM. This antibody also effectively neutralized different isolates of HTV-1, achieving a 50 % neutralization at 0.46 mu g/ml.