Longitudinal determination of hepatitis B surface antigen-specific B lymphocyte frequency in healthy high responder adults after booster vaccination.

OBJECTIVES

The influence of booster vaccination on hepatitis B surface antigen (HBsAg)-specific B lymphocytes in humans has not been well characterized. Considering the low frequency of circulating B cells specific for HBsAg in vaccine high responder subjects, determination of this frequency at different time intervals after booster dose injection may provide invaluable information for evaluation of immune response to rHBsAg and identification of the most appropriate timing for isolation of specific B cells and generation of human monoclonal antibodies.

METHODS

Peripheral blood mononuclear cells (PBMCs) were isolated from 7 healthy high responder adults at 1, 2, 4, 8 and 16 weeks following administration of booster vaccination with an rHBsAg. The cells were transformed with Epstein-Barr virus and cultured at different cell densities over a feeder of human fetal foreskin fibroblasts. Following transformation, total hIg and HBsAg-specific antibody were screened in culture supernatant using ELISA, and primary frequency of specific B cells was calculated by limiting dilution assay based on Poisson analysis. Actual frequency was determined taking into consideration the percent of B cells in each PBMC population and efficiency of EBV transformation.

RESULTS

The mean frequencies of specific B cells after booster vaccination were found to be 1/13,462, 1/3,318, 1/5,224, 1/8,861 and 1/10,714 for the specified time intervals, respectively. Significant differences were observed between the frequencies of samples collected at all time intervals with the exception of week 1 versus weeks 8 and 16, week 2 versus week 4, and week 8 versus week 16.

CONCLUSIONS

Our results may provide an indirect measure for immunological memory and may help optimize immunization strategies for novel vaccines and generate human monoclonal antibodies.