5) ng, which is 3.9 (1.8) log10 ng lower than the controls. Moreover, in the presence of HepeX-B, HBsAg remained undetectable (< 0.1 ng) throughout the 48 hours of the experiment. Fitting of the full kinetics data with the in vitro model (Supporting Material, Equations 10 and 11) indicated that HBV-Ab17 or HBV-Ab19 give rise to a partial blocking of HBsAg release with effectiveness of 99.98% or 98.8%, respectively. A minimal estimate of the effectiveness in blocking release in the presence of HepeX-B is 99.998%. Similar results were obtained by estimation of HBsAg at 48 hours using the analytical solution (Supporting Material, Equations 12 and 13). In order to verify whether the
antibodies to HBsAg have a prolonged effect on HBsAg release from PLC/PRF/5 cells, we repeated the experiment, but after the first 24 hours, we replaced the supernatants with fresh medium and continued measuring the HBsAg levels in supernatants for an additional 24 hours (Fig. 5B). AZD3965 supplier During the first 24 hours,
when either control medium or antibodies to HBsAg were present, the kinetics were similar to the first experiment. After we replaced the supernatants with fresh medium at 24 hours, the HBsAg levels in the new supernatants started from 0 and the kinetics of HBsAg secretion from control cells (treated with medium only) was slightly faster, probably due to a Sirolimus in vivo larger number of cells per well relative to the first 24 hours. In contrast, cells that were treated initially with HBV-Ab17 (undetectable HBsAg during first 24 hours) showed slower (-)-p-Bromotetramisole Oxalate kinetics of HBsAg secretion (i.e., de novo increase in supernatants) also after the antibodies were removed from the medium. Fitting of the kinetics with the model indicated a delay of 2.6 hours in HBsAg
release from the cells and also a slow decline, with a half-life of 6.9 days, of the effectiveness in blocking release from an initial 99.99% at 24 hours. Similarly, a delay of 10.7 hours and a slow decline of the blocking release effectiveness with a half-life of 11.5 days were observed for the cells initially treated with HepeX-B. By modeling viral dynamics during in vivo and in vitro treatment with antibodies to HBsAg, this study reveals a novel antiviral mechanism of antibodies to HBV. Apart from the “conventional” antiviral activities against viral particles in the circulation, anti-HBs antibodies are internalized in liver cells and exert intracellular antiviral activities with prolonged blocking of viral particles release from infected cells. Both experimental approaches—in vivo infusions of HepeX-B and in vitro treatment of cells with the same antibodies—produced concordant results with a reduction of viremia and/or HBsAg titers, as well as a prolonged antiviral effect after anti-HBs administration. The combination of two human antibodies—HBV-Ab17, recognizing a conformational epitope, and HBV-Ab19, recognizing a linear epitope on HBsAg—had an additive effect.