10 Aliquots of cell culture supernatants were added to wells coated with P2D3 monoclonal anti-HBs,20 and the detection was with peroxidase-conjugated monoclonal anti-HBs (D2H5).21 The HBsAg in supernatants was quantitated in nanograms per milliliter, based on a parallel testing of eight standard amounts of HBsAg between 5 and 2000 ng, which were included in the same run of the assay. Details of the western blot and ELISA methodology are given in the Supporting Material. Mathematical modeling of the antiviral effect of the antibodies on viral kinetics in patients was performed by extension of the standard model by Neumann et al.22 Metformin datasheet to examine the

dynamics of HBsAg particles and the hepatitis B virions in the serum, as measured by HBV DNA, and a number of possible antiviral effects. In addition, we developed a model in order to simulate the in vitro kinetics of supernatant HBsAg produced by PLC/PRF/5 cells in culture and the possible effects

of antibodies on that process. Both models, their parameters’ estimates and fitting procedures are explained in detail in the Supporting Material. Analysis of viral kinetics after a single infusion of 40 mg HepeX-B showed a rapid HBV DNA decline starting 0.5 hours after initiation of infusion and continuing throughout the 8-hour infusion period, reaching 2.5-3.3 log10 copies/mL reduction from baseline with a half-life of 0.33-0.53 selleck chemical hours (Fig. 1 and Table 1). A parallel HBsAg decline to undetectable levels (<0.125 ng/mL) was observed in all three

patients, with a half-life of 0.09-0.19 hours and maximal decline of 4.3-4.6 log10 copies/mL relative to baseline. Nonlinear fitting of the HBV DNA and of HBsAg kinetics to a viral dynamics model (Supporting Material, Equations 1-4) allowed us to test various hypotheses for the antiviral mechanism of HepeX-B (Fig. 2). First, blocking de novo infection (1 ≥ η > 0) cannot be the major antiviral mechanism because it can only result in a viral decline slope of the order of the learn more loss rate of infected cells, half-life larger than 1 day, which is not in agreement with the rapid viral decline observed here. Second, accelerated loss of infected cells (k > 1) or blocking of virion production and/or release from infected cells (0 < εV ≤ 1) by themselves are also not sufficient explanations, because the expected decline would follow the clearance rate of serum HBV virions. The half-life of HBV virions (ln(2)/cv) ranges between 3-24 hours, as found in previous studies of HBV kinetics,15, 23-26 which is too slow compared to the very rapid decline observed during HepeX-B infusion (Fig. 1). Third, assuming an accelerated clearance of HBV virions from circulation (aV > 1) cannot by itself explain the rapid decline in serum HBV DNA (Fig. 2). The observed half-life of the order of 0.33-0.53 hours gives a minimal (maximal) estimate of accelerated clearance of HBV particles of aV = 5.7 (72.