Description: CD81 is a required receptor for Hepatitis C virus (HCV) infection of human hepatocytes in vitro . We generated several high affinity anti-human CD81 monoclonal antibodies (mAb) that demonstrated potent, specific and cross-genotype inhibition of HCV entry. One of these mAbs, K04, was administered to human liver chimeric mice before or after HCV infection to determine its ability to prevent HCV infection or spread of HCV infection, respectively. All vehicle control mice established HCV infection, reaching steady-state levels of serum HCV RNA by day 21. Pretreatment of mice with K04 prevented HCV infection in all mice (n=5). Treatment of mice with mAb K04 every 3 days for 21 days, starting at 6 h post-infection resulted in effective inhibition of virus spread. In three mice that were sacrificed on day 24, serum HCV levels remained detectable, below the limit of quantification (LOQ), indicating that infection was established but virus spread was blocked by the anti-CD81 mAb. In five additional mice that were followed for a longer time, virus remained detectable, below LOQ, until days 24 and 30 in four out of five mice. In the fifth mouse, viral load was quantifiable, but reduced to 64-fold below the mean viral load in vehicle control at day 24. In addition, two out of five mice cleared the infection by day 30 and one mouse had undetectable virus load from day 6 onwards. These results demonstrate that CD81 is required for HCV infection and virus spread in vivo , and that anti-CD81 antibodies such as K04 may have potential as broad spectrum antiviral agents for the prevention and for the treatment of HCV infection. This article is protected by copyright. All rights reserved.