All PCR products were gel-eluted and sequenced (GATC Biotech, Konstanz, Germany). The numbering used to identify the mutations in the mED43 viral sequence refers to the ED43 consensus sequence published in GenBank (accession number GU814265).21 The envelope sequence of the virus in mHK6a-infected mice was compared to the sequence of chimeric LY2835219 cell line HK6a/JFH1 virus (GenBank accession number FJ230883).15 However, because the full-length sequence of the HK6a genome has not been published yet, our numbering refers to the published H77 sequence (GenBank accession number AF009606). To validate the new batch of polyclonal

antibodies (H06), its capacity to neutralize the autologous H77C strain was compared to that of a preparation obtained from the same patient in 2003 (H03).16 Therefore, we injected 1 mg of purified H06-IgG per gram body weight into three uPA+/+-SCID mice that harbored human hepatocytes in their liver (chimeric mice) and challenged them three days later with H77C virus. Previous experiments had demonstrated that this was the optimal dose and schedule for nAbs to achieve protection.16 The viral challenge with 104 IU of mH77C per mouse is sufficient to induce a robust infection in all tested control animals.16, 22 The HCV RNA titers in two untreated control mice during week 1-4 postchallenge are

shown in Table 1. A weekly follow-up of the viral RNA levels in the plasma BGB324 concentration of three chimeric mice loaded with H06-IgG showed that 2 weeks after injection of the mH77C virus all animals remained HCV negative (<1,500 IU/mL) (Table 1, Fig. 1). cAMP Between the second and third week two animals died spontaneously, but the remaining third mouse remained HCV-negative until week 5 (<750 IU/mL), after which this animal also died spontaneously.

To further evaluate the potency of H06-antibodies in neutralizing the autologous virus, we challenged three chimeric mice, pretreated with H06-IgG as described above, with a 10-fold higher dose of the mH77C virus (105 IU/mouse). As shown in Table 1, all three animals tested HCV RNA-negative (<1,500 IU/mL) 1 week after injection of the virus. However, 1 week later two animals scored HCV RNA-positive. Viremia in both these animals progressively increased until week 3, reaching levels comparable to nontreated control mice. The third animal remained negative (<600 IU/mL) throughout the 6-week observation period. Although the H06-antibody pretreatment could not protect two out of three chimeric mice from being infected by a 10-fold higher viral challenge, in the weeks that followed the viral titers rose much slower that in untreated mice (Fig. 1). After validation of the polyclonal anti-HCV antibodies obtained from Patient H in 2006, we investigated whether passive immunization with H06-antibodies could protect chimeric mice from a challenge with HCV strains other than the autologous H77C.