HCVpp were incubated for 1 hour with plasma at a 1:100 dilution or mAb at a final concentration of 50 or 10 g/ml and then added in duplicate to Hep3B target cells for 5 to 6 hours before medium was changed. epitope, proving that anti-HCV bNAbs can achieve substantial neutralizing breadth with relatively Chicoric acid few somatic mutations, and identifies HCV envelope variants that favored selection and maturation of an anti-HCV bNAb in vivo. These data provide insight into the molecular mechanisms of immune-mediated clearance of HCV infection and present a roadmap to guide development of a vaccine capable of stimulating anti-HCV bNAbs with a physiologic number of somatic mutations characteristic of vaccine responses. Keywords: Infectious disease Two people who spontaneously cleared hepatitis C virus infection possessed broadly-neutralizing monoclonal antibodies encoded by variable genes with sparse somatic mutations. Introduction Hepatitis C virus (HCV) infects approximately 185 million people worldwide and is a major cause of liver failure and hepatocellular carcinoma (1). With the recent development of potent, oral interferon-free therapies, treatment of HCV infection has improved significantly. However, HCV eradication is unlikely to be achieved with treatment alone. Identification of those with HCV infection is challenging. Therapies are too costly for countries with the highest incidence. Reinfection can occur following treatment, and transmission of drug-resistant HCV is possible (2). The rate of acute HCV infection increased in most US states between 2010 and 2014, following an ongoing epidemic in opioid/heroin use (3C5). This rising epidemic of acute HCV infection in the US gives new urgency to prophylactic vaccine development efforts. Broadly neutralizing human mAbs (bNAbs) capable of neutralizing diverse HCV strains have been isolated from HCV-infected individuals, proving that antibodies can target relatively conserved regions of the two HCV envelope glycoproteins (E1 and E2), despite the enormous genetic diversity of HCV (6C17). Infusion of bNAbs is protective against infection in animal models of HCV (17, 18), and a recent study also showed that Chicoric acid bNAbs could abrogate established HCV infection in a humanized transgenic mouse model (19). Given the efficacy of these bNAbs in blocking HCV infection, the molecular and genetic features of bNAbs and their epitopes may serve as a useful guide for rational HCV vaccine design. Studies of the evolution of HIV-specific bNAbs have enabled an entire field of germline-targeted vaccine designs and stabilization of envelope antigens (20C22). However, studies of the natural evolution of HIV bNAbs still may not be the optimal method for fully understanding the fundamental principles of breadth and potency for bNAbs, because HIV-infected individuals do not clear their infections. In contrast, approximately 30% of individuals who become infected with HCV spontaneously clear the infection (23), even though the viral diversity in HCV-infected individuals is comparable to or exceeds that of the diversity of HIV isolates in HIV-infected subjects (24C27). Spontaneous clearance of HCV has been associated with effective innate Chicoric acid and T cell responses, but we and others have shown that spontaneous clearance is associated with early appearance of broadly neutralizing antibodies against HCV in serum (28, 29). mAbs from individuals with broadly neutralizing serum and clearance of HCV have not been isolated to date, so it is not known whether these mAbs have unique features relative to the mAbs previously isolated from individuals with persistent HCV infection. It is of interest to define the molecular basis for recognition and neutralization of an entire quasispecies of an Rabbit polyclonal to IDI2 antigenically diverse virus like HCV, with subsequent immune-mediated clearance. To study this mechanism, we have followed prospectively a cohort of subjects from a time point prior to infection through the time of their spontaneous clearance of HCV. In this study, we isolated a panel of bNAbs from two of these subjects who spontaneously cleared HCV infection. We characterized the neutralizing breadth of these bNAbs, mapped the targeted epitopes, identified a germline heavy chain variable gene segment that was used by multiple bNAbs, and identified somatic mutations in one bNAb that were critical for breadth of recognition of heterologous envelope variants. We also defined the longitudinal evolution of the virus in the donor of this bNAb, allowing us to show that the bNAb unmutated.