mAbClust with AlphaFold 3 avoids hallucinations to define a quaternary broadly neutralizing HCV epitope.

The hepatitis C virus (HCV) envelope glycoprotein E1E2 heterodimer is the target of broadly neutralizing antibodies (bNAbs). Although prior studies have indicated that E1-dependent bNAbs are associated with spontaneous clearance of HCV, all E1-dependent human monoclonal antibodies (mAbs) have been isolated from individuals with chronic HCV infection. Here, we isolated E1-dependent bNAbs from an individual with high neutralizing antibody breadth who spontaneously cleared HCV, showing that these bNAbs bind to four distinct sites on E1E2. We also developed mAbClust, an algorithm that improves identification of accurate AlphaFold 3 (AF3) structure predictions of antigen-antibody complexes. We used AF3 and mAbClust to generate a high-confidence predicted structure of an E1-dependent bNAb in complex with E1E2, showing that this bNAb binds to a quaternary epitope spanning E1 and E2. This study identifies four neutralizing sites and a quaternary bNAb epitope associated with HCV control, which can guide HCV vaccine design. AF3 with mAbClust could have broad applications for accurate epitope mapping of antibodies.