In this map, the HCVcc strains segregated into six distinct neutralisation clusters. with HCV genotype (GT) 1C6 across 13 HCV strains representing five viral GTs. Using metric multidimensional scaling, we plotted HCV neutralisation onto neutralisation maps. We employed K-means clustering to guide computer virus clustering and selecting representative strains. Results Viruses differed greatly in neutralisation sensitivity, with J6 Picoprazole (GT2a) being most resistant and SA13 (GT5a) being most sensitive. They mapped to six distinct neutralisation clusters, in part composed of viruses from different GTs. There was no correlation between Fgfr2 viral neutralisation and genetic distance, indicating functional neutralisation clustering differs from sequence-based clustering. Calibrating reference viruses representing these clusters against purified antibodies from 496 patients infected by GT1 to GT6 viruses readily identified individuals with remarkable potent and broadly neutralising antibodies. It revealed comparable antibody cross-neutralisation and diversity between specimens from diverse viral GTs, confirming well-balanced reporting of HCV cross-neutralisation across highly diverse human samples. Conclusion Representative isolates from six neutralisation Picoprazole clusters broadly reconstruct the functional HCV neutralisation space. They enable high resolution profiling of HCV neutralisation and they may reflect viral functional and antigenic properties important to consider in HCV vaccine design. Keywords: hepatitis C, HCV, liver, genotype, immunology in hepatology Significance of this study What is already known about this subject? Humoral and cellular responses are critical to protect from chronic HCV contamination. HCV is usually genetically highly diverse. Different assays are used to measure computer virus neutralisation in vitro. What are the new findings? Six reference viruses representing six functionally distinct computer virus neutralisation clusters reconstruct the functional HCV neutralisation space. The virus sequence does not predict mapping to these functional clusters. Functional diversity differs from genetic diversity. How might it impact on clinical practice in the foreseeable future? These reference viruses facilitate the characterisation of the role of antibodies in HCV protection and prioritisation of vaccine candidates. Vaccine candidates addressing viral functional diversity rather than genetic diversity may induce superior protective immunity. Introduction HCV has chronically infected an estimated 71 million people worldwide and is therefore a global health problem.1 Of those who are infected, 50%C80% progress to chronicity and are at risk to develop liver cirrhosis and hepatocellular carcinoma. The introduction of highly effective direct-acting antivirals (DAAs) has revolutionised patient care.2 However, providing access to DAAs for all patients worldwide remains a Picoprazole major public health challenge. In addition, in rare cases, therapy fails due to resistance-associated variants3 and HCV reinfection is possible after treatment-induced cure.4 5 Thus, a prophylactic vaccine is important for control of HCV disease burden. One approach of vaccine development capitalises on the induction of broadly neutralising antibodies (bNAbs) targeting envelope glycoproteins E1 and E2. Multiple studies support an important role of neutralising antibodies for spontaneous HCV clearance.6C9 Moreover, passive immune-prophylaxis studies in animals confirm the importance of antibodies in protecting from HCV infection.10C12 HCV is highly variable and viral isolates are classified into eight genotypes (GTs) and multiple subtypes.13 The envelope proteins are targets of neutralising antibodies and represent the most variable proteins of the virus. For successful vaccine design, it is crucial to elicit cross-protective antibodies against diverse HCV variants, and recent vaccination approaches take this requirement into account. However, use of different experimental systems for quantification of antibody responses complicates comparison of vaccine efficacy. Typically, either infection assays based on retroviral HCV pseudoparticles (HCVpp) or cell culture-derived HCV (HCVcc) particles are used to quantify antibody efficacy. In recent years, several panels of HCVpp or HCVcc were developed.14 In part, these panels include a large number of different E1-E2 proteins.15 However, in some cases, these panels only encompass GT1-derived glycoproteins.16 17 It is unclear if these GT-selective panels adequately report the entire functional diversity of globally sampled HCV, including GT 1 to 7 strains. In other cases, viruses with E1-E2 genes carrying cell culture-adaptive changes, which may influence virus antibody neutralisation, are included.18 Moreover,.