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29/09/2014 HCV • Genetic diversity: difficult to treat and prevent – 7 genotypes; >50 subtypes – High mutation ratequasispecies • Treatment – Direct acting antivirals (DAAs) • Immunotherapeutics for HCV treatment and prevention • MAbs: well tolerated; ideal for HCV patients with liver transplants Hepatitis C virus can accumulate mutations that confer resistance to a novel broadly neutralizing antibody MAb24 Region encoding polyprotein precursor 5’ NTR C Jun Gu 9th Australasian Viral Hepatitis Conferece Alice Springs, September 2014 • Key antigenic component in antibody response NS2 NS3 NS4A NS4B NS5A NS5B (HCVD123.6E11.1B4.1E3) • Elicited to Delta3 immunization – Neutralizing antibodies to E2 are protective in vitro and in vivo – HVR1 elicits isolate specific NAb – Epitope I elicits brNAbs • Cross-reacts with E2661 proteins from 6 major genotypes • Delta3: E2 with three HVRs deleted; lead candidate for a universal HCV vaccine. stem • Inhibit E2-CD81 interaction (IC50 3μg/mL) TM HVR3/igVR Epitope I (aa410-428) MAb24 is a broadly neutralizing antibody and can cross-neutralize HCV from different genotypes 100 IC50 (mg/mL) NS1 • Mouse monoclonal antibody • Direct interaction with host cell receptor CD81 HVR2 E2 Nonstructural proteins Envelope glycoproteins MAb24 • Heterodimerize with glycoprotein E1 via TMD HVR1 E1 Nucleocapsid E2 and E2-specific antibodies E2 3’ NTR Structural proteins MAb24 epitope mapping • Epitope I (residues 412-421) – Highly conserved across genotypes – Target of brNAbs (AP33, 3-11, HCV1) – Low prevalence (2.3%) in HCV infected people 10 1 Q412 0.1 L413 I414 N415 T416 N417 G418 S419 W 420 H421 7a 5a 4a 2a 1b 1a MAb24 (mouse) • Representative of broadly NAbs elicited by Delta3 • Potential as immunotherapeutic AP33 (mouse) 3-11 (rat) HCV1 (human) 1 29/09/2014 1. Cell-free Passaging of HCV in the presence of MAb24 Aim • To determine whether HCV can generate MAb24 resistance in vitro • • HCVcc JC1FLAG G2a with luciferase reporter MAb24 Methods HCV To generate MAb24R virus, we passaged the virus with MAb24, either in cell-free manner or with infected cells Luciferase Activity MAb24 Day 1 Day 2-Day 4 Next Passage Virus was unable to generate escape mutants when passaged in the cell-free methods 2. Passaging with infected cells with MAb24 • HCV alternative transmission mode – Cell-cell spread (Timpe et al.,2008; Brimacombe et al., 2011) Cell free passaging • Advantages 4×106 – Rapid dissemination – Immune evasion IC50 IC90 RLU 3×106 no mAb24 Uninfected 2×106 1×106 • • HCVcc JC1FLAG G2a with luciferase reporter MAb24 HCV Luciferase Activity MAb24 00-1 0-2 3 11-1 1-2 1-3 4 22-1 2-2 2-3 4 33-1 3-2 3-3 4 4 4--1 4-2 4-3 4 55-1 5-2 5-3 4 66-1 6-2 6-3 4 77-1 7-2 4 0 Passage MAb24 potently inhibits cell-free transmission of HCV Day 1 Day 2 to Day 3 Next Passage Passaging with infected cells • Serves as a source for cell-cell spread; In vivo relevance Passaging with infected cells enabled the rapid emergence of MAb24 resistant virus 2X 2.0×107 MAb24 concentration 2x 5X 10X 5x 10x 20X 20x IC50 IC90 RLU 1.5×107 no mAb24 Uninfected 1.0×107 Mutations associated with MAb24 resistance • Sequence analyses on E1E2 structural region from cell-free P9 virus – No changes in E1 – Mutations accumulated exclusively in MAb24 epitope region (epitope I) 5.0×106 • Mutations 0 0--1 0-2 3 11-1 1-2 3 22-1 2-2 3 33-1 3-2 3 4 4--1 4-2 3 55-1 5-2 3 66-1 6-2 3 77-1 7-2 3 88-1 8-2 3 9 9--1 9-2 3 0.0 Passage • P9 virus was able to replicate in the presence of MAb24 at 196μg/mL • P9 virus can confer MAb24 resistance and replicate in the absence of MAb24 • N415D (71%) • N417S (29%) 2 29/09/2014 Mutations associated with MAb24 resistance N415D •Residue N415: important in MAb24 epitope recognition •disrupt the β loop stability of epitope I (Kong et al., 2012) •Enhance CD81 binding (Dhillon et al., 2010) N417S • glycan shift from N417 to N415 (Pantua et al., 2013) • Shield MAb24 epitope by glycan modification Summary • In vitro evidence that HCV can acquire MAb24 resistance by directly modifying epitope residues • Combination immunotherapies needed – MAbs that target different epitope regions of E2 Mutations have been described in chimpanzees treated with brNAb HCV1 (Morin et al., 2012) Acknowledgement Yousef Alhammad Patricia Vietheer Irene Boo Andy Poumbourios Heidi Drummer Viral Fusion Laboratory Funding NHMRC project grant 1020175 ACH2 project grant Reagent Provision Charles Rice Jens Bukh Scholarships Monash University Monash Micromon ASHM Junior Researcher Support Award 2013 Travel Award from 9th Australasian Viral Hepatitis Conference Organizing Committee 3
