JVI Accepted Manuscript Posted Online 27 January 2016 J. Virol. doi:10.1128/JVI.02837-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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A hepatitis C virus envelope polymorphism confers resistance to neutralization

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by polyclonal sera and broadly neutralizing monoclonal antibodies

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Lisa N. Wasilewski1, Ramy El-Diwany1, Supriya Munshaw2, Anna E. Snider1, Jillian K.

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Brady1, William O. Osburn1, Stuart C. Ray1,3, #Justin R. Bailey1

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Division of Infectious Diseases, Department of Medicine1, Department of Oncology3,

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and Carey Business School2, Johns Hopkins University, Baltimore, Maryland 21205.

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running title: Broad resistance to neutralizing antibodies

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abstract word count: 149

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text word count: 4023

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#Address correspondence to: [email protected]

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Center for Viral Hepatitis Research

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Johns Hopkins University

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855 N. Wolfe Street, Suite 530

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Baltimore, MD 21205

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Phone: (410) 614-6087 1

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Abstract

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Hepatitis C virus (HCV) infection is a global health problem with millions of chronically-

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infected individuals at risk for cirrhosis and hepatocellular carcinoma. HCV vaccine

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development is vital in the effort toward disease control and eradication, an undertaking

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aided by an increased understanding of the mechanisms of resistance to broadly

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neutralizing antibodies (bNAbs). In this study, we identify HCV codons that vary deep in

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a phylogenetic tree of HCV sequences and show that a polymorphism at one of these

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positions renders Bole1a, a computationally-derived, ancestral genotype 1a HCV virus,

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resistant to neutralization by both polyclonal HCV-infected plasma and multiple broadly

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neutralizing monoclonal antibodies with unique binding epitopes. This bNAb resistance

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mutation reduces replicative fitness, which may explain persistence of both

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neutralization sensitive and resistant variants in circulating viral strains. This work

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identifies an important determinant of bNAb resistance in an ancestral, representative

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HCV genome, which may inform HCV vaccine development.

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Importance

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Worldwide, more than 170 million people are infected with hepatitis C virus (HCV), the

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leading cause of hepatocellular carcinoma and liver transplantation in the United States.

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Despite recent significant advances in HCV treatment, a vaccine is needed. Control of

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the HCV pandemic with drug treatment alone is likely to fail due to limited access to

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treatment, reinfections in high risk individuals, and potential for resistance to direct

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acting antivirals (DAAs). Broadly neutralizing antibodies (bNAbs) block infection by

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diverse HCV variants and therefore serve as a useful guide for vaccine development,

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but our understanding of resistance to bNAbs is incomplete. In this study, we identify a

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viral polymorphism conferring resistance to neutralization by both polyclonal plasma and

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broadly neutralizing monoclonal antibodies, which may inform HCV vaccine

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development.

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Introduction

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Hepatitis C virus (HCV) vaccine development has been complicated by the

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extraordinary genetic diversity of the virus and rapid viral evolution in infected

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individuals (1-7). The HCV genome is replicated by an error-prone NS5B polymerase

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(8), and past studies have demonstrated that cytotoxic T lymphocytes (CTL) and

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neutralizing antibodies (NAbs) against HCV exert selective pressure that results in

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selection of CTL and NAb escape mutations in the virus (9-15). While viral escape

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mutations allow for continued proliferation in the presence of CTL and NAbs, some of

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these mutations also carry a fitness cost, reducing the replication capacity of resistant

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viral variants (9-11, 16, 17).

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Many NAbs are HCV strain-specific, but broadly-neutralizing human monoclonal

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antibodies (bNAbs) capable of neutralizing multiple diverse HCV isolates have been

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isolated, proving that NAbs can also target relatively conserved regions of the envelope

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(E1 and E2) proteins (11, 18-30). Infusion of bNAbs is protective against infection in

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animal models of HCV (22, 31), and early high-titer bNAb responses against HCV are

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associated with viral clearance in humans (3, 10, 32-35). Unfortunately, resistance to

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bNAbs can also develop, and multiple studies have demonstrated that this resistance

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sometimes results from mutations distant from bNAb binding sites (11, 36-38). Since

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bNAbs may serve as a guide for HCV vaccine development, a more comprehensive

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understanding of resistance to bNAbs is essential. 3

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Previously, our group generated a computationally derived, representative, subtype 1a

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HCV genome known as Bole1a using Bayesian phylogenetics, ancestral sequence

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reconstruction, and covariance analysis (39). We demonstrated that Bole1a is ancestral

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to most circulating genotype 1a HCV strains, it is representative of widely circulating

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strains, and the envelope genes are functional on lentiviral particles (39). This genome

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contains fewer CTL escape mutations than natural circulating strains, since

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phylogenetic reconstruction places more recent, host-specific changes, like escape

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mutations in HLA-restricted CTL epitopes, near the tips of the tree, while Bole1a falls

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near the root (40). This was confirmed in a prior study demonstrating that Bole1a

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contains more intact CTL epitopes than circulating HCV strains (40).

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In contrast to changes near the tips of the tree, changes that occur deeper in the tree,

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closer to the Bole1a sequence, may represent selection that is less host-specific. We

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hypothesized that this could include changes that enhance viral replicative fitness or

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confer resistance to bNAbs. In generation of the Bole1a genome, our analysis predicted

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a single most likely ancestral amino acid at all positions across the genome, but at

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some positions, posterior probabilities of a single ancestral amino acid were relatively

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low, suggesting complex evolution at these positions deep in a phylogenetic tree of

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diverse genotype 1a sequences. We examined 3 of these positions in the genes

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encoding E1 and E2 to determine whether variation at these positions could be

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explained by acquisition of E1E2 bNAb resistance, an increase in viral replicative

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fitness, or both (41).

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Materials and Methods

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Source of mAbs

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CBH-5 (23), HC84.22, HC84.26 (42), and HC33.4 (25) were a gift of Steven Foung

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(Stanford University School of Medicine, Stanford, California). AR3A (22) and AR4A

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(21) were a gift from Mansun Law (The Scripps Research Institute, La Jolla, California,

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USA).

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Source of plasma

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Plasma samples were obtained from the BBAASH (43) cohort (Andrea Cox, Johns

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Hopkins University School of Medicine). Samples from each of the 18 HCV-infected

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subjects who had previously shown at least 50% neutralization by 1:100 plasma dilution

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of at least 2 HCVpp in the 19 HCVpp panel previously described (33) were selected.

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HCVpp neutralization assays

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HCVpp were generated by cotransfection of pNL4-3.Luc.R-E- plasmid and an

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expression plasmid containing the Bole1a HCV E1E2 as described elsewhere (35, 44,

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45). Virus-containing medium was collected at 48 and 72 hours, pooled, and stored at -

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4oC. For infectivity and neutralization testing of HCVpp 8,000 Hep3B cells (American

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Type Culture Collection) per well were plated in flat bottom 96 well tissue culture plates

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and incubated overnight at 37oC. The following day, HCVpp were mixed with either mAb

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(10μg/ml) or heat-inactivated plasma (1:100 dilution) then incubated at 37oC for 1 hour.

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Media was removed from the cells and replaced with 50 µL of HCVpp mixture. The

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plates were placed in a CO2 incubator at 37oC for 5 hours, after which the HCVpp were

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removed and replaced with 100µL of phenol-free Hep3B media and incubated for 72

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hours at 37oC. Media was removed from the cells and 50µL of 1x Cell Culture Lysis

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Reagent (Promega) added and left to incubate for >5 minutes then 45µL from each well

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were then transferred to a white, low-luminescence 96-well plate (Berthold) and

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luciferase activity measured in relative light units (RLUs) in a Berthold Luminometer

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(Berthold Technologies Centro LB960). Pseudoparticle infection was measured in the

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presence of mAb/test plasma (HCVppRLUtest) or nonspecific IgG/HCV-negative normal

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human plasma (HCVppRLUcontrol) at the same dilution. The percentage of neutralization

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was calculated as 100% x [1-(HCVppRLUtest /HCVppRLUcontrol)]. Each sample was tested

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in duplicate. A mock pseudoparticle (no envelope) was used as a negative control.

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Neutralization was tested only for HCVpp with infectivity at least 10X greater than

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typical mock pseudoparticle values.

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Generation of site-directed mutants

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E1E2 site-directed mutants were generated using a QuikChange Lightning Site-Directed

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Mutagenesis Kit (Agilent Technologies).

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HCV NS5A immunostaining

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Human hepatoma Huh7.5.1 cells (a gift of Jake Liang, NIH, Bethesda, Maryland, USA)

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were maintained in DMEM supplemented with 10% fetal bovine serum, 1% sodium

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pyruvate, and 1% l-glutamate. 48 hours after infection, cells were fixed with 4%

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formaldehyde for 20 minutes and then stained for HCV NS5A using primary anti-NS5A

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antibody 9E10 (a gift of Charles Rice, The Rockefeller University, New York City, New

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York, USA) at 1:10,000 dilution in PBS, 3% bovine serum albumin, and 0.3% Triton X-

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100 for 1 hour at room temperature. Cells were washed twice with PBS and stained

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using secondary antibody Alexa Daylight 488–conjugated goat anti-mouse IgG (Life

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Technologies) at 1:500 dilution in PBS, 3% bovine serum albumin, and 0.3% Triton X-

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100 for 1 hour at room temperature. Cells were washed twice in PBS and then stored

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covered in 100μl PBS at 4°C.

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HCVcc neutralization assays

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Huh7.5.1 cells were plated at 10,000 cells per well onto 96-well plates. On the following

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day, antibodies were serially diluted 2.5-fold in growth medium starting from 50μg/ml

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mAb. Controls were performed with a similar dilution series of nonspecific IgG. Viral

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supernatants diluted to fall in a linear range of infectivity were added to antibody

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dilutions in duplicate for 1 hour at 37°C. Medium was removed from cells, and the

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antibody-virus mix added onto cells and incubated overnight. Antibody-virus mix was

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replaced with 100μl fresh medium the following day. After 48 hours, medium was

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removed and cells were fixed and stained. Images were acquired and spot forming units

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were counted in the presence of mAb (HCVccSpotstest) or nonspecific IgG

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(HCVccSpotscontrol)using an AID iSpot Reader Spectrum 219 operating AID ELISpot

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Reader version 7.0. Percent neutralization was calculated as 100% x [1-(HCVccSpotstest

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/HCVccSpotscontrol)]. HCVcc neutralization assays using human plasma were performed

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similarly using 1:100 dilutions of test plasma and HCV-negative normal human plasma.

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HCV E1E2 ELISA

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MAb or plasma binding to E1E2 was quantitated using an enzyme-linked immuosorbent

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assay (ELISA) as previously described (11). 293T cells were transfected with E1E2

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expression constructs. 48 hours post-transfection cell lysates were harvested. Plates

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were coated with 500ng Galanthus nivalis (GNA) lectin (Sigma-Aldrich) and blocked

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with phosphate-buffered saline containing 0.5% Tween 20, 1% non-fat dry milk, and 1%

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goat serum. E1E2 cell lysates were added. mAb or plasma were assayed in duplicate

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2.5-fold serial dilutions, starting at 10µg/ml or 1:100 dilution, respectively. Binding was

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detected using HRP-conjuagated anti-human IgG secondary antibody (BD Pharmingen

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no. 555788). For ELISA performed under denaturing conditions, cells lysates were

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diluted in denaturing buffer (tris-buffered saline with 10% fetal bovine serum, 1.0%

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sodium dodecyl sulfate, and 50mM dithiothreitol) then boiled for 5 minutes. Lysates

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were cooled on ice then added to GNA-lectin coated plates. Assay was completed as

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described above.

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HCV viral load quantitation

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HCV RNA levels in infection supernatants were quantified using a process of RNA

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extraction and utilization of commercial real time reagents (Abbot HCV Real Time

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Assay) migrated onto a research based real time PCR platform (Roche 480 Lightcycler).

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Generation of HCVcc chimeras

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To introduce an AfeI restriction site, HCVcc chimera H77/JFH1 (46), a gift of Jens Bukh

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(Copenhagen University Hospital, Copenhagen, Denmark), was amplified in three

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sections, using primers

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insert_R_new (CACCAGCTGATATAGCGTTTGTAATATGGCGACAGAGTC)

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insert_F_new

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(GGATTCCGATCTACCAGCGCTTTGGAGAACCTCGTAATACTCAATGCAGCATCCC

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TGGCC) back_mid_F (CGGAATATGACCTGGAGCTAATAACATCC)

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backbone_outer_R (GGTGACATGGTAAAGCCCCG)

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Back-mid_R (CCAGGTCATATTCCGGTCTGG)

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Backbone_outer_F (CTGTCGCCATATTACAAACGC)

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This omitted nucleotides 916 to 2579. Amplified sections were re-assembled using In-

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Fusion cloning (Clontech). This backbone was digested with enzyme AfeI (New England

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Biolabs). E1E2 inserts were amplified from library plasmids using primers

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HCVcc_E1E2_1R (GAGGTTCTCCAAAGCCGCCTCCGC) and HCVcc E1E2_1F

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(TGTGCCCGCTTCAGCCTACCAAG). The inserts were cloned into the digested HCVcc

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backbone using In-Fusion cloning.

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To make HCVcc RNA, 2μg plasmid DNA was linearized using XBAI (New England

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Biolabs). Linear DNA was used for in vitro RNA transcription using the T7 MEGAscript

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kit (Ambion). RNA clean-up was performed using RNeasy mini kit (Qiagen) and

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quantified using nanodrop. RNA products were then stored at –80°C.

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To transfect RNA, Nucelofector Kit T was used (Amaxa). 5µg of RNA was transfected

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into 1.8e6 Huh7.5.1 cells and plated in a 6-cm plate. Transfection supernatants were

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collected 4-6 days later, once cells had reached confluency and stored at -80 oC for

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titering by HCV NS5A immunostaining. High-titer transfection supernatants were used

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to infect Huh7.5.1 cells and infection supernatants collected, tittered, and stored at -80

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C for use in infectivity and neutralization experiments.

Western Blotting HCVpp supernatants were concentrated and purified by ultracentrifugation through a

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20% sucrose cushion (123 000g, 2h, 4ºC) with pellets resuspended in TNE buffer.

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HCVpp and E1E2 lysates used in ELISAs were diluted then denatured and run on a 4-

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12% Bis-Tris gel. After transfer, blots were probed with HC33.1.53 (25) (a gift of Dr.

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Steven Foung, Stanford University School of Medicine, Stanford, California) and binding

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was detected with HRP-conjugated anti-human IgG secondary antibody (BD

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Pharmingen no. 555788). HCVpp western blots were also probed with a mouse anti-

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HIV1 p24 (ab9044, Abcam) followed by HRP-conjugated anti-mouse IgG secondary

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antibody (ab97265, Abcam).

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Statistical Analyses

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In Figure 2, percent neutralization values were grouped by individual mutations and a

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one-way ANOVA with a Tukey test was performed to compare the significance of

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changes in neutralization mediated by different amino acid mutations at the same

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position. For each pair of R424S ELISAs, a two-way ANOVA was performed to

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determine the significance of the change in binding between E1E2 lysates.

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Results

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Some polyprotein positions show evidence of deep phylogenetic variation.

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Through the use of Bayesian phylogenetics, ancestral sequence reconstruction, and

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covariance analysis, the most likely ancestral genotype 1a amino acid was predicted at

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each position across the HCV polyprotein, generating the Bole1a genome (39). For the

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majority of polyprotein positions, the most likely ancestral amino acid could be

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determined with very high posterior probability ranging from 0.99 to 1, but for 94 of the

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3012 codons analyzed, the posterior probability of the most likely ancestral codon was

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less than 0.99 (Figure 1A), suggesting complex evolution at these positions deep in the

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phylogenetic tree. We selected three of these positions for further analysis. At 242,

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which falls in HCV E1, leucine (L) was the most likely ancestral amino acid. At 424,

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which falls near the CD81 binding site of E2, arginine (R) was the most likely ancestral

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amino acid. At 570, which falls in the intergenotypic variable region (igVR) (47) near the

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carboxy terminus of E2, asparagine (N) was most likely. We also determined the most 10

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common amino acids at these positions in circulating viral strains (Figure 1B).

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Circulating strains showed significant variability at the 242 and 570 positions, with 4 and

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9 different amino acids observed at each position, respectively, in a reference alignment

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of 390 Genotype 1a sequences. In contrast, the 424 codon is highly constrained, with

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either R or S observed in 387 of 390 circulating strains in the reference alignment.

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Mutation of arginine 424 to serine confers resistance to neutralization by plasma.

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HCV pseudoparticles (HCVpp) with Bole1a E1E2_L242, R424, N570 or Bole1a E1E2

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with other naturally-occurring amino acids at these positions were generated by

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cotransfection of E1E2 expression plasmids with an HIV pNL4-3.Luc.R-E- reporter

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construct. The amino acid polymorphisms to be tested were chosen based on the

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predicted ancestral sequence, the most common amino acids in circulating strains, and

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some additional less frequently observed amino acids. To efficiently measure the effect

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of mutations at each of the three positions in multiple experiments, thirteen HCVpp were

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generated with each mutation alone or combination with mutations at one or both of the

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other two positions. These HCVpp were tested for sensitivity to neutralization by HCV-

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infected plasma samples, and all variants with the same amino acid at 242, 424, or 570

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were grouped for analysis. (Figure 2). Mutation of L242 to valine (V) or methionine (M)

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and mutation of N570 to valine (V) or aspartic acid (D) did not have a significant effect

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on neutralization sensitivity, but mutation of arginine (R) 424 to serine (S) conferred a

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significant increase in resistance to neutralization by plasma. Since variation at

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positions 242 and 570 did not significantly affect Bola1a neutralization sensitivity,

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subsequent experiments focused on variation at the 424 position, with L242 and N570

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held constant. 11

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To confirm the resistance phenotype of S424, Bole1a_R424 and Bole1a_S424 HCVpp

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were tested for neutralization by an additional panel of 19 HCV-infected plasma

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samples (Figure 3A). As in prior experiments, S424 was strongly associated with

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resistance to neutralization (median neutralization 91% for Bole1a_R424 and 44% for

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Bole1a_S424, p=2E-10). Strikingly, for every plasma sample tested, Bole1a_S424 was

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more neutralization resistant than Bole1a_R424, despite the fact that each plasma

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sample was from a different donor and each sample presumably contains polyclonal

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NAbs. This result was confirmed with replication competent cell culture virus (HCVcc)

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expressing either Bole1a_R424 or Bole1a_S424 E1E2 ( Figure 3B). In addition,

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mutation of R424 to S in two natural E1E2 variants isolated from HCV-infected donors

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(1a53 and 1a79) also conferred resistance to neutralization by heterologous HCV-

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infected plasma, and mutation of S424 to R in a third naturally occurring E1E2 variant

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(H77) conferred increased sensitivity to neutralization (Figure 3C). To investigate

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whether changes in neutralization sensitivity observed in Bole1a_R424 and

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Bole1a_S424 might be influenced by incorporation of different amounts of E2 per

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particle, we performed a Western blot using purified HCVpp (Figure 3D). Both E2 and

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the p24 loading control of Bole1a_S424 HCVpp were approximately 2-fold less intense

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than the same proteins from Bole1a_R424 HCVpp, indicating that both HCVpp

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incorporate approximately equal amounts of E2 protein per particle.

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Mutation of arginine 424 to serine confers resistance to binding by antibodies in plasma.

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To elucidate the mechanism of variable neutralization sensitivity between Bole1a_R424

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and Bole1a_S424 variants, the same panel of 19 HCV-infected plasma samples was

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used in E1E2 binding assays. Lysates of cells transfected with Bole1a_R424 or 12

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Bole1a_S424 E1E2 expression constructs were used as the target antigen in enzyme-

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linked immunosorbent assays (ELISAs) with HCV-infected plasma (Figure 4A). Binding

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of plasma antibodies to the S424 E1E2 protein was significantly lower than binding to

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the R424 variant (median OD 1.44 for Bole1a_R424 and 0.0009 for Bole1a_S424,

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p=1E-05), suggesting that mutation of R424 to S confers neutralization resistance by

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reducing binding of NAbs. To confirm that the reduced binding to Bole1a_S424 E1E2

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relative to Bole1a_R424 was not due to a difference in levels of expression of the two

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E1E2 proteins, we performed a Western blot with serial dilutions of both E1E2 lysate

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preparations, confirming that the quantity of each protein used in the ELISAs was

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similar (Figure 4B).

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Mutation of arginine 424 to serine confers resistance to neutralization by a diverse array

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of broadly neutralizing monoclonal antibodies.

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To further investigate the surprising result that an R424S mutation conferred resistance

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to polyclonal sera from numerous donors, we measured sensitivity of replication

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competent cell culture virus (HCVcc) expressing Bole1a_R424 or Bole1a_S424 E1E2 to

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neutralization by a panel of some of the most broadly neutralizing human monoclonal

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antibodies (mAbs) described to date, including CBH-5 (23), HC84.22, HC84.26 (42),

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HC33.4 (25), AR3A(22) and AR4A (21). These mAbs were selected because they are

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broadly neutralizing and also because they bind to distinct epitopes across E2. CBH-5,

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AR3A, HC84.22, and HC84.26 bind to distinct epitopes at or near the CD81 binding site

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of E2, while HC33.4 binds near the amino terminus of E2, and AR4A binds to E1 and

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the carboxy terminus of E2 (Figure 5A). Amino acid 424 is a known binding residue for

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AR3A as determined by alanine scanning mutagenesis (22), but it is not a known 13

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binding residue for any other mAbs in the panel. Despite the differences in E2 binding

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sites of these bNAbs, mutation of R424 to S conferred resistance to each antibody

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(Figure 5B). As shown in Figure 5C, 50% inhibitory concentration (IC50) of each mAb

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against Bole1a_R424 HCVcc was less than 2 µg/mL, while IC50 of the majority of mAbs

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against Bole1a_S424 HCVcc was 50 µg/mL or greater. Similar results were observed

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with HCVpp expressing Bole1a_R424 or S424 E1E2 and with HCVpp expressing R424

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and S424 variants of a naturally occurring HCV strain, 1a53 (Figures 5D and 5E).

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Interestingly, no difference in mAb sensitivity was observed between R424 and S424

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variants of two other naturally occurring HCV strains, 1a79 and H77. Taken together,

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these results indicate that mutation of Bole1a_R424 to S confers resistance to bNAb

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neutralization, regardless of the antibody binding epitope.

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Mutation of arginine 424 to serine confers resistance to binding of broadly neutralizing

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monoclonal antibodies.

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Binding of mAbs to Bole1a_R424 and Bole1a_S424 E1E2 was measured using an

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E1E2 ELISA. MAbs HC84.22, CBH-5, AR3A, and HC33.4 showed greater binding to

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the R424 E1E2 lysate than the S424 E1E2 lysate (Figures 6A and 6B). There was no

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detectable difference in binding of AR4A to either variant, suggesting either that the

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Bole1a_S424 resistance to neutralization by AR4A is not mediated by a reduction in

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binding of the mAb, or that the binding ELISA is not sensitive enough to detect changes

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in binding affinity sufficient to influence neutralization. Taken together, these results

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show a small but consistent reduction in binding of mAbs to S424 E1E2 relative to R424

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E1E2, in agreement with the observed reduction in binding of plasma antibodies to the

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S424 E1E2 protein, suggesting that resistance to bNAb binding is a likely mechanism of

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the observed neutralization resistance of Bole1a_S424.

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Since mutation of R424 to S conferred resistance to binding of mAbs for which 424 is

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not a known binding residue, we tested whether the effect is due to an induced

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structural change in the E1E2 proteins. We were able to test this directly for one mAb,

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HC33.4, since it binds to both native and denatured protein. Binding of HC33.4 to

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Bole1a_R424 and Bole1a_S424 E1E2 protein lysates was measured with the E1E2

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protein in either native or denatured states (Figure 6B). Surprisingly, the difference in

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mAb HC33.4 binding to Bole1a_R424 and Bole1a_S424 E1E2 was significantly more

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pronounced with the protein in a denatured state, suggesting that induction of a

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structural shift may not be the mechanism by which mutation of R424 to S reduces

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binding of HC33.4 Rather, R424 could be a previously undetected HC33.4 binding

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residue.

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We also measured binding of four mAbs to R424 and S424 variants of 1a53, 1a79, and

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H77 E1E2 proteins (Figure 6C). Introduction of S424 into 1a53 E1E2 reduced binding

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of all four mAbs, which is consistent with the observed resistance to these mAbs

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conferred to 1a53 HCVpp by S424. For 1a79 and H77 E1E2, S424 conferred

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resistance to binding by some mAbs but not others, which is also consistent with

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minimal resistance to mAb neutralization conferred to 1a79 and H77 HCVpp by S424.

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Bole1a_S424 carries a fitness cost.

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Despite the broad neutralization resistance conferred by S424, both R424 and S424

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variants are abundant in circulating virus populations, suggesting selection pressure 15

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favoring R424 acting in opposition to the bNAb selection pressure favoring S424. We

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examined the relative fitness of Bole1a_R424 HCVcc and Bole1a_S424 HCVcc in the

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absence of antibody (Figure 7). Bole1a_R424 HCVcc showed higher specific infectivity

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than Bole1a_S424 HCVcc (18.9 SFU/105 IU for Bole1a_R424 vs. 5.4 SFU/105 IU for

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Bole1a_S424). Taken together, these results suggest that selection by bNAbs favors

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persistence of S424 variants, while greater replicative fitness favors persistence of

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R424 variants.

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Discussion

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In this study, we identified a position in E2, codon 424, that varies deep in a

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phylogenetic tree of diverse genotype 1a sequences, yet is constrained to only two

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possible amino acids, arginine and serine, in circulating strains. Mutation of R424 to S

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at this position in Bole1a, a representative, ancestral HCV virus, confers extremely

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broad resistance to neutralization by both polyclonal HCV-infected sera and a diverse

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array of broadly neutralizing mAbs. We showed that this neutralization resistance is due

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to a reduction in binding of NAbs. We also showed that the neutralization resistant

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Bole1a_S424 HCVcc variant has reduced replicative fitness relative to the R424 variant,

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providing an explanation for persistence of both R424 and S424 polymorphisms in

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circulating viral strains.

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This work supports prior studies showing that HCV resistance to NAbs may arise from

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mutations distant from antibody binding sites (11, 36-38). The resistance conferred to

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Bole1a by S424 is exceptionally broad. This mutation conferred resistance to the

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polyclonal NAbs present in every plasma sample tested from dozens of unique donors,

16

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as well as a panel of bNAbs selected specifically because their binding epitopes are

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well-characterized and distant from each other. This work provides evidence that

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selective pressure from commonly expressed bNAbs and competing pressure for high

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replicative fitness may explain some amino acid changes deep in phylogenetic trees of

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diverse HCV sequences. Analysis of phylogenetically ancestral evolution may be a

369

useful method to identify bNAb resistance mutations that are immunologically relevant

370

at a population level.

371

It is interesting that the R424S mutation in Bole1a reduces binding of most mAbs and

372

neutralization of all mAbs given that 424 is a known binding residue only for mAb AR3A.

373

R424 may be a relatively minor, previously undetected binding residue for these mAbs

374

with an unexpectedly significant influence on neutralizing activity. Alternatively, this

375

mutation could influence glycosylation of the protein, although this seems less likely as

376

it does fall at the first or third position of an N-linked glycosylation consensus sequence.

377

It is also possible that the mutation alters the structure of E2, given the known

378

structurally flexibility of the E2 protein in the region of 424 (48, 49). We were able to

379

show for mAb HC33.4 that the reduction in binding to Bole1a_S424 was present even

380

when Bole1a_R424 and Bole1a_S424 E1E2 were denatured, making it less likely that

381

the effect is mediated by a shift in structure. These binding experiments are technically

382

challenging, however, and the possibility of mutation-mediated structural shifts remain

383

an intriguing possibility that warrant further investigation.

384

This study showed consistent and broad neutralization resistance conferred to Bole1a

385

by R424S. The magnitude of the change in resistance to polyclonal serum antibodies

386

was not large, but it was similar to the resistance described in a prior study of HCV 17

387

neutralization escape (37). The effect of R424S was most striking for neutralization of

388

Bole1a HCVcc by broadly neutralizing monoclonal antibodies (Figure 5C), as a more

389

than 100-fold increase in resistance to some bNAbs was observed. It is particularly

390

interesting that the resistance conferred by S424 varied between Bole1a and other

391

E1E2 variants. Work to understand the influence of combinations of polymorphisms on

392

neutralization resistance of E1E2 is ongoing. A better understanding of polymorphisms

393

modulating neutralization sensitivity of representative genomes like Bole1a is critical as

394

work continues to identify the most representative and immunogenic HCV variants for

395

inclusion in HCV vaccines.

396

In conclusion, we have identified a neutralization resistance polymorphism using a

397

unique strategy which identifies amino acid changes deep in a phylogenetic tree of

398

diverse HCV sequences. This R424S polymorphism confers extremely broad resistance

399

to neutralization by both polyclonal HCV-infected sera and a diverse array of broadly

400

neutralizing mAbs with distinct binding epitopes. The neutralization resistant variant

401

S424 has reduced replicative fitness which may explain the persistence of both S424

402

and the neutralization sensitive R424 variant in the population. A more complete

403

understanding of determinants of bNAb resistance in candidate vaccine antigens like

404

Bole1a should help to guide HCV vaccine development.

405

Funding Information

406

The funders had no role in study design, data collection and interpretation, or the

407

decision to submit the work for publication.

18

408

This study was supported by NIH grants 1K08 AI102761 (Justin Bailey), U19 AI088791

409

(Stuart Ray), and R37 DA013806 (Stuart Ray) and the Johns Hopkins University Center

410

for AIDS Research grant 1P30AI094189 (Justin Bailey).

411 412

Acknowledgements

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We would like to thank the faculty members and staff of the Viral Hepatitis Center at

414

Johns Hopkins University School of Medicine as well as participants in the Baltimore

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Before-and-After Acute Study of Hepatitis (BBAASH). Thanks also to Jeffry Quinn for

416

help measuring HCVcc viral loads.

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A Hepatitis C Virus Envelope Polymorphism Confers Resistance to Neutralization by Polyclonal Sera and Broadly Neutralizing Monoclonal Antibodies.

Hepatitis C virus (HCV) infection is a global health problem, with millions of chronically infected individuals at risk for cirrhosis and hepatocellul...
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